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Regualtion of intracellular pH in cultured postnatal rat hippocampal neurons : the potential role of… Cheng, Yen May 2005

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R E G U L A T I O N O F I N T R A C E L L U L A R p H IN C U L T U R E D P O S T N A T A L R A T H I P P O C A M P A L N E U R O N S : T H E P O T E N T I A L R O L E O F A V O L T A G E - G A T E D P R O T O N C O N D U C T A N C E by Y E N M A Y CHENG B.Sc., The University of British Columbia, 2003 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE F A C U L T Y OF GRADUATE STUDIES (Physiology) THE UNIVERSITY OF BRITISH COLUMBIA August, 2005 © Yen May Cheng, 2005 11 ABSTRACT First studied in snail neurons, voltage-gated proton conductances (gH+) have since been described in a number of cell types. While their presence in mammalian neurons has not been formally shown, previous evidence from our laboratory suggests that a gH+ may act to limit the internal acid load imposed by anoxia in rat hippocampal neurons. Thus, in the present study, the potential role of a gH+ in intracellular pH (pH;) regulation was examined in cultured postnatal rat hippocampal neurons by i) measuring the changes in [Ca 2 +] t and pHj evoked by membrane depolarization in neurons loaded with Ca - and/or pH-sensitive ratiometric fluorophores and ii) attempting to isolate H + currents in neurons voltage-clamped in the whole-cell configuration. All experiments were performed under nominally HC0 37C02-free, HEPES-buffered conditions. Consistent with previous reports, under control conditions (3 mM K + 0 , 2 mM C a 2 + 0 , pH 0 7.35, 37°C), exposure to 25 - 139.5 mM [K + ] 0 caused reversible increases and decreases in [Ca2+]i and pHj, respectively. Under 0 C a 2 + 0 conditions, the same stimuli failed to affect [Ca2+]j but caused increases in pHj that were dependent on [K + ] 0 and, thus, membrane voltage. Consistent with the properties of gH+s in other cell types, the rise in pHj was sensitive to Z n 2 + and was dependent on the transmembrane pH gradient (ApH m e r nb). Increasing ApH m e mb by treatment with the protonophore FCCP prior to high [K + ] 0 exposure enhanced both the rise in pHi and the inhibitory effects of Zn , suggestive of increased acid extrusion via a gH+. Under 0 Ca 0 , pH 0 7.8 conditions, the inhibitory effects of Z n 2 + at any given ApH m e mb were further enhanced, consistent with a pH0-dependent inhibition of the putative gH+ by Zn 2 + . Additionally, under conditions designed to isolate H + currents, voltage-dependent outward currents that appeared to show some selectivity for protons were recorded from hippocampal neurons. However, perhaps due to technical issues related to the study of H + currents, and in contrast to the results of the Ill microspectrofluorimetric studies, the currents were not sensitive to Z n 2 + or temperature. Nonetheless, together these results suggest that a gH+ may be present in rat hippocampal neurons and may contribute to H + efflux under depolarizing conditions. iv TABLE OF CONTENTS Page Abstract ii Table of Contents iv List of Tables vii List of Figures viii Acknowledgements x 1. Introduction 1 1.1. Physiology, pathophysiology and pH 1 1.2. pHj and neuronal excitability 2 1.3. Regulation of pHj in rat hippocampal neurons 3 1.3.1. Acid extrusion mechanisms 4 1.3.2. Acid loading mechanisms 5 1.3.3. Other mechanisms that may affect pH, 5 1.4. Voltage-gated proton conductances 7 1.4.1. General properties 7 1.4.1.1. Historical perspective 7 1.4.1.2. Varieties of gH+s 8 1.4.1.3. High proton selectivity 8 1.4.1.4. Small unitary conductance 9 1.4.1.5. Strong dependence on temperature 10 1.4.1.6. Voltage-dependent gating 11 1.4.1.7. Modulation of voltage-dependent gating by pH 12 1.4.1.8. Inhibition by polyvalent metal cations 12 1.4.1.9. Physiological modulators 14 1.4.2. Functions and properties in specific cell types 15 1.5. Overview and obj ectives 16 2. Materials and Methods 21 2.1. Neuronal cultures 21 2.2. Microspectrofluorimetry 22 2.2.1. S olutions and test compounds 22 2.2.2. Ratiometric dyes and cell-loading 23 2.2.3. The ratiometric method and experimental set-up 24 2.2.4. Calculation of pH, and [Ca2+]s 26 2.2.4.1. BCECF 26 2.2.4.2. HPTS 28 2.2.4.3. SNARF-5F 29 2.2.4.4. Fura-2 30 2.2.5 Experimental manoeuvres 30 2.2.6. Analysis of microspectrofluorimetric data 31 V 2.2.6.1. Steady-state pHi and [Ca2+]\ changes 31 2.2.6.2. Initial rates of increase of pH, 32 2.3. Electrophysiology 33 2.3.1. Solutions and chemicals 33 2.3.2. Experimental set-up and electrophysiological recordings 34 2.3.3. Experimental protocols 36 2.3.3.1. Current-clamp recordings 36 2.3.3.2. Steady-state voltage-clamp recordings 36 2.3.3.3. Tail current recordings 37 2.3.3.4. Simultaneous microspectrofluorimetry and electrophysiology 37 2.3.4. Analysis of electrophysiological data 37 2.4. Additional statistical procedures 39 3. Results 55 3.1. Microspectrofluorimetric studies in intact neurons 55 3.1.1. Depolarizing conditions cause a fall in pHj in the presence of external Ca 2 + . . . 55 3.1.1.1. pH; and [Ca2+]j responses to high [K + ] 0 exposure 55 3.1.1.2. pHj and [Ca2+]j responses to depolarization induced by veratridine 56 3.1.1.3. Role of a plasmalemmal Ca 2 + , H+-ATPase 57 3.1.2. Depolarization in the absence of external C a 2 + causes an intracellular alkalinization 59 3.1.2.1. pHj and [Ca2+]j responses to high [K + ] 0 and veratridine under 0 C a 2 + 0 conditions 60 3.1.2.2. Measurements of membrane potential with changing [K + ] 0 61 3.1.2.3. Contributions of known proton efflux pathways to the rise in pHj 61 3.1.3. Contributions of a gH+ to the H + efflux caused by depolarization under 0 C a 2 + 0 conditions 63 3.1.3.1. The rise in p H and its inhibition by Z n 2 + are sensitive to the transmembrane pH gradient 63 3.1.3.1.1. The effects of Z n 2 + i n neurons with a normal ApHmemb 63 3.1.3.1.2. The effects of Z n 2 + i n neurons with an enhanced A p H m e r n b 64 3.1.3.2. The effects of removing extracellular Z n 2 + with DTPA 66 3.1.3.3. The effects of increased pH 0 67 3.1.3.4. The effects of reducing temperature 68 3.1.4. Summary of microspectrofluorimetric findings 68 3.2. Electrophysiological studies in neurons patch-clamped in the whole-cell configuration 69 3.2.1. Characterization of currents recorded under conditions designed to isolate H + currents 69 3.2.1.1. Depolarizing voltage-steps evoke outward currents that slowly run-up... 69 3.2.1.2. Contribution of ions other than protons to the current 70 3.2.1.3. The effect of changing ApHmemb on F r e v 71 3.2.1.4. Gating kinetics 72 3.2.2. Contribution of a gH+ to the outward currents 73 3.2.2.1. The effects of extracellular Z n 2 + 73 3.2.2.2. The effects of increased temperature 73 3.2.3. Summary of results of electrophysiological studies 74 vi 4. Discussion 121 4.1. The pHj response to depolarization is dependent on the presence of external C a 2 + 121 4.1.1. TheroleofaCa 2 + ,H + -ATPase 121 4.1.2. The rise in pHj observed in the absence of external Ca is not due to known rat hippocampal neuron H + efflux pathways 122 4.2. A gH+ likely contributes to voltage-dependent H + efflux from rat hippocampal neurons '24 4.3. Estimation of current density from H+fluxes 127 4.4. Mediators of Zn2+-insensitive, voltage-dependent H + efflux under 0 C a 2 + 0 conditions 128 4.5. The depolarization-evoked outward current is carried in part by protons 129 4.6. Comparison of outward currents with H + currents from other cell types 130 4.6.1. Current run-up 130 4.6.2. Gating kinetics 131 4.6.3. Sensitivity to Zn 2 + and changes in temperature 132 4.7. Technical considerations and future directions 133 4.8. Conclusions and functional implications 135 5. References 141 6. Appendix 154 vii LIST OF TABLES Page Table 1.1 Comparison of varieties of H + currents 18 Table 2.1 Compositions of experimental solutions commonly used in microspectrofluorimetric studies 40 Table 2.2 List of pharmacological agents used in microspectrofluorimetric experiments 41 Table 2.3 Composition of solutions used in whole-cell voltage-clamp experiments designed to isolate H + currents 42 Table 3.1 Magnitudes of high [K+]0-induced changes in [Ca2+]j and pHj in the presence of external C a 2 + 75 Table 3.2 Comparison of pH, measurements made with BCECF, HPTS and SNARF-5F in the presence of external C a 2 + 76 Table 3.3 The effects of the PMCA inhibitor eosin B on the pHj and [Ca2+]j responses to 75 mM [K + ] 0 in the presence of external C a 2 + 77 Table 3.4 Magnitude and time course of the internal alkalinizations induced by high [K + ] 0 in the absence of external C a 2 + 78 Table 4.1 Comparison of outward currents recorded in rat hippocampal neurons with varieties of H + currents from other cell types 138 viii L I S T O F F I G U R E S Page Figure 1.1 Plasmalernmal pHj regulating mechanisms in rat hippocampal neurons and the consequences of their activation on pHj 19 Figure 2.1 Schematic representation of the optical equipment used with neurons single-loaded with a dual-excitation fluorophore 43 Figure 2.2 Schematic representation of the optical equipment used to measure pHj and [Ca 2 +]i in hippocampal neurons co-loaded with the dual-emission dye SNARF-5F and the dual-excitation dye fura-2 45 Figure 2.3 Full in situ calibration experiment for BCECF 47 Figure 2.4 In situ calibration of HPTS 49 Figure 2.5 In situ calibration of SNARF-5F 51 Figure 2.6 Experimental parameters measured to assess the p H and [Ca2+]j responses to depolarizing conditions 53 Figure 3.1 [Ca2+]j and p H i responses evoked by a 5 min exposure to 75 mM [K + ] 0 in the presence of external C a 2 + 79 Figure 3.2 [Ca ]j and pHj responses to veratridine in the presence of external Ca 81 Figure 3.3 Simultaneous measurements of pHj and [Ca2+]j in response to 75 mM [K + ] 0 in neurons co-loaded with SNARF-5F and fura-2 83 Figure 3.4 The effects of the Ca 2 + , H+-ATPase inhibitor, eosin B, on the [Ca2 +], and p H i responses to 75 mM [K + ] 0 in the presence of external C a 2 + 85 Figure 3.5 [Ca2+]j and pHj responses to 75 mM [K + ] 0 in the absence of external Ca 2 + . . . 87 Figure 3.6 [Ca 2 +]i and p H i responses to veratridine in the absence of external C a 2 + 89 Figure 3.7 The changes in p H and Vm evoked by high [K + ] 0 in the absence of C a 2 + 0 are both dependent on [K + ] 0 91 Figure 3.8 The rise in pHj evoked by high [K + ] 0 under 0 C a 2 + 0 conditions is not due to N a + / H + exchange or H + efflux via TTX-sensitive voltage-gated Na + channels 93 Figure 3.9 The effect of Z n 2 + on high [K+]0-induced rises in pHj in neurons with a normal ApHmemb 95 ix Figure 3.10 The effect of Zn on high [K ]0-induced increases in pHj in neurons with a ApHmemb increased by pre-treatment with FCCP 97 Figure 3.11 The rise in pHj observed under depolarizing conditions in the absence of external Ca 2 + , and its inhibition by Z n 2 + , are dependent on ApH m e m b 99 2_|_ Figure 3.12 The effects of removing extracellular Zn with the membrane impermeant chelator, DTP A 101 Figure 3.13 The effects of increased pH 0 on the rise in pH, and the inhibitory effects of Z n 2 + 103 Figure 3.14 Effects of reducing temperature on the internal alkalinization evoked by high [K + ] 0 under 0 C a 2 + 0 conditions 105 Figure 3.15 Depolarizing voltage steps evoke outward currents that slowly run-up 107 Figure 3.16 The outward current is likely not carried by CI or Mg 2 +ions 109 Figure 3.17 Tail currents and mean reversal potentials (F r e v) of outward currents measured at a range of transmembrane pH gradients (ApHmemb) I l l Figure 3.18 The time course of activation is best fit by a double exponential function... 113 Figure 3.19 Time constants of activation and deactivation of the outward currents 115 Figure 3.20 Lack of effect of Z n 2 + on the outward currents 117 Figure 3.21 Lack of effect of increasing temperature on the outward currents 119 Figure 4.1 Depolarization causes increases in pHj in cells with low internal buffering capacity 139 X ACKNOWLEDGEMENTS First and foremost, I am truly indebted to Dr. John Church for his endless gifts of patience, guidance and support; and whose wisdom and relentless pursuit of excellence are a marvel to behold. I would also like to extend my gratitude to the members of my supervisory committee, Drs. Steven Kehl, David Mathers and Edwin Moore, for their offerings of time, knowledge and words of encouragement throughout the course of my research. I am grateful to my lab-mates and friends, Tony Kelly and Claire Sheldon, for sharing their experience and for generating a warm and supportive working atmosphere - I could not have asked for more. For their contributions to the work presented in this thesis, additional thanks must be extended to Bjorn Vegsund, Claire and Abdoullah Diarra, who performed the veratridine and HPTS experiments. To the members of the Department of Cellular and Physiological Sciences, and especially to Dr. Tony Pearson, Logan Lee, Anu Khurana and Daniel Kwan, thank you for your camaraderie and support. To Chris Jen - thank you for your friendship, care and understanding. Most importantly, to my family, Frank, Leng, Ming and Shawn, thank you for your unconditional love and support. Financial support was provided by a National Sciences and Engineering Research Council of Canada - Canada Graduate Scholarship (Master's) and a Michael Smith Foundation for Health Research Junior Graduate Studentship to myself; and an operating grant to Dr. John Church from the Heart and Stroke Foundation of B.C. and Yukon. 1 1. INTRODUCTION 1.1. Physiology, pathophysiology and pH The H + ion is one of the most potent of intrinsic neuromodulators, with nanomolar changes in its concentration causing significant alterations in neuronal function (Kaila and Ransom, 1998). This sensitivity to protons is due to the fact that the ionization state of weak acids and bases -including peptides and proteins - is highly dependent on pH (Boron, 2004). Conversely, neuronal activity itself can result in changes in pH, as a result of metabolic activity or the transmembrane movement of acid equivalents via various activity-dependent pathways (reviewed by Chesler, 1990, 2003). As a result, many physiological events in the brain are associated with fluctuations in pH (reviewed by Chesler and Kaila, 1992; Chesler, 1990, 2003). For example, exposure to glutamate receptor agonists or electrical stimulation of the CA1 region of the rat hippocampus causes an increase in extracellular pH (pH 0; Krishtal et al., 1987; Gottfried and Chesler, 1994; Smith et al., 1994; Paalasmaa and Kaila, 1996) and fall in intracellular pH (pH;; Trapp et al., 1996b). These events are often followed by a long-lasting extracellular acidosis and the gradual return of pHj to normal resting levels (Voipio and Kaila, 1993; Paalasmaa and Kaila, 1996; reviewed by Chesler, 2003). Associations between pH and pathophysiological events in the brain have also been described. For example, anoxia induces a transient intracellular acidification followed by an alkalinization in rat hippocampal neurons (Diarra et al., 1999; Sheldon and Church, 2002). Intracellular acidosis has been described as a general response to ischemia and the changes in pHi and pH 0 that occur in response to cerebral ischemia have been ascribed both neurotoxic and neuroprotective effects, highlighting the complex relationship between pH and neuronal function (reviewed by Lipton, 1999; Yao and Haddad, 2004). For example, in hippocampal slices, mild extracellular acidosis has been shown improve neuronal recovery by delaying the 2 onset of hypoxic spreading depression-like depolarizations (Tombaugh, 1994). In contrast, more severe reductions in pHi exacerbate damage in energy-depleted neurons (reviewed by Siesjo et al., 1996; Upton, 1999). 1.2. p H j a n d n e u r o n a l exc i t ab i l i t y As mentioned above, electrical activity can cause both intracellular and extracellular pH shifts in the central nervous system (CNS) (also see Chesler and Kaila, 1992; Chesler, 2003). However, while it is well established that marked changes in pH 0 occur in response to neuronal activity and can influence the events that initiated them, it is only in recent years that the ability of activity-evoked shifts in pH, to reflect and, in turn, influence neuronal function has been recognized. In addition, the pH; of mammalian central neurons is very dependent on pH 0 , such that the effects of changes in pH 0 on neuronal excitability may, in part, be due to concomitant changes in p H (Ou-Yang et al., 1993; Church et al., 1998; Bouyer et al , 2004). Many of the first studies showing that membrane depolarization causes a fall in pHj were performed on large invertebrate neurons and in multiple preparations, including snail neurons, the acidification has been found to depend on Ca influx (Meech and Thomas, 1980, 1987). It was later shown that the Ca2+-dependent fall in pHj largely originates from the activation of a Ca2+-extruding, H+-importing plasmalemmal Ca 2 + , H+-ATPase (PMCA; Schwiening et al., 1993; Schwiening and Willoughby, 2002). Cytosolic acidification of vertebrate neurons also occurs in response to depolarizing stimuli such as excitatory synaptic transmission, elevation of [K + ] 0 , or exposure to excitatory amino acids (reviewed by Chesler, 2003). In several mammalian neuronal preparations, including hippocampal CAI neurons, these reductions in pHj. have also been found to depend on C a 2 + influx (Werth and Thayer, 1994; Trapp et al., 1996a, b; Willoughby and Schwiening, 2002) 3 and consequent increases in the production of metabolic acids such as CO2 and lactate (Wang et al., 1994; Zhan et al., 1998) and, as observed in snail neurons, activation of a PMCA (Trapp et al., 1996a, b; Wuetal., 1999). The potential importance of the shifts in pHi caused by neuronal activity is underscored by the fact that many factors critical to neuronal function are sensitive to pHj, such as voltage-gated (reviewed by Tombaugh and Somjen, 1998), ligand-gated (reviewed by Traynelis, 1998) and gap-junctional (reviewed by Spray and Scemes, 1998) conductances. For example, decreases in pHj inhibit afterhyperpolarizations mediated by Ca2+-dependent K + channels in rat hippocampal neurons (Church et al., 1998; Kelly and Church, 2004), with consequent effects on neurotransmitter release, postsynaptic integration of synaptic potentials and neuronal firing behaviours (Storm, 1990). Additionally, changes in pHj can modulate the extent of dye- and electrotonic-coupling between rat hippocampal CA1 neurons by modulating gap junctional conductances (e.g. Church and Baimbridge, 1991), effects which may have important roles in the initiation or termination of epileptiform activity (Prince and Connors, 1986; Bennett and Zukin, 2004). In addition to the modulation of plasma membrane conductances, changes in pHi can also affect other neuronal processes, e.g. the activities of enzymes and second messenger systems (Vignes et al., 1996; Chen et al , 2000; Ross et al., 2001), as well as transport and buffering mechanisms for ions such as Ca (Dipolo and Beauge, 1982; Mullins et al., 1983; Doering and Lederer, 1993; Hoyt and Reynolds, 1998) and transmitters such as glutamate (reviewed by Takahashi et al., 1997). 1.3. Regulation of pHj in rat hippocampal neurons Due to the important role it plays in determining function and viability, cells have developed various means to regulate pHj (see Roos and Boron, 1981; Chesler, 2003). However, it is only 4 relatively recently that the mechanisms that contribute to pHi homeostasis in mammalian central neurons have started to be elucidated. The most extensive studies of pHj regulating mechanisms in mammalian central neurons have been performed on hippocampal neurons. To date, three electroneutral mechanisms have been found to play a role in pHj regulation in rat hippocampal neurons and these can be categorized as either 'acid extruders' or 'acid loaders'. 1.3.1. Acid extrusion mechanisms To date, rat hippocampal neurons have been found to possess two major acid extrusion pathways, both of which are dependent on external Na +. First, a Na + /H + exchanger (NHE; Raley-Susman et al., 1991; Baxter and Church, 1996), which exchanges extracellular Na + for intracellular H + (see Fig. 1. 1^ 4). Although NHEs have been implicated in pHj regulation in many cell types, and are typically inhibited by amiloride and amiloride analogues (reviewed by Bevensee and Boron, 1998), these drugs have no effect on functional NHE activity in the hippocampus (Raley-Susman et al., 1991; Schwiening and Boron, 1994; Baxter and Church; 1996; Bevensee et al., 1996). Although harmaline, a hallucinogenic alkaloid, has been reported to inhibit amiloride-insensitive NHE (Raley-Susman et al., 1991; Ou-yang et al., 1993), the fluorescence characteristics of this compound preclude its use in studies where pH, is measured using 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) or other microspectrofluorimetric probes (Ou-yang et al., 1993; Schwiening and Boron, 1994; Baxter and Church, 1996). Thus, as yet, there are no known pharmacological inhibitors of NHE activity in hippocampal neurons suitable for use in microspectrofluorimetric studies. The second acid extruder in rat hippocampal neurons is a Na+-dependent Cl /HC0 3 " exchanger (see Fig. 1.1C; Schwiening and Boron, 1994; Baxter and Church, 1996; Bevensee et al., 1996; Brett et al., 2002). In invertebrate preparations, this is the predominant acid-extruding 5 mechanism and mediates the exchange of 1 extracellular Na + ion for 1 intracellular C f ion for each pair of neutralized intracellular protons (reviewed by Thomas and Schwiening, 1998). Similar to invertebrate neurons, the Na+-dependent C1/HC0 3" exchanger in hippocampal neurons has an absolute requirement for external Na + and H C 0 3 \ and for internal CI", and is inhibited by stilbene derivatives such as 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS; Schwiening and Boron, 1994; Baxter and Church, 1996; Bevensee et al., 1996). 1.3.2. Acid loading mechanisms The predominant acid loader in rat hippocampal neurons is an alkali-extruding Na+-independent CI/HCO3" exchanger that imports external CI in exchange for internal HC0 3 " (see Fig. 1.15; Baxter and Church, 1996; Brett et al., 2002). Initial studies suggested that hippocampal neurons do not possess an acid-loading CI /HC0 3~ exchanger, based on the insensitivity of pHj to the removal of external CI (Raley-Susman et al., 1991). It has since been shown, however, that this exchanger can operate in reverse, as an acid-extruder, under very acidic conditions (e.g. pHj < 6.5) in a manner that is sensitive to DIDS, CI and HC0 3", but not Na + (Baxter and Church, 1996; reviewed by Bevensee and Boron, 1998). Furthermore, its function as an alkali extruder under physiological conditions was confirmed when it was shown that pHj recovery from base-loading in rat hippocampal neurons is inhibited by DIDS and/or the removal of CI „, and can occur in the absence of N a + 0 (Brett et al., 2002). 1.3.3. Other mechanisms that may affect pHj In addition to the three electroneutral pHj regulating mechanisms described above, pHj in rat hippocampal neurons may be influenced by several other H+-translocating pathways, although 6 these are not usually considered 'pHj regulating mechanisms'. First, as noted above (Section 1.2), the PMCA is a potential pathway through which large changes in pHj can occur in response to normal neuronal activity. Second, rat hippocampal neurons have recently been found to express Na + /HC0 3" cotransporters (Schmitt et al., 2000; Cooper et al., 2005) that can, depending on the membrane potential (Vm), increase or decrease pHj (see Fig. 1 .ID). Although it remains to be determined whether Na +/HC0 3~ cotransport contributes to pHj (or [Na+]0 regulation under normoxic conditions (Schwiening and Boron, 1994; Baxter and Church, 1996; Rose and Ransom, 1997), it may contribute to acid loading during O2 deprivation (Yao et al., 2003). Third, a variety of plasmalemmal transport mechanisms for neurotransmitters (e.g. glutamate), peptides, and metal ions include acid equivalents (i.e. H + , OH and/or H C 0 3 ) in their transport cycle and can therefore affect pH; (e.g. Daniel and Kottra, 2004; Kanai and Hediger, 2004; Mackenzie and Hediger, 2004). Lastly, recent evidence is consistent with the possibility that a voltage-dependent H + extrusion pathway may exist in rat hippocampal neurons. For example, anoxia, which is associated with membrane depolarization (Tanaka et al., 1997; Lipton, 1999), can be associated with a Zn2+-sensitive increase in pHj that is not attributable to the activity of other known pHj regulating mechanisms and which may help to alleviate the detrimental falls in pHj that might otherwise occur under this condition (Diarra et al., 1999). Additionally, under normoxic conditions where NHE and Na+-dependent C1/HC0 3" exchange are inhibited, hippocampal neurons recover faster from intracellular acid loads under depolarizing conditions - an effect that is inhibited by Z n 2 + (Sheldon and Church, 2002). Together, these results suggest that rat hippocampal neurons have an acid extrusion mechanism that is activated by membrane depolarization and is sensitive to Zn , consistent with the activity of a voltage-gated proton conductance (gH+). Indeed, this H + efflux pathway may be especially important during ischemia, 7 given findings that soon after the onset of anoxia, NHE activity is inhibited (Sheldon and Church, 2004) while a potential acid loader (Na+/HC0 3" cotransport) may be activated (Yao et al., 2003). The possible contribution of a gH+ to p H regulation in rat hippocampal neurons will thus be the focus of this thesis. 1.4. Voltage-gated proton conductances The putative gH+ in rat hippocampal neurons may be mediated by a mammalian, voltage-gated H+-selective channel. Since their first description in snail neurons (Thomas and Meech, 1982), voltage-gated proton channels have been identified in a number of mammalian, non-neuronal cell types (for reviews, see DeCoursey, 1998, 2003). Although the molecular identities of voltage-gated proton channels remain unknown, a number of their properties distinguish them from other, more classical ion channels and H + transporters. Indeed, although distinct H + currents can be recorded from a variety of cell types, there is some controversy over whether voltage-gated proton channels are actually ion channels at all. As such, I have chosen in this thesis to use the broader term 'voltage-gated proton conductance' to refer to voltage-gated proton channels. 1.4.1. General properties 1.4.1.1. Historical perspective Following the initial description of voltage-dependent H + currents and associated intracellular pH changes in snail neurons by Thomas and Meech (1982), Byerly et al. (1984) proceeded to thoroughly characterize the electrophysiological properties of voltage-gated H + currents in voltage-clamped snail neurons. H + currents were also described shortly thereafter in axolotl oocytes (Barish and Baud, 1984). Snail neurons and newt oocytes would remain the only cells 8 known to express gH+s until 1991, when H + currents were described in rat alveolar epithelial cells (DeCoursey, 1991). Since then, the list of mammalian non-neuronal cell types with H + currents has steadily grown. Although a mammalian neuronal gH+ has not yet been described, the difficulty in finding cells that do not have H + currents to serve as heterologous expression systems (see Banfi et al., 2000) suggests that cell types that have not been systematically explored may also express gH+s. 1.4.1.2. Varieties of gH+s Although all gH+s share the same basic characteristics, they are not necessarily identical in all preparations and, based on criteria established by DeCoursey (1998, 2003), can be divided into five varieties (Table 1.1). The most striking difference between the types of gH+s lies in their gating kinetics. The time constants of current activation and deactivation (indicated by x a ct and Ttaii) range from a few milliseconds in invertebrate neurons, to tens of seconds in mammalian epithelial cells. The type x (oxidase-related) H + currents may or may not arise from a distinct type of gH+. Initially, they were proposed to be mediated by a gH+ that is only active when NADPH oxidase is functional (Banfi et al., 1999). Recent evidence now suggests, however, that type x H + currents likely arise from a shift of type p (phagocyte) gH+s into a distinct gating mode upon stimulation with activators of NADPH oxidase (DeCoursey et al., 2000, 2001; Cherny et al., 2001). The properties of type x H + currents remain poorly characterized. 1.4.1.3. High proton selectivity The relative permeability of gH+s to protons and other cations can be established by comparing the reversal potential (Vrev) of H + currents to the equilibrium potential for H + , EH, or by 9 comparing the slope of the relationship between VKV and the transmembrane p H gradient ( A p H m e m b ) to the theoretical slope defined by the Nernst equation. In most studies, differences have been reported between VKV and E H , and the slope of Vrev versus A p H m e m b is often 40-50 mV/unit change in A p H m e m b , sometimes even <40 mV/unit, instead of the theoretical 58 mV/unit (at 20°C; reviewed by DeCoursey, 2003). Nevertheless, because [H+]i under the electrophysiological recording conditions typically employed to characterize H + currents is usually several orders of magnitude lower than that of the predominant cation, the relative permeability, as estimated by the Goldman-Hodgkin-Katz equation, of H + to other cations is often >106 (reviewed by DeCoursey, 2003). Although this relative permeability is very high, it should not be considered to reflect the finite permeability of gH+s, as the deviations of Vrev from EH are generally considered to be due more to experimental error in measurements of VreY, contributions of leak current(s), or imperfect pHj control, rather than a permeability to other cations (DeCoursey, 2003). The high selectivity of gH+s for protons suggests that the mechanism of conduction may be via a chain of hydrogen bonds formed between amino acid side chains in membrane proteins, rather than a water-filled pore (Nagle and Morowitz, 1978). 1.4.1.4. Small unitary conductance Early attempts to resolve the unitary conductance of gH+s from current variance analysis met with limited success, due in part to poor signal-to-noise ratios (S/N ratio <1). Rough estimates ranged from ~10 fS at p H i 6.0 in human neutrophils (DeCoursey and Cherny, 1993) to 90 fS at pHj 5.5 in human skeletal myotubes (Bernheim et al., 1993). Recently, the very slow gating of the gH+ in human eosinophils was exploited to improve the S/N ratio to > 100 and distinct excess fluctuations ascribable to gH+ gating were observed (Cherny et al., 2003). The unitary conductance estimated from these recent findings is 30-40 fS at pHj 6.5 (Cherny et al., 2003). 10 The conductance was almost fourfold greater at pHj 5.5 (140 fS) and, although unitary currents could not be recorded at physiological pH values, single channel-like currents were observed at pHj <5.5 near t^hreshold ( t n e m o s t n e g a t ive voltage at which appreciable macroscopic currents are seen) with amplitudes of 7-16 fA (Cherny et al., 2003). Estimates of the unitary conductance of gH+s are very small relative to other ion channels and, based on their conductance alone, gH+s cannot be distinguished from H + carriers (DeCoursey, 2003). However, that H + current fluctuations have recently been observed (Cherny et al., 2003) provides evidence of gating, supportive of the description of gH+s as ion channels. 1.4.1.5. Strong dependence on temperature Both the open-channel conductance and gating kinetics of gH+s exhibit a very strong sensitivity to temperature (Byerly and Suen, 1989; Kuno et al., 1997; DeCoursey and Cherny, 1998). The Qio value is the relative change in reaction rate for a 10°C increase in temperature. In their study on multiple cell types, DeCoursey and Cherny (1998) found that x a c t and x t a n had Qjo values between 6-9. In comparison, values of Qio for gating in other ion channels are typically in the range of 2 to 4, similar to those of enzymatic reactions (see Hille, 2001), although higher values have been described for C1C-0 chloride channels (Pusch et al., 1997). The temperature sensitivity of the open-channel conductance is more difficult to determine, due to the effects of temperature on gating. At low temperatures, it can often be difficult to attain steady-state H + currents because of their slow activation (x a ct >100 s; DeCoursey and Cherny, 1998). Alternatively, at high temperatures large H + fluxes can result in droop and distortion of the current, due to the resulting changes in pHj and/or pH 0 and, thus, ApHmemb (DeCoursey and Cherny, 1994b; DeCoursey and Cherny, 1998). Nevertheless, estimates of the Qjo for the open-channel conductance have been obtained and are 2.1 in snail 11 neurons (Byerly and Suen, 1989) and between 2.1-3.1 in mammalian non-neuronal cell types, including neutrophils, eosinophils and phagocytes (DeCoursey and Cherny, 1998). In excised patches, the values of Qio are higher (2.8-5.1), presumably due to a reduced tendency for H + depletion to occur (DeCoursey and Cherny, 1998). The Q i 0 values for H + conductances are greater than that of the conductances described in 20 other studies of ion channels (see DeCoursey and Cherny, 1998). It is possible, therefore, that H + conduction via a gH+ is energetically more demanding than ion permeation through other ion channels, perhaps reflecting conduction via a hydrogen-bonded chain mechanism (DeCoursey, 2003). 1.4.1.6. Voltage-dependent gating The voltage-dependency of gH+ gating is not absolute, and is strongly modulated by pH0/i (see Section 1.4.1.7). From a deactivated (closed) state, depolarization evokes outward currents that do not inactivate. While H + currents are outwardly rectifying in the steady-state, this is largely due to both the voltage- and pH-dependency of gating, rather than to the intrinsic properties of gH+s themselves (Cherny et al., 1995). Indeed, the instantaneous current-voltage relationship, determined from tail current analysis, shows that open gH+s conduct inward currents nearly as well as outward currents (Cherny et al., 1995). Although gH+s do not inactivate, decay or droop of H + currents is sometimes observed in response to large or prolonged depolarizations that result in large H + fluxes (e.g. Thomas and Meech, 1982; DeCoursey, 1991; DeCoursey and Cherny 1993, 1994b; Demaurex et al., 1993; Kapus et al., 1993a; Morihata et al., 2000). However, as mentioned in Section 1.4.1.5, H + current droop is largely due to the effects of H + depletion and thus, increased pHj, which leads to a positive shift of EH and a reduction in the outward driving force for protons (DeCoursey and Cherny, 1994; DeCoursey, 2003). 12 1.4.1.7. Modulation of voltage-dependent gating by pH A defining feature of gH+s is their sensitivity to pH. In every cell where H + currents have been observed, the position of the voltage-activation curve is shifted to more negative potentials by increases in pH 0 or decreases in pHj, i.e. conditions where E\\ is more negative (reviewed by DeCoursey, 1998). A systematic study of H + currents recorded over a wide range of pHj and pH 0 values in rat alveolar epithelial cells showed that the position of the voltage-activation curve is determined by ApH m e mb, rather than pHj or pH 0 independently (Cherny et al., 1995). Thus, in rat alveolar epithelial cells, the position of the voltage-activation curve, defined by threshold can be estimated as (Cherny et al., 1995) threshold = (40 ApH m emb + 20) mV {Equation Ll) Although qualitatively similar pH dependencies are observed in other cell types, these relationships have not been described quantitatively. The functional implication of the pH-dependency of gating is that gH+s will function primarily as acid extrusion mechanisms. 1.4.1.8. Inhibition by polyvalent metal cations One of the difficulties of trying to determine the molecular identity of gH+s is that there are no highly selective and potent inhibitors of H + currents. A variety of organic agents appear to partially inhibit H + currents. For example, tetraethylammonium+ (TEA +) has been reported to moderately reduce H + currents in some preparations (Byerly et al., 1984; Meech and Thomas, 1987; Bernheim et al., 1993). However, robust H + currents have also been recorded with externally applied T E A + (Barish and Baud, 1984; Mahaut-Smith, 1989) and with symmetrical isotonic T E A + solutions (DeCoursey and Cherny, 1993; Gordienko et al., 1996). The effects of T E A + are therefore obscure and unlike those of a simple channel blocker. Additionally, the 13 ability of many organic agents to affect gH+s has been suggested to stem from their properties as weak bases and, thus, their ability to alter pHj and/or pH 0 (DeCoursey, 2003). Aside from organic agents of questionable potency and selectivity, all gH+s studied to date are sensitive to inhibition by polyvalent metal cations. Indeed, the sensitivities of gH+s to externally applied C d 2 + and Z n 2 + are generally considered as defining characteristics of gH+s. DeCoursey's (1998) compilation of studies where the inhibitory effects of two or more polyvalent cations were compared in single cell types resulted in the following "consensus" potency sequence: C u 2 + ~ Z n 2 + > N i 2 + > C d 2 + > C o 2 + > M n 2 + > Ba 2 + , Ca 2 + , M g 2 + ~ 0 There have also been reports that the trivalent cations L a 3 + , G d 3 + and A l 3 + also inhibit H + currents (Thomas and Meech, 1982; Meech and Thomas, 1987; Eder et al. 1995; Schrenzel et al., 1996). Whether Ba 2 + , C a 2 + and M g 2 + are weak inhibitors or completely ineffective remains questionable; in snail neurons these cations were completely ineffective (Byerly et al., 1984; Byerly and Suen, 1989), while some effect of one or more of these ions has been observed in human skeletal myotubes (Bernheim et al., 1993) and murine microglia (Eder et al., 1995). In contrast, marked effects of externally applied Zn can be observed at <1 uM in some cell types (e.g. Gordienko et al., 1996; Banfi et al., 1999; Cherny and DeCoursey, 1999), although higher concentrations (> 100 uM) are often used (e.g. Byerly and Suen, 1989; DeCoursey and Cherny, 1993; Demaurex et al., 1993; Kapus et al., 1994; Kuno et al., 1997; Banfi et al., 2000). The primary site of action of Z n 2 + appears to be on the external side of the membrane (Peral and Ilundain, 1995; Cherny and DeCoursey, 1999); internal application of Zn via the patch pipette results in only a mild inhibition of the gH+ in rat alveolar epithelial cells (Cherny and DeCoursey, 1999). 14 Inconsistent with the mode of action of conventional ion channel blockers, polyvalent metal cations likely do not inhibit gH+s by occluding a pore. Rather, metal cations shift the voltage-dependence of gating towards more positive voltages and slow channel opening; they may also reduce the maximal conductance, although this is only seen at high concentrations (Cherny and DeCoursey, 1999). The effects of polyvalent metal cations may be due to a charge screening effect on the gH+, as proposed for the actions of C a 2 + (Frankenhaeuser and Hodgkin, 1957), whereby the interaction of a polyvalent metal cation with negatively charged groups on the external side of the membrane alters the apparent voltage perceived by the gH+. The effects of metals in several studies suggest, however, that the inhibitory effects of polyvalent metal cations are not always sufficiently described by charge screening alone (reviewed by DeCoursey, 2003) and the precise mechanism of block remains unclear. The inhibitory effects of metals on gH+s are very dependent on pH 0 , such that increasing pH 0 increases their apparent potency (Cherny and DeCoursey, 1999). It has been suggested that the steep pH 0 dependence of inhibition by Z n 2 + is due to strong competition between H + and 94-Zn for an external binding site on gH+s, perhaps consisting of two or three titratable groups such as histidine residues (Cherny and DeCoursey, 1999). 1.4.1.9. Physiological modulators Although not considered essential for their function, there are reports that H + efflux through gH+s can be modulated by various physiological mediators (for a review, see DeCoursey, 2003). Arachidonic acid (AA), which may have a role in the activation of NADPH oxidase (Bromberg and Pick, 1983; Henderson et al., 1993), enhances H + currents in a variety of mammalian non-neuronal cell types (e.g. DeCoursey and Cherny, 1993; Kapus et al., 1994; Gordienko et al., 15 1996; Schrenzel etal., 1996). The role of intracellular free C a 2 + ions on H + currents is somewhat controversial. Byerly et al. (1984) found that changes of intracellular C a 2 + concentration ([Ca2+]j between 0.1 and 10 uM) had no effect on H + currents in snail neurons. In contrast, a pH,-dependent enhancement of + 2"1" H currents by increases in [Ca ]\ may occur in phagocytes (e.g. Gordienko et al., 1996; Schrenzel etal., 1996). H + currents are also enhanced by phosphorylation. In relatively intact cells (i.e. permeabilized-patch clamp), the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) increases H + currents, perhaps through second messenger pathways (Calonge and Ilundain, 1996; DeCoursey et al , 2000, 2001; Mankelow et al., 2003). However, ATP is not required for H + efflux via gH+s, as similar H + currents are observed in various cell types with or without ATP in the pipette solution (Kapus et al., 1993a; Schrenzel et al., 1996). 1.4.2. Functions and properties in specific cell types Due to their strong sensitivity to pH0/j and their efficient conduction of protons, gH+s are well-suited to contribute to the regulation of intracellular pH by acting, under conditions appropriate for activation, as acid extruders. For example, in excitable cells such as snail neurons, gH+s are likely to open in response to a single action potential and have been suggested to act as a major pathway by which these cells recover from the internal acid load associated with depolarization, C a 2 + influx and the activation of an acid-loading PMCA (Meech and Thomas, 1987; Schwiening et al., 1993; Schwiening and Willoughby, 2002). gH+s may have a similar function in human skeletal myotubes, although the gH+ in this cell type has a slower activation (Bernheim et al., 1993) and may therefore require a train of action potentials before sufficient activation occurs 16 for effective H + efflux. Whether a gH+ contributes to acid efflux from mammalian neurons in response to electrical activity remains to be determined. The physiological function of gH+s in non-excitable cells has been most extensively studied in phagocytes, such as macrophages, eosinophils, neutrophils and microglia. An in-depth discussion of the role of gH+s in pHj regulation in these cell types is beyond the scope of the present study and the reader is directed to an excellent review by DeCoursey (2003). Nevertheless, in these cell types H + currents may help to regulate pHj and membrane potential during the respiratory burst, at which time activated NADPH oxidase leads to the production of two intracellular protons and the efflux of two electrons, resulting in membrane depolarization and a fall in pHj (Cross and Jones, 1991). In the presence of Z n 2 + or C d 2 + , pHj falls dramatically in response to NADPH oxidase activity (Henderson et al., 1988), leading to the suggestion that gH+s are mediating the efflux of H + during the respiratory burst. 1.5. Overview and objectives Due to the close relationship between pH and neuronal function, cells have developed several processes to regulate pHj within narrow limits. Recent evidence suggests that, in addition to the pH; regulating mechanisms described to date, rat hippocampal neurons may possess an additional acid extruding mechanism that is voltage-dependent and sensitive to Z n 2 + and may therefore be a putative gH+. This possible acid efflux pathway may play an important role in pHj regulation in hippocampal neurons under pathological conditions such as anoxia, during which other acid extrusion mechanisms, such as NHE, are inhibited. Thus, this thesis will examine the potential role of a gH+ in pHj regulation in cultured rat hippocampal neurons. In initial experiments, ratiometric measurements utilizing pH sensitive fluorophores were employed 17 under normoxic conditions to characterize the changes in pHj evoked by exposure to depolarizing agents. Additionally, in an attempt to record H + currents associated with the depolarization-induced pHj changes, whole-cell patch-clamp recordings were performed in a separate series of experiments, under conditions designed to isolate H + currents. 18 Table 1.1. Comparison of varieties of H + currents Y¥ current type n (neuron) o (oocyte) e (epithelial) p (phagocyte) x (oxidase related) Gated by V, ApHmemb V, ApHmemb V, ApHmemb V, ApHmemb V, pHo, p H i , A A ? , N A D P H oxidase activity? x a c t (at +60 m V ) Fast Medium Slow Slow Slow xtaii (at -40 m V ) Fast Medium Medium Slow Very slow Cells expressing Snail neuron Frog and newt oocytes Rat alveolar epithelium, frog proximal tubule Microgl ia , neutrophils, eosinophils, mast cells, macrophages, basophils, H L -60, T H P - 1 , C H O , P L B cells Eosinophils, neutrophils, P L B cells Abbreviations: V, voltage; A p H m e m b , transmembrane pH gradient (pH 0 - pHj); A A, arachidonic acid; T a c t , activation time constant; x taii> tail current time constant. The categories of x are arbitrary, because time courses do not always follow a single exponential, depend on pHj, and are obtained from studies that were not all performed at the same pHi. The categories of xa c t are based on criteria established by DeCoursey (1998), where 'fast' means a time constant of a few milliseconds, 'medium' means a few hundred milliseconds, and 'slow' means seconds. A similar approach is used in the description of x t a i i , where 'fast' means a few milliseconds, 'medium' means under a hundred milliseconds, 'slow' is a few hundred milliseconds and 'very slow' means seconds. Table adapted from DeCoursey (2003). 19 Fig. 1.1. Plasmalemmal pH; regulating mechanisms in rat hippocampal neurons and the consequences of their activation on pHj. Na + /H + exchange, Na+-independent CT/HC0 3 " exchange and Na+-dependent C17HC03~ exchange are electroneutral whereas Na +/HC0 3~ cotransport may be electroneutral or electrogenic. A, increased Na + /H + exchange activity causes an increase in pHi (H + efflux) and Na + influx. B, Na+-independent C17HC03" exchangers extrude HCO3" ions in exchange for extracellular Cl" ions, thereby causing a fall in pHj. C, In-dependent C17HC03" exchange activity produces a rise in pHj. D, electroneutral (1:1 transport stoichiometry) Na + /HC0 3 " cotransporters act to increase pHj and cause Na + influx. Na + /HC0 3 " cotransporters with either 1:2 or 1:3 transport stoichiometrics are electrogenic. Because, under physiological conditions, the reversal potential of lNa +:2HC0 3~ cotransport (—70 mV) is close to the normal resting membrane potential (Fm), membrane depolarization (i.e. Vm<-7Q mV) promotes inward Na + /HC0 3 " cotransport, leading to increases in pHj whereas membrane hyperpolarization (i.e. Fm>-70 mV) promotes outward transport and a fall in pHj. In contrast, the reversal potential of lNa + :3HC0 3 cotransport is —26mV: at rest (i.e. Fm>-26mV), outward lNa + :3HC0 3 cotransport predominates and transport activity elicits falls in pHj whereas at depolarized membrane potentials (i.e. Fm<-26mV), inward Na + /HC0 3" cotransport contributes to rises in pHj. 20 A B C D N a + / H + exchange Na* A 0 G H* N a + -independent C I 7 H C 0 3 " exchange cr A V HC0 3 " N a + -dependent CI7 H C O a " exchange HCO3 c r ^ " ^ ^ " H* 1:1 HCO3 N a + / H C 0 3 " cotrans 1:2 Vm< -70 mV / \ V m > -70 mV * 'M 2HCO3 2HC0 3 " \ Na* 1 Na ' port 1:3 V m > -26 mV / \ V m < -26 mV 3HC0 3 " 3HC0 3 " Influence on pH: t t t I t 21 2. M A T E R I A L S A N D M E T H O D S 2.1. Neuronal cultures Primary cultures of postnatal rat hippocampal neurons were prepared as described previously (Diarra et al , 1999), with slight modifications. Two to four day old postnatal Wistar rats (Animal Care Centre, University of British Columbia) were anesthetised with 3% halothane in air and decapitated. Brains were rapidly removed and collected in ice-cold Leibovitz's L-15 medium (Invitrogen Canada Inc., Burlington, ON) supplemented with 34 mM glucose (L-15/G). The hippocampi were then removed, collected in ice-cold L-15/G and enzymatically dissociated by incubation for 15 min at 37°C in L-15/G containing 1 mg/ml papain (from papaya latex; Sigma-Aldrich Canada Ltd., Oakville, ON) and 25 |ig/ml DNase I (type II from bovine pancreas; Sigma-Aldrich Canada Ltd.). Following this, the L-15/G was replaced with Dulbecco's Modified Eagle Medium F-12 (DMEM-F12; Invitrogen Canada Inc.) supplemented with 29 mM NaHC0 3 (pH 7.4 at 36°C after equilibration with 5% C0 2 ) , 10% fetal bovine serum (Sigma-Aldrich Canada Ltd.), 100 U/ml penicillin and 100 u.g/ml streptomycin (Penicillin-Streptomycin Solution; Sigma-Aldrich Canada Ltd.). Mechanical dissociation of the hippocampi was then performed using fire-polished Pasteur pipettes with tips of diminishing diameters. Following the determination of the neuronal concentration with a hemocytometer, the resulting cell suspension was diluted appropriately in order to plate neurons onto 18 mm glass 5 2 coverslips (Fisher Scientific Co., Ottawa, ON) at a density of 6 - 8 x 10 neurons/cm . Coverslips were pre-coated with poly-D-lysine (100 ug/ml, dissolved in 0.15 M Borate; Sigma-Aldrich Canada Ltd.) and laminin (16.7 u.g/ml; Sigma-Aldrich Canada Ltd.). Neurons were allowed to settle and adhere to the substrate for 1.5 - 2 h before coverslips were rinsed in DMEM-F12 and transferred to 12 well culture plates. After 24 h, the growth medium was completely changed to Neurobasal Medium A (Invitrogen Canada Inc.) supplemented with B-27 22 Supplement (Invitrogen Canada Inc.), 0.5 mM glutamine (Invitrogen Canada Inc.), 100 U/ml penicillin and 100 ug/ml streptomycin. Glial proliferation was inhibited 48 h after initial plating by adding 10 uM cytosine-P-arabinofuranoside hydrochloride (Sigma-Aldrich Canada Ltd.) and cultures were fed every 3 -4 days by half-changing the existing medium with fresh Neurobasal Medium A. Each coverslip consisted mainly of hippocampal neurons with a maximum of 15% of cells being glial. Neuronal cultures were used 7-14 days after plating. 2.2. Microspectrofluorimetry 2.2.1. Solutions and test compounds The compositions of the nominally HC0 37C02-free, HEPES-buffered media commonly used in experiments are shown in Table 2.1. Unless otherwise noted, all solutions were prepared at room temperature (~ 22°C ) and titrated to pH 7.48 with 10 M NaOH in order to achieve a final pH of 7.35 at 37°C (see Baxter, 1995). Experiments were performed at 37°C, unless otherwise stated. Corning 240 and 440 pH meters (Corning Inc., Corning, NY), calibrated daily, were used to measure the pH of all solutions. For Ca2+-free media (Solution 2; Table 2.1), CaCL. was omitted, [Mg2 +] was increased to 3.5 mM and 200 uM ethylene glycol-bis(|3-aminoethyl ether) N, N, N', TVMetraacetic acid (EGTA, dissolved in 0.5 M Tris base) was added. In experiments using Ca2+-free, Na+-free medium (Solution 3; Table 2.1), TV-methyl-D-glucamine (NMDG) was employed as a substitute for Na + in Ca2+-free medium and the solution was titrated with 10 M HCL To depolarize neurons, solutions containing elevated (25 - 139.5 mM) [K+] were prepared by equimolar substitution of KC1 for NaCl in standard HEPES-buffered and Ca2+-free solutions, or for N M D G in 0 C a 2 + - 0 Na + medium. Lastly, in solutions containing 250 uM Z n 2 + , MgS04 was replaced with MgCb and NaH2P04 was omitted; the approximate concentration of free Z n 2 + was 23 50 uM in the presence of 200 uM EGTA. A list of pharmacological agents used is presented in Table 2.2. All salts and test compounds were obtained from Sigma-Aldrich Canada Ltd. 2.2.2. Ratiometric dyes and cell-loading All ion-sensitive fluorescent indicators were obtained from Molecular Probes Inc. (Eugene, OR). In the majority of experiments, pHj measurements were made with the dual-excitation ratiometric fluorophore, BCECF. In some experiments, the dual-excitation ratiometric probe 8-hydroxypyrene-l,3,6-trisulfonic acid (HPTS; trisodium salt) or the dual-emission seminaphthorhodafluor ratiometric indicator SNARF-5F carboxylic acid (SNARF-5F) were 2+ used to measure pHj. Changes in [Ca ]i were measured using the dual-excitation dye fura-2. Except for HPTS, all fluorophores were bulk-loaded into neurons in their hydrophobic, uncharged, membrane-permeable acetoxymethyl ester (AM) forms. Upon entry into the cytosol, these A M esters are hydrolysed by esterases, intracellularly trapping the dyes in their hydrophilic, polyanionic free acid forms. B C E C F - A M and SNARF-5F-AM were prepared in anhydrous dimethyl sulfoxide (DMSO) as 1 and 10 mM stock solutions, respectively, and stored at -60°C. Fura-2-AM was dissolved in chloroform and divided into aliquots so that, after vacuum evaporation of the chloroform, 30 ng of fura-2-AM remained in each vial. Vials were stored at -60°C and, on the day of use, fura-2-AM was prepared in DMSO as a 1 mM stock. In general, cells were loaded in the dark in standard loading medium (Solution 4; Table 2.1), consisting of standard HEPES-buffered saline, pH 7.35 at room temperature, with the iso-osmotic replacement of 3 mM NaCl with NaHC03. When needed, neurons were loaded in this medium with 2 uM B C E C F - A M for 30 min at room temperature or 5 uM fura-2-AM for 1 h at 30°C; following the incubation period the neurons were transferred to fresh, dye-free loading 24 medium for 15 min to allow for complete de-esterification of the intracellular dye. To load neurons with HPTS, which is unavailable in A M ester form, cells were incubated for 2 - 4 h at 36°C in culture medium containing 20 mM HPTS, after which they were perfused (2 ml/min) with the initial experimental solution at 37°C for 15 min prior to the start of an experiment. In this method, HPTS is presumably taken up by endocytosis and, following washout of the dye from the external medium, the entrapped dye is released from endocytic vesicles, resulting in a uniformly loaded cytosol (see Straubinger et al., 1990; Fushimi and Verkman, 1991). Lastly, in experiments in which [Ca 2 + ]i and pH- were measured simultaneously in the same cells, neurons were dual-loaded with fura-2 and SNARF-5F. To do this, neurons were first incubated in standard loading medium with 5 uM fura-2-AM for 30 min at 30°C; 10 uM SNARF-5F and 0.10% Pluronic acid F-127 (Sigma-Aldrich Canada Ltd.) were then added to the loading medium and the cells allowed to incubate for a further 30 min before transfer to fresh loading medium for de-esterification. Following dye-loading, a coverslip with its attached neurons was placed face-up into a temperature-controlled perfusion/recording chamber, supported from below by a 22 mm glass coverslip and an O-ring rimmed with high vacuum silicone grease. 2.2.3. The ratiometric method and experimental set-up The ratiometric method for measuring intracellular ion concentrations takes advantage of the differential sensitivities to the ion of interest of two different wavelengths within a fluorophore's absorption or emission spectrum. For example, in the case of the dual-excitation dye, BCECF, the intensity of fluorescence emission during excitation at 488 nm is directly related to pH, whereas emission during excitation at 452 nm is relatively pH insensitive; thus, as pH increases, the ratio of emitted fluorescence during excitation at 488 nm to 452 nm also increases. 25 Alternatively, for the dual-emission probe, SNARF-5F, increases in pH cause a fall in the intensity of fluorescence emission measured at 550 nm and a rise at 640 nm; the ratio of fluorescence emissions measured at 550 nm to 640 nm therefore decreases with increases in pH. In the case of each dye, the fluorescence emissions are from the same cell volume; therefore, the ratio of intensities emitted at two different excitation or emission wavelengths will not, in principle, be susceptible to artefacts caused by changes in optical path length, local probe concentration, illumination intensity and/or photobleaching (Bright et al., 1987, 1989; Tsien, 1989; Silver etal., 1992). Microspectrofluorimetric measurements were made using a fluorescence ratio-imaging system (Atto Instruments Inc., Rockville, MD; Carl Zeiss Canada Ltd., Don Mills, ON) fitted with two intensified charge-coupled device cameras (Atto Instruments Inc.). A schematic diagram of the equipment used for the determination of pHj or [Ca ]j in neurons single-loaded with the dual-excitation dyes BCECF (or HPTS) or fura-2, respectively, is presented in Fig. 2.1 and includes a description of the excitation and emission filters employed with each fluorophore. Following excitation with UV light at the appropriate wavelengths, the resulting BCECF, HPTS or fura-2 fluorescence emissions were measured by a single intensified charge-coupled device camera (Camera 1; Fig. 2.1). Data were obtained simultaneously from multiple neuronal somata selected as individual regions of interest (ROIs). To provide an indication of background fluorescence, data were also acquired from a region free of cellular elements. Minimization of dye photobleaching and/or UV light-induced damage to the neurons was achieved by restricting the exposure of cells to light to periods of data acquisition with a computer-controlled high-speed shutter. Additionally, when possible, neutral density filters (Chroma Technology Corp., Rockingham, VT) and a variable intensity lamp control (AttoArc, Carl Zeiss Canada Ltd.) were used to reduce the intensity of incident light at each excitation 26 wavelength. Camera gains remained constant for the duration of an experiment and were set to maximize image intensity and minimize the possibility of camera saturation. Intensities of emitted fluorescence at each excitation wavelength were digitized at 8-bit resolution and ratio pairs were acquired at 1 - 15 s intervals through the course of an experiment. In some experiments, measurements of [Ca2+]j and pHj were made simultaneously from neurons co-loaded with fura-2 and SNARF-5F, respectively. To accommodate the concurrent use of a dual-excitation and a dual-emission dye, a second intensified charge-coupled device camera and a second dichroic mirror were added to the imaging system, as illustrated schematically in Fig. 2.2. Filter selection was based on the published in vitro spectra of fura-2 and seminaphthorhodafluor dyes (Martinez-Zaguilan et al., 1991; Liu et al., 2001). In experiments using neurons co-loaded with fura-2 and SNARF-5F, ratio pairs for each dye were collected by alternating between the excitation and emission modes; each automated cycle took -1.5 s to complete, including a ~0.5 s delay between collecting fura-2- and SNARF-5F-derived ratio pairs, and was repeated every 2 - 15 s during the course of an experiment. 2~r~ 2.2.4. Calculation of pHt and [Ca ]t 2.2.4.1. BCECF Raw intensity data of fluorescence emissions collected at >520 nm for each excitation wavelength (488 and 452 nm) from ROIs placed on individual neuronal somata were corrected for background fluorescence prior to calculation of the background-corrected BCECF emission intensity ratio (BU^IBUsi)- Only those neurons able to retain BCECF for the duration of an experiment, as judged by Esi values (see Bevensee et al., 1995), were analysed. The one-point high [K+]/nigericin technique was used to convert BI^IBEsi values into pHj values. At the end of each experiment, BCECF-loaded neurons were perfused with a pH 7.00, high [K+] solution 27 (Solution 5, Table 2.1) containing 10 uM nigericin (Sigma-Aldrich Canada Ltd.; see Baxter and Church, 1996). Nigericin is a carboxylic ionophore that equilibrates cytoplasmic and extracellular [K+] and, in doing so, equilibrates pH; to pH 0 (see Thomas et al., 1979). The advantage of this method is that it allows for a one-point calibration of each cell or coverslip (i.e. neuronal population) examined. For each cell, the average BI^IBUsi ratio value obtained during the calibration period was used to normalize all other experimentally-derived BI^BUsi ratio values, which were then converted to pHj using the equation pH = pKa + log [ ( R n - R n(min))/(Rn(max) - R n ) ] (Equation 2.1) where R n is the BI^BI^i ratio value normalized to pH 7.00, R n ( m i n ) and Rn(max) are the minimum and maximum obtainable values for the normalized ratio (i.e. at low and high pH values, respectively) and pKa is the -log of the dissociation constant of BCECF. R n ( m i n ) , Rn(max) and p./va values were derived from non-linear least squares regression fits to normalized BU$%/Bl452 ratio values vs. pH data obtained from full in situ calibration experiments (Fig. 2.3A). In such experiments, neurons were exposed to 10 uM nigericin-containing, high [K+] media (Solution 5, Table 2.1) titrated to a range of pH values (pH 5.5 - 8.5 in 0.5 pH unit increments). The average BUwIBhn ratio value at each pH was normalized to the value obtained at pH 7.00 and plotted as a function of pH (Fig 2.35). The upper and lower asymptotes of this curve represent the Rn(max) and Rn(min), respectively, while the cytosolic pH on the abscissa corresponding to the point of inflection of the curve represents the measured pKa of BCECF (Baxter and Church, 1996). The calibration parameters were not sensitive to changes in temperature or the "age" of the hippocampal neurons used (data not shown). Full calibration experiments were performed whenever the mercury arc lamp was replaced. For the 7 full calibration experiments used in analyzing all experiments, the values for Rn(max), Rn(min) and pKa 28 (mean ± s.E.M.) were 2.00 ±0.02, 0.51 ± 0.01 and 7.31 ± 0.02, respectively. Nigericin can adhere to perfusion tubing and/or recording chambers and, by acting as an acid-loading K + / H + exchanger, can alter pHj (Richmond and Vaughan-Jones, 1997; Bevensee et al., 1999). Thus, after every experiment in which nigericin was employed, perfusion lines were replaced and the imaging chamber was decontaminated by soaking first in ethanol, then in 20% Decon 75 (BDH Inc., Toronto, ON) and rinsed vigorously with water (see Richmond and Vaughan-Jones 1997; Bevensee et al., 1999). 2.2.4.2. HPTS Raw fluorescence emission intensities measured at >520 nm at each excitation wavelength (452 and 380 nm) were corrected for background fluorescence prior to calculation of the background-subtracted HPTS emission intensity ratio (BI^/Bhw). Again, the one-point high [K+]/nigericin technique was used at the end of an experiment to normalize BI^IBhw ratio values to pH 7.00, which were then converted to pHj values using the equation pH = [p^a + logF380min/max] + log[(Rn - Rn(min))/(Rn(max) - Rn)] (Equation 2.2) where R n is the BUnlBhm ratio normalized to pH 7.00; p^ a is the -log of the apparent dissociation constant for HPTS; Rn(max) and Rn(min) are the maximum and minimum obtainable values for the normalized ratio; and F380m\n/mwi is the ratio of the normalized background-subtracted fluorescence intensities at the acidic and basic extremes while exciting the dye at 380 nm. As described for BCECF, full calibration experiments with HPTS (Fig. 2.4) were used to obtain the parameters in Equation 2.2. For the 5 full calibration experiments used to analyze all HPTS experiments, the values of pKa + logi^ SOmin/max, Rn(max) and Rn(min) (means ± s.E.M.) were 8.06 ±0.10, 11.65 ± 0.99 and 0.16 ± 0.08, respectively. 29 2.2.4.3. SNARF-5F Raw fluorescence emission intensities measured at 550 and 640 nm (excitation wavelength constant at 488 nm) were corrected for background fluorescence prior to calculation of background-subtracted SNARF-5F emission intensity ratios (BISSQIBIMQ). Similar to BCECF and HPTS, a one-point calibration was performed at the end of each experiment using the high-[K+]/nigericin technique, and the average BI^/BI^o ratio value obtained during the calibration was used to normalize the experimentally-derived BISSQIBI^Q ratio values to pH 7.00. At the end of some experiments at 37°C, I was unable to obtain stable ratio values for SNARF-5F during the calibration period. In these situations, the experimental data were normalized with ratio values obtained under identical experimental and optical conditions from a fresh coverslip of neurons (from the same batch of cultures) loaded with SNARF-5F and exposed to high-[K+] calibrating medium at pH 7.00. A similar instability of SNARF-5F ratio values, characterized by irregular increases and decreases in background-subtracted 550 and 640 nm emission intensities, respectively, was also sometimes experienced during full calibration experiments at 37°C when pH values were <7.00 (Fig. 2.5). The reasons for this atypical behaviour, which was observed in neurons loaded with SNARF-5F in the presence or absence of fura-2, remain unclear, although similar difficulties have been experienced by others using SNARF-derivatives (e.g. Bassnett et al., 1990; Martinez-Zaguilan et al., 1991; Seksek et al., 1991; Blank et al , 1992; Boyarsky et al., 1996; Seksek and Bolard, 1996). Nonetheless, reproducible calibration parameters at 37°C were obtained and normalized ratio values were converted to pH- using the equation pH = pKa + lOg F640m i n/m ax " log [(Rn - Rn(min))/(Rn(max) " Rn)] {Equation 2.3) where R n is the background-subtracted SNARF-5F fluorescence ratio normalized to pH 7.00; pKa is the -log of the dissociation constant of the fluorophore; Rn(max) and Rn(min) are the 30 maximum and minimum obtainable values for the normalized ratio; and F640rnin/max is the ratio of fluorescence measured at 640 nm for low pH (pH 5.5) to that for high pH (pH 8.5) (see Buckler and Vaughan-Jones, 1990). As described for BCECF and HPTS, the parameters in Equation 2.3 were obtained from pH titration curves derived from full calibration experiments (Fig 2.5). There were no differences between parameters obtained from full calibration experiments using neurons single-loaded with SNARF-5F and those using neurons co-loaded with SNARF-5F and fura-2. For the 5 full calibration experiments used to analyze all SNARF-5F data, the values of pKa + log F640min/max, Rn(max) and Rn(min) (mean ± s.E.M.) were 6.92 ± 0.08, 1.83 ± 0.13 and 0.34 ± 0.03, respectively. All conversions of normalized background-subtracted fluorescence ratios of BCECF, HPTS and SNARF-5F to pHj values were performed with the appropriate equation and calibration parameters using Visual Basic macros (written by Dr. K. Baxter and/or Dr. C. Sheldon) running in Excel 2002. 2.2.4.4. Fura-2 Due to the known difficulties of calibrating fura-2-derived ratio values in terms of absolute [Ca ]i values, particularly when pHi is changing (see Church et al., 1998), the effects of 2+ experimental manoeuvres on [Ca ]j are presented here as changes in fura-2 derived BI^IBIno 9+ ratio values, rather than absolute [Ca ]j values. 2.2.5. Experimental manoeuvres Prior to the application of any test manoeuvre, coverslips with fluorophore-loaded neurons were perfused at 2 ml/min in the presence or absence of C a 2 + 0 for 5 - 10 min, in order to establish 31 baseline values of pHj and [Ca2+]j. The pH- and/or [Ca2+]j response to depolarization was then assessed by the application of a depolarizing agent (i.e. high [K + ] 0 or veratridine) for a fixed duration of time, in most instances, 5 min. In cases where the effects of treatments such as eosin B or Z n 2 + on the pHi or [Ca2+]i responses were assessed, neurons were pre-treated with the agent of interest for a fixed period of time before depolarization, during which time the test agent was also present. 2.2.6'. Analysis of microspectrofluorimetric data 2.2.6.1. Steady-state pHj and [Ca' ]x changes 2+ The basic parameters used to compare the pH- and [Ca ]• responses to depolarizing conditions in the presence and absence of external C a 2 + are shown in Fig. 2.6. With respect to the pHj response (Fig 2.6.4), in the presence of external Ca , the magnitude of the fall in pH- (A) is the difference between the pre-depolarization steady-state pH; value and the lowest pHj value observed in response to the depolarization. In experiments using neurons dual-loaded with SNARF-5F and fura-2 to measure pH- and [Ca2+]j simultaneously, the time required for the initiation of the fall in pH- (B), which is the time between the beginning of the depolarizing stimulus and the point at which pHj begins to decrease, was also measured. In terms of the pHj 2"F • response to depolarization in the absence of Ca 0 , the magnitude of the internal alkalinization (C) is the difference between the pHj value immediately prior to the depolarization and the maximum pH; value reached in response to the depolarization. With respect to the [Ca2+]i response to depolarization (Fig. 2.65), the magnitude of the rise in [Ca2+]- (D) is the difference between the BI334/BI3&0 ratio value prior to the depolarization and the maximum 5/334/5/3so ratio value reached during the stimulus. The magnitude of the [Ca2+]i recovery during the depolarizing stimulus (E) is the difference between the maximum 32 5 / 3 3 4 / 5 / 3 8 0 ratio value and that reached at the end of the stimulus. In experiments where pHj and [Ca ]i were measured simultaneously in neurons dual-loaded with SNARF-5F and fura-2, the time required for the initiation of the rise in [Ca2+], (F) was also measured and is the time between the beginning of the depolarization and the point at which the 5 / 3 3 4 / 5 / 3 3 0 ratio value begins to increase. 2.2.6.2. Initial rates of increase of pHj * 2+ In some experiments under 0 Ca 0-conditions, the initial rate of pHj increase in response to depolarization with high [K + ] 0 was measured. To calculate rates, data points were fitted, using the least squares method (SigmaPlot v.8.02, Jandel Scientific, San Rafael, CA), to a single exponential function of time having the format pHj = a + b( 1 -ect) (Equation 2.4) where a, b, and c are the exponential parameters. The differentiated form of Equation 2.4 provides the rate of change of pHi dpHj/d^ = bc(e"ct) (Equation 2.5) from which the initial rate of rise of pHj (dpHj/d/1) was calculated (see Baxter and Church, 1996; Smith etal., 1998). Unless otherwise stated (see Section 2.4, below), experimentally-evoked changes in pHj, [Ca2+]j and rates of rise of pHi measured in response to depolarization were assessed with unpaired, two-tailed Student's t tests. Errors are expressed as S . E . M . and n refers to the number of neuronal populations (i.e. coverslips) from which data were obtained. Significance was assumed when P < 0.05. 33 2.3. Electrophysiology 2.3.1. Solutions and chemicals To determine the degree of membrane depolarization upon exposure to elevated [K + ] 0 , conventional whole-cell current clamp recordings were performed with the same HEPES-buffered bath solutions used in the microspectrofluorimetric studies, titrated to pH 7.35 at 37°C (see Section 2.1; Table 2.1). For these experiments, the pipette solution consisted of (mM): 145 potassium methylsulfate (KMeS0 4), 10 KC1 and 10 HEPES, pH 7.35 with an osmolality of -290 mOsm/kg H2O (measured with a uOsmette osmometer; Precision Systems, Inc., Natick, MA). The compositions of the bath and pipette solutions designed for the isolation of H + currents in conventional whole-cell voltage-clamp recordings are presented in Table 2.3. Solutions were designed to minimize the concentration of ions permeant through other ion channels (e.g. Na + , K + , Cl); tetramethylammonium methanesulfonate (TMAMeSOa) was chosen as the major constituent as it has been shown consistently to allow for the recording of H + currents in other cell types (see DeCoursey and Cherny, 1994, 1998; Cherny and DeCoursey, 1999). A 1 M stock solution of TMAMeSC>3 was prepared by neutralizing tetramethylammonium hydroxide (TMAOH) with methanesulfonic acid (MeSOsH). Bath and pipette solutions were prepared in ultra pure water and, unless otherwise stated, titrated at room temperature to pH 7.5 and 6.0, respectively, with 2 M T M A O H . When ZnCl2 was added to the bath solution, E G T A was omitted. In experiments where [Cl ] 0 = 20 mM, 12 mM TMAC1 iso-osmotically replaced TMAMeS03. In experiments where [Mg 2 + ] 0 = 20 mM, 16 mM Mg(OHh iso-osmotically replaced TMAMeS03. The osmolarities of the bath and pipette solutions, as measured with an uOsmette osmometer, were -300 and -290 mOsm, respectively. All salts 34 were obtained from Sigma-Aldrich Canada Ltd. MeSOaH was obtained from Fisher Scientific Canada Ltd. 2.3.2. Experimental set-up and electrophysiological recordings Patch pipettes were pulled in three-stages with a Sutter P-87 electrode puller (Sutter Instrument Co., Novato, CA) from thin walled borosilicate glass tubing (1.5 mm o.d. x 1.1 mm i.d.; World Precision Instruments Inc., Sarasota, FL) and coated with Sylgard 184 (Dow Corning Corp., Midland, MI). Electrical contact with the pipette solution was achieved by a 1 mm diameter Ag/AgCl pellet attached to a silver wire surrounded by a wax-sealed Teflon tube (Axon Instruments Inc., Union City, CA). Only patch pipettes with a resistance of 2.5 - 4.5 M Q were used. For whole-cell current-clamp experiments, the reference electrode was a Ag/AgCl pellet connected to the bath through a 3 M KC1, 4% agar bridge; for whole-cell voltage-clamp experiments, the reference electrode was connected to the bath via an agar bridge made with standard HEPES-buffered solution (Solution 1; Table 2.1). The conventional whole-cell patch clamp technique was used (Hamill et al., 1981); a hyperpolarizing voltage step (5 mV) was used to monitor increases in pipette resistance observed upon contact with a cell and the brief application of negative pressure was followed by the formation of a tight seal (> 1GQ). Whole-cell recordings could be made following the application of light suction, resulting in an input resistance (Rm) >200 M Q . Cultured rat hippocampal neurons were visualized under phase contrast with a Zeiss Axiovert 135 epifluorescence microscope (Carl Zeiss Canada Ltd.) and chosen for study based on the following morphological criteria: a smooth, non-granular pyramidal shaped soma 12-20 urn in diameter, a single major process (presumably an apical dendrite) projecting from one pole of the soma and the presence of two or more smaller process (basal dendrites) at the opposite pole (see 35 Schwiening and Boron, 1994). The patch pipette was positioned on a chosen neuron using a Sutter MP285 motorized micromanipulator (Sutter Instrument Co.). Seals were formed in standard HEPES-buffered solution and the zero-current potential established just before the pipette came into contact with the cell. For voltage-clamp experiments, measurements indicated that the combined correction for the liquid junction potentials at the initial pipette-bath interface and subsequent bath-reference electrode interface amounted to <1 mV; thus, no corrections for junction potentials have been applied to the data. Current-clamp recordings were made with an Axoclamp 2 amplifier (Axon Instruments Inc.) while voltage-clamp recordings utilized an Axoclamp 200B amplifier (Axon Instruments Inc.). All signals were low-pass filtered at 2 - 5 kHz and digitized at 5 kHz using a Digidata 1322A controlled by pClamp software (v.8, Axon Instruments Inc.). In voltage-clamp experiments, which were often as long as 2 h, series resistance (Rs), whole-cell capacitance (Cm) and R\n would sometimes vary with time and were thus closely monitored for the duration of the experiment; only cells with changes <25% in these parameters were considered for analysis. The rather high Rs ( >50 M Q ) often encountered in voltage-clamp experiments may have been due in part to the high concentration of MES buffer used in the pipette solution (T. Kelly and Y . M . Cheng, unpublished observations), and was compensated by 50 - 85% with the series resistance compensation circuitry of the amplifier. In experiments performed at elevated temperature, cells were kept under constant perfusion and the temperature of the bath solution was monitored and maintained by automatic temperature controllers connected to the solution reservoirs and an in-line solution heater (Harvard Apparatus Canada, Saint-Laurent, QC). 36 2.3.3. Experimental protocols 2.3.3. L Current-clamp recordings To determine the degree of depolarization associated with exposure to high-[K+]0, current-clamp recordings were made from neurons in the whole-cell configuration, at 37°C. The degree of depolarization upon exposure to increased [K + ] 0 was quantified as the difference between the initial Vm and that observed when the cell was bathed in high [K+] solution. 2.3.3.2. Steady-state voltage-clamp recordings Recordings of outward currents from cultured neurons were, unless otherwise stated, performed at room temperature and did not commence until at least 10 min after acquiring the whole-cell patch configuration, to allow for the equilibration of the cytosol with the pipette solution. During the first 5 min of this period, neurons were continuously perfused with the T M A M e S 0 3 -based bath solution and the holding potential (Vh) was gradually increased in 5 mV increments from an initial value of -70 mV to -20 mV (see Mahaut-Smith, 1989). Ten min after establishment of the whole-cell configuration, outward currents were elicited by 8 s depolarizing voltage steps from Vh = -20 mV to +60 mV in 20 mV increments. Leak currents were subtracted using the P/N leak subtraction protocol in pClamp. Including the leak subtraction protocol, the total time required to record a family of currents was -6.5 min. Following the acquisition of a family of currents, the cell was allowed to rest at Vh = -20 mV for -3.5 min (during which time any changes in Rs, Cm or R-m were assessed) before the family of depolarizing voltage steps was reapplied and the cycle repeated. Families of currents were thus recorded at 10 min intervals. In 2+ situations where a test treatment was applied (e.g. Zn ), comparisons were made between families of currents recorded consecutively, after current run-up (see Results, Section 3.2.1.1.). 37 2.3.3.3. Tail current recordings To assess the contribution of protons to the outward currents recorded under voltage-clamp conditions, tail current analysis was performed. Under various combinations of pH 0 and pHj, families of tail currents were evoked by first activating the outward current with a 4 s conditioning pulse from = -20 mV to +60 mV followed by 250 ms test pulses to +40 mV to -60 mV in 20 mV increments. Leak currents were subtracted using the P/N leak subtraction protocol in pClamp. 2.3.3.4. Simultaneous microspectrofluorimetry and electrophysiology In some experiments, measurements of pHj were made with BCECF in patch-clamped neurons. These experiments were performed at room temperature using the TMAMeS03-based bath and pipette solutions (Table 2.3). Neurons were either bulk-loaded with 2 pM B C E C F - A M (see Section 2.2.) prior to patching or, in later experiments, BCECF was loaded via the patch pipette by including 100 uM of the free-acid form of BCECF in the pipette solution. Fluorescence measurements were made as described above (see Sections 2.2.3 and 2.2.4.1). One-point calibrations of BCECF-derived BIw%IBI<x2 ratio values to pH 7.0 were achieved using the ratio values obtained from bulk-loaded, unpatched neurons on the same coverslip exposed to high-[K+]/nigericin calibrating medium or, in the case of neurons loaded with BCECF via the patch pipette, with ratio values obtained from the calibration of bulk-loaded neurons on a fresh coverslip of sister neurons. 2.3.4. Analysis of electrophysiological data For each family of outward currents, the peak current at each voltage was measured as the average current over the last 20 ms of the voltage step. The amplitude of tail currents was 38 measured isochronally for all voltages -1-1.5 ms after the beginning of the test pulse. Analysis of activation kinetics was performed by fitting the current record with a double exponential I(i) = A0exp(-t/xact( i)) + A i exp(-t/xact(2)) + C (Equation 2.6) where A Q and A- are the magnitudes of the rapid and slow current components, respectively, C is a constant, t is the time after initiation of the voltage step, and xact(i) and T a c t(2) are the time constants of the rapid and slow phases of current activation, respectively. In some cases, the activation time course could be fit to the sum of an exponential and a linearly rising component, described by I(i) = A-exp(-t/xact) + mt + C (Equation 2. 7) where A is the magnitude of the exponential component, t is the time after initiation of the voltage-step, T a c t is the time constant of the exponentially rising component, m is the slope of the linearly rising phase and C is a constant. The tail current time constant, x t aii , was obtained by fitting the current with a single exponential I(t) = A0exp(-t/xtaii) + C (Equation 2.8) where A 0 is the amplitude of the decaying part of the current, t is the time after start of the voltage-step and C is the steady-state current (DeCoursey and Cherny, 1998). Data were analyzed in pClamp, Origin v.7 (OriginLab Corp., Northampton, MA), and/or Microsoft Excel 2002 and are presented as means ± S.E.M. Curve-fitting was performed both in pClamp and Table Curve 2D v.5 (Jandel Scientific), n values correspond to the number of neurons from which data were obtained. Unless otherwise stated, statistical comparisons were 39 performed using Student's two-tailed Mest, paired or unpaired as appropriate, with significance assumed atP < 0.05. 2.4. Additional statistical procedures In some instances the relationship between two values needed to be assessed (e.g. the magnitude of the alkalinization observed under 0 Ca 0 depolarizing conditions and the transmembrane pH gradient). In these situations, simple linear regression was performed and significance (i.e. a trend, or slope ^ 0) assumed when P <0.05. Additionally, it was sometimes desirable to compare two regression lines. Thus, overall tests of coincidence were performed whereby the variability of the data when fit to separate regression lines was compared to that when the data were fit to a single regression line, culminating in the quantification of the improvement of fit obtained by fitting the data separately as the F test statistic (Glantz, 2002). Significance was assumed when F > F c r i t , P < 0.05. 40 Table 2.1. Compositions of experimental solutions commonly used in microspectrofluorimetric studies Standard 0 C a 2 + 0 C a 2 + - Standard High [K +]-HEPES- 0Na + loading nigericin buffered (NMDG) _Q) (2) (3) (4) (5) NaCl 136.5 136.5 - 133.5 -NaHC0 3 - - - 3.0 -KC1 3.0 3.0 3.0 3.0 -CaCl 2 2.0 - - 1.0 1.0 NaH 2 P0 4 1.5 1.5 - 1.5 1.5 MgS0 4 1.5 3.5 3.5 1.5 1.5 D-glucose 10.0 10.0 10.0 10.0 10.0 HEPES 10.0 10.0 10.0 10.0 10.0 E G T A - 0.2 0.2 - -N M D G - - 138.0 - -NaGlu - - - - 10.0 K G l u - - - - 130.5 Titrated with 10 M NaOH 10 M NaOH 10MHC1 10 M NaOH 1 0 M K O H All concentrations are shown in mM. High [K ]-nigericin solutions (Solution 5) used for the in situ calibration of pHj-sensitive dyes were titrated to a range of pH values (pH -5.5 to -8.5 in 0.5 pH unit increments) with 10 M K O H and contained 10 uM nigericin. Abbreviations: EGTA, ethylene glycol-bis (P-aminoethyl ether) N, N, N', A '^-tetraacetic acid; HEPES, 4-(2-hydroxyethyl)piperazine-l-ethanesulfonic acid; N M D G , A^-methyl-D-glucamine; Na Glu, sodium gluconate; K Glu, potassium gluconate. 41 Table 2.2. List of pharmacological agents used in microspectrofluorimetric experiments Compound: action Stock Solvent Test Storage Concentration Concentration DTPA: membrane-impermeant heavy metal chelator (employed to remove extracellular Zn 2 + ) Eosin B: Ca 2 + , H+-ATPase inhibitor FCCP: protonophore (employed to reduce pHj) 20 mM 1 mM Tetrodotoxin: inhibitor of voltage-gated 1 mM Na + channels TMAmine: weak base (employed to increase pH-) 0 C a 2 + media Ultra-pure H2O Ethanol Ultra-pure H2O 0 C a 2 + -0Na + media 1.5 mM 20 uM 1 uM 1 uM 5 mM 4°C -60°C* 4°C Veratridine: persistently activates voltage-gated Na + channels 20 mM Ethanol 20 uM -20°C In the absence of a specified stock concentration, test compounds were added directly to experimental media on the day of use and no storage temperature is indicated. A Milli-Q UF Plus Reagent Grade Water Purification System (Millipore, Mississauga, ON) was used to obtain ultra-pure H2O. *, FCCP stock solution was prepared daily and stored at -60°C until immediately prior to use; due to poor stability, solutions containing FCCP were either used within -3 h or discarded in favour of fresh solution. Abbreviations: DTPA, diethylenetriaminepentaacetic acid; FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; TMAmine, trimethylamine hydrochloride. 42 Table 2.3. Compositions of solutions used in whole-cell voltage-clamp experiments designed to isolate H + currents Bath Solution pH7.5 Pipette Solution pH6.0 T M A M e S 0 3 74.0 109.0 MgCl 2 4.0 4.0 BAPTA - 1.0 E G T A 1.0 -MES - 50.0 HEPES 100.0 -Titrated with 2 M T M A O H 2 M T M A O H All concentrations are shown in mM. Abbreviations: BAPTA, l,2-Bis(2-aminophenoxy)ethane-Af A^'iV-tetraacetic acid; EGTA, ethylene glycol-bis (P-aminoethyl ether) N, N, N', N'-tetraacetic acid; HEPES, 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid; MES, 2-morpholinoethanesulfonic acid; TMAMeS0 3 , tetramethylammonium methanesulfonate. A I M stock solution of T M A M e S 0 3 was prepared by neutralizing tetramethylammonium hydroxide with methanesulfonic acid. When the bath pH was reduced to pH 6.5, 100 mM MES replaced HEPES. 43 Fig. 2.1. Schematic representation of the optical equipment used with neurons single-loaded with a dual-excitation fluorophore. Light (solid and dotted lines) provided by a 100 W mercury arc lamp was band-pass filtered through alternating interference excitation wavelength filters (A,ex(i) and ^ ex(2))- The filtered light then reflected off a long-pass dichroic mirror (Dichroic 1), passed through the objective (Zeiss LD Achroplan, n.a. 0.6, 40x) and excited the fluorophore loaded into neurons. Resulting fluorescence emissions (bold dashed line) at each excitation wavelength passed through Dichroic 1 and a long-pass emission filter (Xem) prior to detection by an intensified charge-coupled device camera (Camera 1). The filter sets used to measure fluorescence emitted from neurons loaded with BCECF, HPTS or fura-2 are described in the table below the diagram. LP indicates a long-pass filter. Ratios of the background-subtracted emission intensity detected following excitation at the first excitation wavelength (5/ex(i)) to that following excitation at the second excitation wavelength (6/ex(2)) were measured for each fluorophore. Optical filters were obtained from Chroma Technology Corp. (Rockingham, VT). Adapted from Sheldon et al., 2004a. 44 Lamp Excitation Filters | A« X (1 ) e^x(2) Camera 2 Emission Filter Cell Objective Xe, Dichroic 1 Dichroic 2 (not used) m Emission Filter Camera 1 Excitation Dichroic 1 Emission Measured Filters Filter Ratio A,eX(i)(nm) X e x ( 2 ) (nm) Aem (nm) BIex(i)/BIeK(2) BCECF 488 ± 5 452 ± 5 510 520 LP HPTS 452 ± 5 380 ± 5 510 520 LP 5 / 4 5 2 / 5 / 3 8 0 fura-2 334 ± 5 380 ± 5 395 420 LP 5 / 3 3 4 / 5 / 3 8 0 45 Fig. 2.2. Schematic representation of the optical equipment used to measure pHj and [Ca2 +]i in hippocampal neurons co-loaded with the dual-emission dye SNARF-5F and the dual-excitation dye fura-2. Light from a 100 W Hg arc lamp was band-pass filtered at 488 ± 5 nm (for SNARF-5F) or alternately at 334 ± 5 and 380 ± 5 nm (for fura-2). The filtered light then reflected off a 505 nm long-pass dichroic mirror (Dichroic 1), passed through the objective (Zeiss L D Achroplan, n.a. 0.6, 40x) and excited the fluorophores loaded into cells. Resultant SNARF-5F fluorescence emissions passed through Dichroic 1, were split by a second dichroic mirror centred at 605 nm (Dichroic 2) and passed through 640 ± 5 nm or 550 ± 40 nm band-pass emission filters before being detected by Cameras 1 and 2, respectively. Fura-2 fluorescence emissions passed through Dichroic 1, were reflected by Dichroic 2, passed through a 550 ± 40 nm band-pass emission filter and were detected by Camera 2. Adapted from Sheldon et al., 2004b. Cell Lamp Excitation Filters 334 nm 380 nm 488 nm Camera 2 Emission Filter 550 nm Objective Dichroic 1 505 nm Dichroic 2 605 nm Emission Filter 640 nm Camera 1 47 Fig. 2.3. Full in situ calibration experiment for BCECF. A, a full calibration experiment in which cells loaded with BCECF were exposed at room temperature (~22°C) to nominally HCO3 /CO2 - free, HEPES-buffered, high [K+] solutions containing 10 uM nigericin at the pH 0 (and, thus, pHj) values indicated above the record. The trace represents the mean background-subtracted BI^IBUn ratio values normalized to 1.00 at pHj 7.0 (R n) from 18 cultured postnatal rat hippocampal neurons recorded simultaneously on a single coverslip. B, plot of R n against pHi. The curve is the result of a non-linear least squares regression fit to Equation 2.1. For this particular calibration, the values of Rn(max), Rn(min) and pKa were 1.95 and 0.48 and 7.26, respectively. Error bars represent S.E.M. (n = 4); where absent, error bars lie within the symbol area. 49 Fig. 2.4. In situ calibration of HPTS. A, a full calibration experiment in which cells loaded with HPTS were exposed at room temperature (~22°C) to nominally H C 0 3 7 C 0 2 - free, HEPES-buffered, high [K+] solutions containing 10 uM nigericin at the pH 0 (and, thus, pHj) values indicated above the record. The trace represents the mean background-subtracted normalized ratio value (R n) in 4 cultured postnatal rat hippocampal neurons recorded on a single coverslip. B, plot of R n against pHj. R n was calculated by normalizing the average background-subtracted ratio (S/452/5/380) values for all neurons on a given coverslip to the BImlBhw ratio value in the same neurons at pH 7.00. The curve is the result of a four-parameter sigmoid fit to Equation 2.2. For this particular calibration, the values of Rn(max), Rn(min) and pKa + logF380min/max were 11.28, 0.13 and 8.04, respectively. Error bars represent S.E.M. (n = 4). 51 Fig. 2.5. In situ calibration of S N A R F - 5 F . A, a calibration experiment performed at 37°C where neurons dual-loaded with SNARF-5F and fura-2 were exposed to nominally H C O 3 / C O 2 -free, HEPES-buffered, high [K+] solutions containing 10 u.M nigericin at the pH values indicated above the trace. The record shows the mean normalized SNARF-5F-derived ratio value (Rn) of 14 cultured postnatal rat hippocampal neurons on a single coverslip. Due to the instability of the ratio during the latter part of the exposure to pH 7.00 medium, the ratios used for normalization to pH 7.00 were taken prior to the point indicated by the arrow. B, plot of R n vs. pHj. The curve is the result of a non-linear least squares regression fit to Equation 2.3. For this particular calibration, the values of pKa + log F640min/max, Rn(max), and Rn(min) were 7.14, 1.49 and 0.32, respectively. Error bars represent S.E.M. (n = A). 53 Fig. 2.6. Experimental parameters measured to assess the pH- and [Ca 2 +]- responses to depolarizing conditions. A, the pHi response of 12 neurons on a single coverslip to 5 min high [K + ] 0 in the presence and absence of C a 2 + 0 . In the presence of C a 2 + 0 , the magnitude of the fall in pHj (A) is shown. In the absence of C a 2 + 0 , the magnitude of the increase in pHj (D) is shown. B, the [Ca2+]i response, represented by fura-2-derived ratio values (BIi^/BI^o), of 10 neurons on a single coverslip to 5 min high [K + ] 0 in the presence and absence of C a 2 + 0 . The break in the trace represents a 15 min gap in the record. Shown are the magnitude of the initial rise in [Ca2+]- (D) and the magnitude of the recovery during the stimulus (E). In experiments using neurons dual-loaded with SNARF-5F and fura-2 to measure pHj and [Ca2+]j simultaneously, additional measurements included the time required for the initiation of the fall in pHj (B) and the time required for the initiation of the rise in [Ca ]j (F). See text for specific limits of parameters A -F. 55 3. RESULTS 3.1. Microspectrofluorimetric studies in intact neurons Using cultured postnatal rat hippocampal neurons loaded with ion-sensitive fluorophore(s), I initially characterized the changes in steady-state p H and [Ca2+]j that occurred in response to depolarizing conditions in the presence of external C a 2 + and examined the contribution of the PMC A to the changes observed. The pHj and [Ca2+]j responses to depolarization were then re-9+ examined in the absence of external Ca , to investigate any possible pHj changes independent of C a 2 + influx, such as H + efflux via a gH+. Lastly, the effects of variables shown to affect gH+s in other cell types on the pHj response to depolarization under 0 C a 2 + 0 conditions were examined. 3.1.1. Depolarizing conditions cause a fall in pHj in the presence of external Ca2+ 3.1.1.1. pHj and [Ca2+]t responses to high [Kf]a exposure In neurons bathed in nominally HC0 37C02-free, standard HEPES-buffered medium and loaded 9+ + with fura-2, the [Ca ], changes evoked by 5 min exposures to high [K ] 0 (25 - 75 mM) were characterized by two phases (Table 3.1). A 5 min exposure to 75 mM [K + ] 0 (Fig. 3.1/4), for example, induced an initial rapid rise in [Ca2+]j of 0.69 ± 0.09 BI334/BI3&0 ratio units (n = 9), with the peak [Ca 2 + ]i reached at 1.1 ± 0 . 2 min after the start of the depolarization. In the continued presence of 75 mM [K + ] 0 , [Ca2+]i recovered by 0.34 ± 0.05 ratio units, a 47.40 ± 0.04% recovery. Qualitatively similar responses were evoked by 5 min applications of 25 and 50 mM [K + ] 0 (see Table 3.1; Fig. 2.65), and the magnitudes of both the initial increase in [Ca 2 + ]i (P <0.01; r = 0.63; n = 21) and the recovery of [Ca2+]i during exposure to high [K + ] 0 (P <0.001, r = 0.73; « = 21) were found to be dependent on [K +] 0 . Parallel experiments performed on neurons loaded with BCECF showed that pHi falls in response to high [K + ] 0 exposure in the presence of external C a 2 + (Table 3.1; Fig. 3.15). In 56 response to 75 mM [K +] 0 , for example, neurons with a resting pH- value of 7.34 ± 0.04 (n = 9) exhibited a fall in pHj of 0.51 ± 0.03 pH units, with pH- reaching its minimum value at 4.60 ± 0.45 min after the start of the depolarization. Qualitatively similar responses were observed with 25 and 50 mM [K + ] 0 (see Table 3.1; Fig. 2.6,4), and the magnitude of the fall in pH- during exposure to high [K + ] 0 was dependent on [K + ] 0 (P O.05; r = 0.48; n = 21). These results confirm that depolarization in the presence of external C a 2 + evokes increases and decreases in [Ca2+]j and pHj, respectively. In addition, the magnitudes of these changes are dependent on [K + ] 0 and thus, the degree of membrane depolarization. 3.1.1.2. pHj and [Ca2+]j responses to depolarization induced by veratridine [K+]0-dependent, membrane depolarization-independent, changes in pHj have previously been reported in some non-neuronal cell types and ascribed to the activity of a putative K + / H + exchanger (Bonanno, 1991; Wilding et al., 1992; Graber and Pastoriza-Munoz, 1993) or to a plasmalemmal K7H-ATPase (Yanaka et al., 1991; Ikuma et al., 1998). To assess whether the high [K+]0-induced changes in pHj (and [Ca2+]j) observed in cultured rat hippocampal neurons in the presence of C a 2 + 0 reflected membrane depolarization or changes in H + transport via a In-dependent pathway, veratridine was used as an alternate method of depolarization. Veratridine causes membrane depolarization by activating voltage-gated sodium channels and preventing their inactivation (Valkina et al., 1993; Khodorov et al., 1994; Zhan et al., 1998), and its use in the present experiments has the added benefit of allowing depolarization to be imposed at a constant [Na+]0, avoiding the potential modulation of Na+-dependent pHj regulating mechanisms. In response to a 2 min exposure to 20 uM veratridine in the presence of C a 2 + 0 , [Ca2+]i responded in the same biphasic manner seen in response to high [K + ] 0 exposure, i.e. with an initial rapid increase in BI334/BI380 ratio values followed by a partial recovery in the continued 57 presence of veratridine to a new plateau level (Fig. 3.2). Additionally, as seen with depolarization evoked by high [K +] 0 , 20 uM veratridine evoked a fall in pHj (measured with BCECF) of 0.46 ± 0.12 pH units (n = 5; Table 3.2; Fig. 3.2). A lack of recovery of pHi (and [Ca2+]i) was observed following treatment with veratridine, which may be attributed to the toxic effects of this depolarizing agent (Ramnath et al., 1992). As the relative degree of depolarization due to high [K + ] 0 and veratridine exposure was not assessed, no quantitative comparisons between the magnitudes of the [Ca2+]j and pHi changes observed in response to high [K + ] 0 and veratridine exposure are made; nonetheless, that the responses to the two modes of depolarization are similar supports the hypothesis that the [Ca2+]i and pHj responses to high [K + ] 0 are due to the effects of depolarization and not H + influx via a K+-dependent pathway or changes in [Na+]0 due to replacement with K + . Similarly, Sheldon and Church (2002) have shown in acutely dissociated adult rat hippocampal CA1 neurons that the P-type K +/H +-ATPase inhibitors omeprazole and SCH 28080 did not affect the ability of high [K + ] 0 to increase the rate of pHj recovery from internal acid loads. 3.1.1.3. Role of a plasmalemmal Ca2+, hf-ATPase In various neuronal and non-neuronal cell types (e.g. Gatto and Milanick 1993; Schwiening et al., 1993; Khodorov et al., 1995; Wu et al., 1999), as well as in rat hippocampal slices (Smith et al., 1994; Trapp et al., 1996b; Zhan et al., 1998), the intracellular acidification observed in response to depolarization (with high [K +] 0 , veratridine, iV-methyl-D-aspartate (NMDA), electrical stimulation, and/or ouabain) has been attributed to the activation of a PMCA by the depolarization-evoked rise in [Ca ]j. Thus, I proceeded to assess whether a PMCA plays a role in the pHj and [Ca ]j responses to high [K ] 0 exposure in my preparation of cultured postnatal rat hippocampal neurons. 58 It has been reported that BCECF, a fluorescein analogue, inhibits PMCA activity in red blood cells (7C5 0 = 100 uM; Gatto and Milanick, 1993) and it has been suggested that BCECF may not be an appropriate indicator to use for measurements of pHi in cells that possess PMCAs (Willoughby et al., 1998). Experiments were thus repeated using two non-fluorescein based pH-sensitive dyes, HPTS and SNARF-5F, to confirm the validity of the aforementioned pHj changes measured with BCECF in response to depolarization in the presence of C a 2 + 0 . The magnitudes of the pHj responses to approximately 2 min exposures to 25 mM [K + ] 0 or 20 pM veratridine measured with HPTS, which has been suggested not to inhibit PMCA activity (Willoughby et al , 1998), were similar to those measured with BCECF (Table 3.2; Fig. 3.2). Additionally, in neurons loaded with SNARF-5F (resting p H 7.38 + 0.07; n = 12), a 2 min exposure to 75 mM [K + ] 0 evoked a fall in pHj of 0.54 ± 0.03 pH units (Table 3.2; Fig. 3.3), a value not significantly different (P >0.05) from the fall in pHj of 0.47 ± 0.03 (n = 9) pH units evoked by the same stimulus in BCECF-loaded neurons. Thus, the pHi response to depolarizing conditions in cultured hippocampal neurons in the presence of C a 2 + 0 does not appear to be markedly different when measured with BCECF, HPTS or SNARF-5F. In agreement, others have successfully examined the activity of the PMCA in neurons loaded with BCECF (e.g. Trapp et al., 1996b; Wu et al , 1999). If a PMCA indeed contributes to the fall in pHj evoked by high [K + ] 0 in the presence of C a 2 + 0 , pHj should start to decrease after, and certainly not before, the rise in [Ca2+]i. To assess the temporal relationship between the pHj and [Ca ]j changes, measurements of pHj with 94-SNARF-5F were made simultaneously with fura-2 measurements of [Ca ]j in the same cells (see Materials and Methods, Section 2.2). The pHj and [Ca ]i responses of a single neuron to a 2 min exposure to 75 mM [K + ] 0 in a representative neuron are shown in Fig. 3.3A. Inversion of the pHi trace (Fig. 3.35) highlights the fact that [Ca 2 +]i increased prior to any change in pHj. 59 Indeed, simultaneous measurements of pHi and [Ca 2 +]i in 52 individual neurons on 12 coverslips co-loaded with SNARF-5F and fura-2 indicated that the rise in [Ca2+]j evoked by 75 mM [K + ] 0 consistently started to occur before the fall in pHi (0.09 ± 0.02 min vs. 0.29 ± 0.3 min; P O.01, paired Mest), consistent with the intracellular acidification being dependent on C a 2 + influx and the subsequent secondary activation of an acid-loading PMCA. Lastly, the effects of the non-selective PMCA inhibitor, eosin B, on the pHj and [Ca 2 +]i responses to high [K + ] 0 were assessed. In the presence of 20 pM eosin B, the magnitude of the rise in [Ca 2 +]i observed in response to a 5 min application of 75 mM [K + ] 0 was significantly greater (P <0.01; n = 6) than that observed under control conditions (Table 3.3; Fig. 3.44). Conversely, in the presence of eosin, the 30.09 ± 0.05% recovery of [Ca2+]j and the 0.24 ± 0.02 unit fall in pHj observed during exposure to 75 mM [K + ] 0 were both significantly smaller (P <0.05 and P <0.0001, respectively) than the respective changes observed under control conditions (n = 6; Table 3.3; Fig. 3.45). These results indicate that eosin B reduced C a 2 + efflux and H + influx during the depolarization in the presence of C a 2 + 0 and are consistent with previous findings in rat hippocampal neurons (Trapp et al., 1996b) and other cell types (Gatto and Milanick, 1993; Wu et al , 1999) that the intracellular acidification observed during depolarization in the presence of external C a 2 + 0 is due, at least in part, to the activity of a PMCA. 3.1.2. Depolarization in the absence of external Ca causes an intracellular alkalinization In the presence of extracellular Ca , it is possible that under depolarizing conditions, H efflux via a voltage-dependent pathway, such as a gH+, may be masked due to the activity of the acid-loading PMCA. Thus, the pHj response to depolarization was re-examined under 0 Ca 0 60 conditions whereby pHj changes independent of C a 2 + influx' and PMCA activity might be observed. 3.1.2.1. pHj and [Ca2+]j responses to high [lC]0 and veratridine under 0 Ca2+0 conditions In contrast to the large increases in [Ca2+], observed in the presence of external C a 2 + , no appreciable changes in BI334/BI3&0 ratio values were observed in response to 5 min exposures to high [K ] 0 under 0 Ca 0 conditions (Fig. 3.5,4), indicating that most, if not all, of the increases in [Ca2+]j described in Section 3.1.1 were due to C a 2 + influx. Figure 3.55 shows a representative trace of the pH- response to 5 min 75 mM [K + ] 0 in the absence of external Ca 2 + . Under these conditions, neurons with a resting pHj of 7.15 ± 0.03 (n = 37) showed a rise in pH- of 0.17 ± 0.01 pH units that began at 0.40 ± 0.07 min and reached a maximum at 5.33 ± 0.10 min after the start of the depolarization (Table 3.4). Qualitatively similar responses were observed for 25, 50 (see Fig. 2.6), and 139.5 mM [K + ] 0 (Table 3.4), and the magnitude of the rise in pHj was found to be significantly associated with the [K + ] 0 used to depolarize the neurons (n=49; P O.01; r = 0.37). As was done in the presence of external C a 2 + (see Section 3.1.1.2), the effects of 20 uM veratridine on pHj were also assessed under 0 C a 2 + 0 conditions, to ensure that the increases in pHj observed in response to high [K + ] 0 were due to the effects of membrane depolarization and not a K+-dependent H + transport pathway or the effects of changing [Na+]0. In response to a 5 min exposure to 20 uM veratridine in the absence of external Ca 2 + , pHj began to increase at 0.36 ± 0 . 1 0 min from a resting value of 7.05 ± 0.08, rising by 0.29 ± 0.04 pH units at the end of 5 min (n = 9 BCECF-loaded neuronal populations; Fig. 3.6). A similar 0.32 ± 0.06 pH unit increase in pHj was evoked by 20 uM veratridine in HPTS-loaded neurons under Ca2+0-free conditions (n = 3; Fig. 3.6). No recovery of pHi was observed following washout of veratridine; 61 likely reflecting the toxic effects of the alkaloid compared to high [K + ] 0 (see Section 3.1.1.2). That both high [K + ] 0 and veratridine evoke an intracellular alkalinization under 0 C a 2 + 0 conditions suggests that most, if not all, of the rise in pHj is due to the effects of voltage, rather than H + efflux via K + - or Na+-dependent H + transport pathways. 3.1.2.2. Measurements of membrane potential with changing [lC]0 The results detailed above suggest that the rises in pHj observed in response to high [K + ] 0 in the absence of C a 2 + 0 are likely due to the effects of changes in membrane voltage. To examine directly the membrane depolarizations evoked by high [K + ] 0 , electrophysiological experiments were performed where cultured hippocampal neurons were current-clamped in the conventional whole-cell configuration and perfused with the same high [K + ] 0 solutions used to depolarize neurons in the microspectrofluorimetric studies (Fig. 3.7A). Under resting conditions (i.e. 3 mM [K+] 0), Vm was -62 ± 1 mV (n = 31). As would be predicted for a Vm dependent on the transmembrane K + electrochemical gradient, as the value of log[K+] 0 increased, the magnitude of the change in membrane potential also increased, ranging between 36.5 ± 1.5 to 64.3 ± 1 . 6 mV for 25 and 139.5 mM [K + ] 0 , respectively (P O.0001; r = 0.88). Thus, Vm and the magnitude of the rise in pHj observed under 0 C a 2 + 0 conditions decrease and increase, respectively, with increasing [K + ] 0 (Fig 3.75), supportive of the hypothesis that the internal alkalinizations are due to H + efflux via a voltage-dependent pathway. 3.1.2.3. Contributions of known proton efflux pathways to the rise in pHt Under the nominally-HC03 /CO2 free, HEPES-buffered, 37°C conditions used, a N H E has been reported to be the major acid extruding pathway in rat hippocampal neurons (Raley-Susman et al., 1991; Baxter and Church, 1996). To examine whether the rise in pHj observed in response to 62 high [K ] 0 in the absence of external Ca was due to NHE activity, the effects of the removal of extracellular Na + on high [K+]0-induced changes in pHi were assessed. Under 0 C a 2 + 0 conditions, exposure to 0 Na + , NMDG-substituted medium (pH 0 constant at 7.35) caused a rapid fall in intracellular pHj of 0.28 ± 0.04 pH units, from a resting pHj of 7.12 ± 0.04 (n = 7; Fig. 3.8/4). In order to restore pH, to near control levels, the weak base TMAmine (5 mM) was then added to the 0 N a + 0 perfusion medium, resulting in a rise in pH, to 7.20 ± 0.05, followed by a slow decrease to pHi 7.11 ± 0.03. At this point, a 5 min exposure to 75 mM [K + ] 0 evoked a rise in p H of 0.16 ± 0.03 pH units, which was not significantly different from that observed under control (NaVcontaining), 0 C a 2 + 0 conditions (P >0.05; see Table 3.4). These results suggest that NHE activity is not mediating the voltage-dependent increases in pHj, consistent with findings that the antiporter is not directly affected by changes in membrane potential (Raley-Susman et al., 1991; Demaurex et al., 1995). Additionally, Sheldon and Church (2002) have shown in rat hippocampal neurons that the enhancement of pH; recovery from internal acid loads by high [K + ] 0 is not dependent on [Na+]0, supportive of a Na+-independent, voltage-dependent H + efflux pathway. It has been suggested that highly H + permeable (Mozhayeva and Naumov, 1983), non-inactivating, tetrodotoxin (TTX)-sensitive voltage-gated Na + (Nav) channels may provide a voltage-dependent pathway for transmembrane H + movement in frog nerve fibres (Valkina et al., 1993, 1995; Khodorov et al., 1994). Though not formally assessed, this may also be true in rat hippocampal neurons, which possess a TTX-sensitive, non-inactivating Na v current (French et al., 1990; Taylor, 1993) that is enhanced by increased external potassium (Somjen and Muller, 2000), i.e. conditions used in this study. Thus, to assess whether H + efflux via non-inactivating, TTX-sensitive Na v channels contributes to the depolarization-evoked rise in pH, observed in rat hippocampal neurons, experiments were repeated in the presence of T T X (Fig. 63 3.85). Under 0 C a 2 + 0 conditions, in the presence of 1 pM TTX, the rise in pH, of 0.12 ± 0.04 pH units observed in response to a 5 min exposure to 75 mM [K + ] 0 was not significantly different from that seen under control conditions (P > 0.05; n = 3). In summary (Fig. 3.8C), the results suggest that the internal alkalinization evoked by high [K + ] 0 in the absence of external C a 2 + is not due to H + extrusion via either NHE or non-inactivating, TTX-sensitive Na v channels and may therefore be due to an additional, as yet undefined, voltage-dependent H + efflux pathway. 3.1.3. Contributions of a gH+ to the ht efflux caused by depolarization under 0 Ca2+0 conditions To assess the possibility that the pHj response to depolarizing conditions in the absence of 2+ + external Ca may be mediated by a voltage-dependent H conductance, the effects of high micromolar concentrations of Z n 2 + and changing the transmembrane pH gradient and temperature, variables known to modulate gH+s in other cells types, on the intracellular alkalinization were examined. 3.1.3.1. The rise in pHt and its inhibition by Zn2+ are sensitive to the transmembrane pH gradient 3.1.3.1.1. The effects ofZn2+ in neurons with a normal ApHme,„b Paired experiments were performed where neuronal populations (i.e. coverslips) were exposed first to 75 mM [K ] 0 for 5 min in the absence of external Ca , allowed to recover, then exposed to 75 mM [K ] 0 for a second time in the presence of 250 pM Zn (Fig. 3.9). The resting pH; prior to the control depolarization was 7.11 ± 0.03, while the pHj value prior to the 24* depolarization in the presence of Zn was 7.08 ± 0.02 (n = 10). While the magnitude of the rise in pHj appeared to be slightly smaller in the presence of Zn (0.22 ± 0.03 vs. 0.18 ± 0.02 pH 64 units), the difference was not significant (P >0.05). The same was true for the initial rate of increase in pHi (0.0013 ± 0.0003 vs. 0.0012 ± 0.02 pH units/s; P >0.05). A cursory interpretation of these results would suggest that the rise in pHj evoked by 75 mM [K + ] 0 in the absence of Ca 0 is not sensitive to Zn and, therefore, is not due to the activity of a gH+. However, upon closer examination it became apparent that both the magnitude of the rise in pH-under control conditions (and the inhibitory effect of Zn 2 +) were enhanced in cells with a lower resting pHj and thus, a greater ApH m e m b (cf Fig 3.9A and B). Indeed, the magnitude of the rise in pHj measured under both control conditions (n = 37, r = 0.71, P < 0.0001) and in the presence of Z n 2 + (n = 10, r = 0.74, P < 0.05) was dependent on the value of the ApHm emb immediately prior to the application of 75 mM [K + ] 0 (see Fig. 3.11,4). These properties are consistent with the dependence of gH+ gating on the ApHm emb (Kapus et al., 1993a; Cherny et al., 1995) and 2_|_ indicate that any investigation into the effects of Zn (or any other treatment) must take into account the effect of the ApH m e m b. These findings are considered further in Section 3.1.3.1.2 and Fig. 3.11. 3.1.3.1.2. The effects of Zn in neurons with an enhanced ApHmemb In order to further assess the dependence of the rise in p H i and the effect of Z n 2 + on the A p H m e m b , experiments were repeated under conditions of enhanced A p H m e m b . The mitochondrial uncoupler and protonophore FCCP was used to lower pHj prior to exposure to high [K + ] 0 , thereby increasing the A p H m e m b (see Buckler and Vaughan-Jones, 1998). In response to a 5 min pre-treatment with 1 uM FCCP, p H - fell by 0.57 ± 0.04 p H units to 6.62 ± 0.03, from a resting value of 7.19 ± 0.03 (n = 25; Fig. 3.10). Under the p H 0 7.35 conditions used, this represents a ApHmemb of 0.62 ± 0.03 p H units, which is significantly greater than the 0.20 ± 0.02 p H unit ApHmemb observed in neurons without FCCP pre-treatment (P <0.0001). Following treatment 65 with FCCP, and after a brief interval to allow membrane potential and [Ca 2 +]i to recover (see Buckler and Vaughan-Jones, 1998; Nowicky and Duchen, 1998; Park et al., 2002), a 5 min exposure to 75 mM [K + ] 0 resulted in a rise in pHj of 0.47 ± 0.02 pH units (Fig. 3.1&4; n= 11), a significantly larger alkalinization than that observed in neurons with a normal ApHmemb (0.17 ± 0.01 pH units; n = 31;P <0.0001). The rise in pHi evoked by 75 mM [K + ] 0 in FCCP pre-treated cells was considerably smaller in the presence of 250 pM Z n 2 + , 0.34 ± 0.02 pH units, than in its absence (5 O.0001; n = 14; Fig. 3.105). Linear regression analyses of data obtained from neurons with normal and enhanced ApHmembS, show that under control conditions (i.e. in the absence of Zn 2 +) both the magnitude of the rise in pH; (P O.0001; r = 0.87; Fig. 3.11) and the initial rate of rise of pHj (P O.05; r = 0.49) are dependent on the value of ApHmemb immediately prior to the application of 75 mM [K +] 0; similar analyses indicate that these relationships still hold in the presence of Z n 2 + (P <0.0001, r = 0.89; P O.01, r = 0.62, respectively; Fig. 3.11). Overall tests of coincidence of the regression lines describing data obtained in the absence and presence of Z n 2 + indicated that the two sets of data are significantly different (5 < 0.01). Results similar in all aspects were also observed in response to depolarization with 139.5 mM [K + ] 0 (data not shown). 2+ To summarize, under 0 Ca 0 conditions the rise in pHj and the rate of rise of pHj observed in response to high [K+]0-induced membrane depolarization is directly dependent on the value of the ApHmemb immediately prior to the stimulus, consistent with increased H + extrusion at greater ApHmembS due to the increased driving force for H + movement and an increased and more rapid activation of gH+s. Furthermore, the inhibitory effect of Z n 2 + on the rise in pH, and the rate of rise of pHj is also dependent on the ApHmemb, such that the degree of inhibition is larger at a greater ApHmemb, consistent with an increasing availability of gH+s for Zn to inhibit at larger ApHmembS. 66 3.1.3.2. The effects of removing extracellular Zn2+ with DTPA To examine from which side of the plasma membrane Z n 2 + was acting to inhibit the rise in pHj, neurons were exposed to 75 mM [K + ] 0 for 5 min in the presence of both 250 u.M Z n 2 + and the membrane impermeant heavy metal cation chelator, DTPA (1.5 mM) (Peral and Ilundain; 1995). Under these conditions, in neurons with a ApH m e m b of 0.25 ± 0.06 pH units (n = 3), 75 mM [K + ] 0 induced a rise in pHi of 0.27 ± 0.06 pH units (Fig. 3.12,4), which was not significantly different from that observed under control conditions (i.e. in the absence of Zn 2 + ) in neurons with a similar A p H m e m b (0.19 ± 0.04; n = 10; P > 0.05). Treatment with DTPA alone did not cause a significant change in the magnitude of the rise in pHj evoked by 75 mM [K + ] 0 in the absence of Ca 0 (P > 0.05; n = 3; data not shown). To ensure that the result with DTPA was not simply due to the complete chelation of all the Z n 2 + in the solution (i.e. preventing any Z n 2 + entry into cells), experiments were also performed where neurons were exposed first to 250 U.M Z n 2 + and 75 mM [K + ] 0 and then, partly into the depolarization, DTPA was added (Fig. 3.125). This protocol allowed for an initial depolarization-induced entry of Z n 2 + into the cells (reviewed by Colvin et al., 2000), via Na + /Ca 2 + exchange (Sensi et al., 1997), glutamate receptor-operated channels (Sensi et al., 1997; Cheng and Reynolds, 1998), and/or voltage-gated C a 2 + channels (Sensi et al., 1997; Kerchner et al., 2000), prior to the rapid removal of the extracellular Z n 2 + with DTPA. Under these conditions, pHj initially increased in response to 75 mM [K + ] 0 at a rate of 0.0006 ± 0.0002 pH units/s (n = 4). Following the application of DTPA and, thus, the removal of extracellular Z n 2 + , the rate of rise of pHj rose to 0.0017 ± 0.0004 ± pH units/s, significantly greater than that observed in the absence of DTPA and, thus, the presence of external Z n 2 + (P < 0.05). The results of both types of experiments with DTPA are consistent with those of previous studies that found that the inhibitory site of action of Zn on gH+s is extracellular (Peral and Ilundain, 1995; Cherny and DeCoursey, 1999). 67 3.1.3.3. The effects of increased pH0 The dependence on pH 0 of the rise in pHj evoked by 75 mM [K+]„ and its inhibition by Z n 2 + were assessed by increasing the pH 0 under which experiments were performed. In cells with a resting pH; of 7.27 ± 0.03 under 0 Ca 0 conditions at pH 0 7.35 (n = 16; Fig. 3.13/4), perfusion 94-with pH 0 7.8, 0 Ca o media caused a rise in pH, to 7.46 ± 0.02 pH units, resulting in a ApHmemb (0.34 ± 0.02 pH units) that was significantly greater than that observed under pH 0 7.35, control conditions (P O.0001; see Section 3.1.3.1.1.). Under pH 0 7.8 conditions, a 5 min exposure to 75 mM [K + ] 0 evoked a further rise in pHj of 0.23 + 0.02 pH units. Comparisons of the relationships between the magnitudes of the high [K+]0-evoked rises in p H and ApHmemb under p H 0 7.35 and 7.8 conditions indicate that they are not significantly different (P >0.05) and can be fit by a single line (P < 0.0001; r = 0.83; Fig. 3.135). Thus, the results of experiments performed at p H 0 7.8, combined with those from experiments performed at p H 0 7.35, indicate that the magnitude of the rise in pHj evoked by depolarization in the absence of external C a 2 + is not dependent on either pH 0 or pHi directly, but rather on ApHmemb, a finding that is consistent with the ApHmemb-dependent gating properties of gu+s in other cell types. Similar to the results obtained under pH 0 7.35 conditions, the magnitude of the 75 mM [K+]0-evoked rise in pHi was also attenuated (P < 0.01) by 250 pM Z n 2 + under pH 0 7.8, 0 C a 2 + 0 conditions in a ApHmemb dependent manner (n = 15; P O.0001; r = 0.63; Fig. 3.135). However, the degree of inhibition by Zn appeared to be greater in neurons exposed to high-pH0 media, even after the effects of ApHmemb had been taken into account. Indeed, comparison of the alkalinizations observed under pH 0 7.35 and 7.8 conditions in neuronal populations with a ApHmemb between 0.25 - 0.35 pH units shows that, while there is no difference between the magnitudes of the rises in pHj observed under control conditions, the inhibitory effect of Z n 2 + is significantly greater under pH 0 7.80 conditions (P < 0.01; Fig. 3.13C). Thus, the ability of Z n 2 + 68 to reduce the rise in pH- evoked by high [K + ] 0 is sensitive to changes in pH 0 , in addition to ApHmemb, consistent with the properties of gH+s described elsewhere (Cherny and DeCoursey, 1999). 3.1.3.4. The effects of reducing temperature To assess whether the rise in pHj observed under depolarizing conditions in the absence of external Ca was temperature dependent, experiments were repeated at room temperature (~22°C). Under these conditions, resting pH- was 7.36 ± 0.02 (n = 5 coverslips), significantly greater than that observed in same-day control experiments performed on sister cultures at 37°C (7.19 ± 0.08 ; n = 4; P <0.04). Thus, to examine the effects of temperature, cells with a ApH m e m b between 0.05 - 0.15 pH units were selected. In these neurons, a 5 min exposure to 75 mM [K + ] 0 at 37°C evoked a rise in pHj of 0.13 ± 0.02 pH units (n = 1 neurons); a significantly smaller increase of 0.04 ± 0.01 pH units was observed at room temperature in = 6 neurons; P O.0001; Fig. 3.14,4,5). A similar inhibitory effect of reduced temperature on the initial rate of rise in pHj was observed in these neurons (P O.0001; Fig. 3.14Q. The sensitivity of the rise in pHj observed under 0 C a 2 + 0 , depolarizing conditions to reductions in temperature is consistent with the reported temperature sensitivity of gH+s in other cell types (Kuno et al., 1997; DeCoursey and Cherny, 1998). 3.1.4. Summary of microspectrofluorimetric findings The pH- response to depolarization with high [K + ] 0 or veratridine is dependent on C a 2 + 0 . In the presence of external C a 2 + , depolarization evokes a fall in pHj that is secondary to the concomitant increase in [Ca2+]i, and is likely the result of plasmalemmal C a 2 + , H+-ATPase 69 activity. In the absence of external Ca 2 + , the same depolarizing stimuli evoke a rise in pHj that does not appear to reflect H + efflux via the acid-extruding pathways previously described in rat hippocampal neurons. The rise in pHj is sensitive to extracellular Z n 2 + , the A p H m e m b and temperature in a manner consistent with the properties of gH+s described in other cell types. 3.2. Electrophysiological studies in neurons patch-clamped in the whole-cell configuration The possibility that a gH+ may be present in rat hippocampal neurons and contribute to the rise in pHj observed under depolarizing conditions in the absence of C a 2 + 0 was explored further by attempting to isolate an underlying H + current using voltage-clamp methods. Initially, the properties of outward currents recorded from cultured postnatal rat hippocampal neurons patch-clamped in the conventional whole-cell configuration under conditions designed to allow for the isolation of H + currents were characterized. The second stage of the electrophysiological studies then assessed the potential contribution of a gu+ to the current. 3.2.1. Characterization of currents recorded under conditions designed to isolate currents 3.2.1.1. Depolarizing voltage-steps evoke outward currents that slowly run-up Under control conditions (~22°C, pH 0 7.5, pHj 6.0, Vh = -20 mV), 8 s depolarizing steps of varying magnitude applied 10 min after establishing the whole-cell configuration evoked families of outward currents with a peak current of 24.6 ± 4.4 pA at +40 mV (n = 22; Fig. 3.15/4). Interestingly, following a brief rest period (see Materials and Methods, Section 2.3.3.2), a subsequent series of voltage-steps elicited a family of currents of greater amplitude (peak current at +40 mV = 50.3 ± 6.9 pA, n = 17). Indeed, when each series of voltage steps were repeated at 10 min intervals, the size of the outward currents continued to increase such that at 110 min after establishing the whole-cell configuration, the magnitudes of the currents were 70 approximately five-fold greater than observed at the beginning of the experiment (Fig. 3.15,4,5). The voltage at which currents could be recorded also became more negative, shifting from 20 to 0 mV between 10 and 110 min. The time-course of this current-run up is summarized in Fig. 3.15C. Due to the large increases in current amplitudes associated with the run-up, particularly in the first hour of an experiment, test procedures were not performed until two (usually three) consecutively recorded current families of similar magnitudes were observed. This typically occurred -1.5 h after establishing the whole-cell configuration, after which the run-up appeared to be mostly complete (see Fig. 3.15Q. A marked run up of H + currents has been described in other cell types, such as snail neurons (Byerly et al., 1984) and rat alveolar epithelial cells, where H + currents tended to become larger, and activate faster and at more negative potentials, as experiments progressed (Cherny et al., 1995). 3.2.1.2. Contribution of ions other than protons to the current Under control conditions, the concentration of permeant ions in the bath and pipette solutions were minimized to facilitate the recording of H + currents (for a review on solution choices for recording H + currents, see DeCoursey and Cherny, 1994b). Nonetheless, there remained the 2+ - -\-possibility that ions remaining in the solutions, namely Mg , CI and/or T M A could be contributing to the outward currents. To examine this possibility, the effects of changing the transmembrane concentration gradients of Mg and CI on the currents were tested. Increasing [CI ] 0 from 8 to 20 mM ([CI ] i = 8 mM) did not cause a significant change in the magnitudes of the outward currents measured after completion of current run-up (Fig. 3.16,4). Increasing [Mg 2 + ] 0 to 20 mM ([Mg 2 +]i = 4 mM) also failed to affect the outward currents (Fig. 3.165). Thus, it appears that the currents are not carried either by CI or Mg . 71 The potential contribution of T M A + to the outward currents was not formally examined. However, in initial experiments performed with CsMeS03- rather than TMAMeS03-based solutions in = 11), outward currents that exhibited a similar run-up and magnitude to those recorded using T M A + were observed. That the outward currents can still be observed in the absence of T M A + suggests that this large cation is an unlikely carrier of the outward current. 3.2.1.3. The effect of changing ApHmemb on Vrev To directly assess the selectivity of the outward current for H + , tail currents were recorded under different ApHmembS and the effects on Vrew examined. Under control conditions (ApH m e m b = 1.5 pH units), after a 4 s conditioning pulse to +60 mV, voltage-steps to +40 to -60 mV in 20 mV increments elicited tail currents that reversed at -22 ± 2 mV (n = 7; Fig. 3.17/4). The relationship between VKV and ApH m e m b is shown in Fig. 3.175; VKy varied by -10 mV/pH unit change in ApHmemb (r = 0.97; P < 0.05), suggesting that at least part of the current may be due to H + efflux. This relationship is, however, much less steep than the theoretical slope of 58 mV/pH unit change in ApHmemb if the current were only carried by protons. Nevertheless, it is not uncommon for the F r e v of H + currents to diverge from the theoretical values; slopes of 23 - 45 mV/pH unit change in ApHmemb have been reported and attributed, in part, to a poor control of pHj and, thus, ApHmemb (DeCoursey, 1998, 2003). A similar difficulty may contribute to the results presented here. Measurements of pHj in patch-clamped neurons loaded with BCECF indicated that bulk cytosolic pHi only falls to 6.52 ± 0.12 pH units when the pipette pH is 6.0 (pH 0 7.5). As ApHmemb is calculated using the absolute pH of the bath and pipette solutions, this leads to an overestimation of ApHmemb- These matters are considered further in the Discussion. 72 3.2.1.4. Gating kinetics The time course of the outward currents appeared to have two phases, a short, rapidly rising phase, followed by a long, slow increase that continued for the rest of the depolarization. A maximal steady-state current was rarely observed during the 8 s time course of the depolarizing steps employed, especially at the higher voltages. The outward currents exhibited time courses that followed either a double exponential or the sum of a single exponential with a linearly rising component (cf. Fig. 3.15,4, right panel with Fig. 3.20,4, left panel). While there is a great deal of variability in the gating kinetics of H + currents observed among various cell types (see DeCoursey, 2003), a single exponential (with or without a linearly rising component) has consistently been used to describe the gating kinetics of H + currents in cells such as snail neurons (Byerly et al., 1984), human eosinophils (DeCoursey et al., 2001), human neutrophils, rat alveolar epithelial cells and mammalian phagocytes (DeCoursey and Cherny, 1998; Cherny and DeCoursey, 1999). However, in rat hippocampal neurons, the slow rising phase of the outward currents appeared to follow an exponential time course approximately 60% of the time and, overall, the currents were fitted better to a double exponential function compared to the sum of a single exponential function with a linearly rising component (Fig. 3.18). Thus, for the sake of simplicity, all currents were fit to a double exponential function, resulting in two activation time constants, one for the fast rising phase (xact(i)) and one for the slow phase (xact(2))-The activation time constants obtained in this way are plotted in Fig. 3.19,4. The time course of the fast rising phase became more rapid at more positive potentials, with xact(i) decreasing e-fold in 36 mV (r = 0.96). Interestingly, the time course of the slow rising phase became slower at more positive potentials, with xact(2) increasing e-fold in 43 mV (r = 0.82). The time constant of deactivation of the outward current (xtan) was obtained by fitting tail currents like those illustrated in Fig. 3.17,4 with a single exponential. This provided a reasonable 73 fit over the voltage-range negative to the threshold for activating the outward currents (i.e. below 0 mV). Deactivation was faster at more negative potentials, with xtaii changing e-fold in 24mV(Fig. 3.195). 3.2.2. Contribution of a gH+ to the outward currents 3.2.2.1. The effects of extracellular Zn2+ To examine the possibility that the outward currents described above are mediated by H + efflux via a gH+, the effects of Z n 2 + were assessed. In contrast to its inhibitory effect on H + currents recorded in other cell types, as well as on the high [K+]0-induced intracellular alkalinization observed in the microspectrofluorimetric studies, the application of Z n 2 + had no effect on the magnitude or time course of the outward currents (n = 5; Fig. 3.20). 3.2.2.2. The effects of increased temperature Due to the high temperature sensitivity of both the conductance and gating kinetics of gH+s, the possibility that putative H + currents might be enhanced if the temperature was increased from ~22°C to 30°C was examined. At the higher temperature, the viability of patch-clamped neurons was reduced, such that experiments were often < 40 min in duration. As a result, comparisons had to be made before run-up was complete, between time-matched currents under control (i.e. room temperature) and 30°C conditions. Regardless, unlike the pronounced effect of temperature on the magnitude and rate of rise of pHj observed with microspectrofluorimetry in response to high [K ] 0 exposure under 0 Ca 0 conditions, there appeared to be little difference in the properties of the outward currents recorded at 20°C and 30°C (Fig. 3.21). 74 3.2.3. Summary of results of electrophysiological studies Outward currents that run-up very slowly were recorded from cultured rat hippocampal neurons under conditions designed to isolate H + currents. The F r e v of the currents varied with the ApHmemb, suggestive of at least a partial involvement of protons as the charge carrying species. There was, however, no effect of temperature or Z n 2 + on the magnitude of the currents, inconsistent with the properties of gH+s described to date in other cell types. 75 Table 3.1. Magnitudes of high [K + ] 0 - induced changes in [Ca 2 +]- and pH- in the presence of external C a 2 + [K + ] 0 Resting [Ca2+]-( 5 / 334 / 5 / 380 ratio) Magnitude of ( 5 / 334 / 5 / 380 ratio values) Resting pHj Magnitude of fall in pHi * (pH units) (mM) Rise in [Ca 2 +]i * Recovery during * 25 0.19 ± 0 . 0 1 (7) 0.39 ± 0.03 (7) 0.07 ± 0.02 (7) 7.27 ± 0.04 (6) 0.39 ± 0.03 (6) 50 0.21 ± 0 . 0 1 (5) 0.69 ± 0.07 (5) 0.24 ± 0.04 (5) 7.33 ± 0.04 (6) 0.46 ± 0.05 (6) 75 0.20 ± 0 . 0 1 (9) 0.69 ± 0.09 (9) 0.34 ± 0.05 (9) 7.34 ± 0.04 (9) 0.51 ± 0 . 0 3 (9) In all cases, high [K ] 0 was applied for 5 min. n values for each measurement are shown in parentheses. *, indicates that there is a statistically significant association (P < 0.05) between the measured parameter and the [K + ] 0 used to depolarize neurons. 76 Table 3.2. Comparison of pHj measurements made with BCECF, HPTS and SNARF-5F in the presence of external Ca 2 + Experimental condition Change in pH, (pH units) measured with BCECF HPTS 25 mM [K + ] 0 -0.42 ± 0 . 0 1 (5) -0.41 ± 0 . 0 3 (5) 20 pM Veratridine -0.46 ± 0 . 1 2 (5) -0.40 ± 0.07 (3) Experimental condition Change in pHj (pH units) measured with BCECF SNARF-5F 75 mM [K + ] 0 -0.47 ± 0.03 (9) -0.54 ± 0 . 0 3 (12) In all cases, measurements of p H were made after approximately 2 min exposures to depolarizing conditions, n values are shown in parentheses and represent the number of neuronal populations or coverslips tested. 77 Table 3.3. The effects of the P M C A inhibitor eosin B on the pHj and [Ca 2 +]j responses to 75 m M [ K + ] 0 in the presence of external C a 2 + Magnitude of T T t T > 1 , D T ,. . . . Magnitude of fall in pHj (BhwlBIm ratio units) 6 . — — : — T ^ T T H ^ — : — : (pH units) Rise in [Ca ]; Recovery during Control (9) 0.69 ± 0.09 0.34 ± 0.05 0.51 ± 0.03 20 pM Eosin B (6) 0.79 ± 0.02* 0.24 ± 0.04* 0.24 ± 0.02* In all cases, 75 mM [K + ] 0 was applied for 5 min. n values are shown in parentheses. [Ca2+]j and pHi measurements were made in fura-2 and BCECF-loaded neurons, respectively, in parallel experiments conducted in sister neurons. *, indicates statistically significant difference between measurements made under control conditions and those made in the presence of eosin B. 78 Table 3.4. Magnitude and time course of the internal alkalinizations induced by high [K+]„ in the absence of external C a 2 + [K + ] 0 (mM) Magnitude of the rise in pHj (pH units) Time required for (min) Start of rise in pHj Maximum pHj 25 (4) 0.10 ± 0 . 0 3 0.81 ± 0 . 3 2 5.76 ± 1.01 50 (4) 0.14 ± 0 . 0 1 0.59 ± 0.27 5.73 ± 0 . 3 3 75 (37) 0.17 ± 0 . 0 1 0.40 ± 0.07 5.33 + 0.10 139.5 (4) 0.24 ± 0.02 0.21 ± 0 . 1 2 4.79 + 0.19 In all cases, high [K + ] 0 was applied for 5 min. n values for each [K + ] 0 are shown in parentheses. There is a statistically significant (P <0.05) association between the each of the measured parameters and the [K + ] 0 used to depolarize the neurons. 79 Fig. 3.1. [Ca 2 +]j and pHi responses evoked by a 5 min exposure to 75 m M [ K + ] 0 in the presence of external C a . Experiments were performed under H C 0 3 7 C 0 2 - f r e e , HEPES-buffered conditions in the presence of 2 mM C a 2 + 0 . A, a 5 min exposure to 75 mM [K + ] 0 caused a rapid increase in [Ca ]j, represented by an increase in 5/334/5/330 ratio values. In the continued presence of high [K + ] 0 , [Ca2+]j recovered by 37% and remained at this level for the remainder of the stimulus. Upon return to resting conditions (i.e. 3 mM [K+]0), [Ca2+]j recovered quickly back to baseline values. The record shows the averaged response of 10 neurons recorded simultaneously on a single coverslip and is representative of 9 experiments. 5 , a 5 min exposure to 75 mM [K + ] 0 evoked a rapid fall in p H . Upon the return to resting conditions, pH, recovered gradually towards its initial baseline value. The record shows the averaged response of 5 neurons recorded simultaneously on a single coverslip and is representative of 9 similar experiments. 80 OQ 1.0 0.8 0.6 OQ 0.4 0.2 75 mM [K + l 0.0 5 min 8 x Q. 75 mM [K + ] 0 5 min 81 Fig. 3.2. [Ca 2 +]i and pH- responses to veratridine in the presence of external C a 2 + . Shown are superimposed records of the effects of 20 uM veratridine on [Ca2+]j and pHj, measured in separate groups of hippocampal neurons loaded with fura-2 and BCECF or HPTS, respectively. Exposure to veratridine evoked a rapid increase in [Ca2+]j (represented by an increase in 5 /334 /5 /380 ratio values) followed by a decline in [Ca2+]- to a new steady-state level; in a total of 5 separate experiments, [Ca2+]j at the end of a 2 min exposure to 20 uM veratridine was 0.84 ± 0.10 5 /334 /5 /330 ratio units above the resting value. In contrast, in 5 populations of sister neurons loaded with BCECF, veratridine evoked a fall in pHj of 0.46 ± 0.12 pH units which failed to recover following washout. A similar reduction in pHj was observed in a separate group of neurons loaded with HPTS (see Section 3.1.1.3. and Table 3.2). 83 Fig. 3.3. Simultaneous measurements of pHj and [Ca 2 +]j in response to 75 m M [ K + ] 0 in neurons co-loaded with S N A R F - 5 F and fura-2. Experiments were performed under HC0 3 " /CCVfree, HEPES-buffered conditions in the presence of 2 mM C a 2 + 0 . A , a 2 min exposure to 75 mM [K + ] 0 evoked a rise in [Ca2+]j (shown in terms of BI^AIBI^Q ratio values) and a fall in pH, similar to the respective changes seen in neurons single-loaded with either fura-2 (see Fig. 3 A A) or BCECF (see Fig. 3.15). B, inverting the pHj trace from (A) highlights the fact that the rise in [Ca2+]j precedes the fall in pHj, consistent with the activation of an acid-loading Ca 2 + , H + -ATPase by elevated [Ca 2 + ] i . The records were obtained from a single neuron and are representative of responses observed in a total of 52 neurons on 12 coverslips. 84 A 1 5 1 75 mM [K + ] n 85 Fig . 3.4. The effects of the C a 2 + , H + - A T P a s e inhibitor, eosin B , on the [Ca 2 + ]- and pH-responses to 75 m M [ K + ] „ in the presence of external C a 2 + . A, under control conditions, a 5 min exposure to 75 mM [K + ] 0 caused an increase in [Ca2+]j (represented by BI^AIBI^Q ratio values) that recovered by -47% in the continued presence of high [K +] 0 . When the treatment was repeated in the presence of 20 uM eosin B, the rise in [Ca2+]j was enhanced while the recovery of [Ca2+]j during the rest of the stimulus was reduced to -30% from -47%. Eosin did not cause appreciable changes in steady-state [Ca 2 +]i or background fluorescence (not shown). The trace is the averaged response of 12 neurons on a single coverslip and is representative of 6 experiments. B, under control conditions, a 5 min exposure to 75 mM [K + ] 0 induced a large fall in pHj that almost completely recovered following the return to resting conditions. Subsequent perfusion with eosin B caused a slight decrease in resting pHj. Once pH- had stabilized, a second 5 min exposure to 75 mM [K + ] 0 evoked a fall in pHj that was significantly smaller than that observed under control conditions. The trace is the averaged response of 11 neurons on a single coverslip and is representative of 6 experiments. 86 20 uM Eosin B 7.3 n 7 5 mM [K+]n 20 u,M Eosin B 87 Fig. 3.5. [Ca 2 + ] i and pH- responses to 75 m M [ K + ] 0 in the absence of external C a 2 + . Experiments were performed under HC0 37C02-free, HEPES-buffered conditions, in the absence of external Ca 2 + . A, a 5 min exposure to 75 mM [K + ] 0 caused no appreciable changes in [Ca2+]j. The trace is the averaged response of the same 6 neurons from which Fig. 3AA was obtained and is representative of 3 experiments. 5, under 0 C a 2 + 0 conditions, a 5 min exposure to 75 mM [K + ] 0 evoked a rise in pHi, which recovered gradually to baseline values upon the return to resting conditions. The trace is the averaged response of the same 5 neurons from which Fig. 3.15 was obtained and is representative of 37 experiments. The magnitude of the rise in pHj was dependent on the [K + ] 0 used to depolarize the neurons (see Table 3.4; Fig. 3.7). 89 Fig. 3.6. [Ca 2 +]j and pHi responses to veratridine in the absence of external C a 2 + . Shown are superimposed records of the effects of 20 pM veratridine on [Ca2+]i and pHj, measured in separate groups of hippocampal neurons loaded with fura-2 and BCECF or HPTS, respectively. Exposure to veratridine in the absence of external C a 2 + failed to affect [Ca2+]j (represented by /J /334 /5 /380 ratio values) and caused pHj to rise in both BCECF- and HPTS-loaded neurons. 91 Fig. 3.7. The changes in Vm and pH- evoked by high [K + ] „ in the absence of C a 2 + 0 are both dependent on [K + ] 0 . A, current-clamp recording of membrane potential from a neuron patch-clamped in the conventional whole-cell configuration, resting Vm = -53 mV. Two min exposures to media of various [K+] caused rapid and reversible changes in Vm. The trace is representative of 31 similar recordings. B, the magnitudes of both the depolarization (A Vm) and the rise in pH-(A pH-) observed under 0 C a 2 + 0 conditions are dependent on [K +] 0 . For every 10-fold change in [K + ] 0 , A Vm changes by 37.65 mV (r = 0.99; P < 0.0001) and A pHj increases by 0.16 pH units (P < 0.0001; r = 0.99; n = 45). Error bars represent S.E.M. [Kl ( m M - ) : 25 50 75 139,5 O m V 20 mV 5 min < 80 60 40 20 10 100 [K + ] 0 (mM) 1000 93 Fig. 3.8. The rise in pHj evoked by high [K +]„ under 0 C a 2 + 0 conditions is not due to Na + /F£ + exchange or H + efflux via TTX-sensitive voltage-gated N a + channels. Experiments were performed under HCO3 /CCVfree, HEPES-buffered conditions, in the absence of external Ca 2 + . A, in neurons with a resting pHj of -7.28, exposure to 0 N a + 0 (NMDG) media caused pHj to fall to -6.90. Subsequent exposure to 0 N a + 0 medium supplemented with 5 mM T M Amine caused pHi to recover to resting levels. Application of 75 mM [K + ] 0 under these conditions evoked a reversible rise in pHj of-0.16 pH units. The trace is the averaged response of 11 neurons on a single coverslip and is representative of 7 experiments. B, in neurons with a resting pHj of 7.25, perfusion with 1 pM T T X evoked a very slight increase in pHj. Exposure to 75 mM [K + ] 0 for 5 min under these conditions evoked a reversible rise in pH; of 0.07 pH units. The trace is the averaged response of 8 neurons on a single coverslip and is representative of 3 experiments. C , comparison of the magnitudes of the rises in p H (A pHj) observed under conditions illustrated in (A) and (B) with control measurements. Removal of extracellular Na + or the presence of 1 pM T T X had no significant effect on the alkalinization induced by 75 mM [K + ] 0 when compared to control measurements (P >0.05 in each case), n values are shown in each bar; error bars represent s.E.M. 0 Ca 2 + 0 N a + n (NMDG) 5 mM TMAmine 75 mM [K + l 5 min 7.40 7 .35 7.30 7 .25 7.20 J 0.20 0 .15 A Q. 0.10 0 .05 0.00 T Control 0 Na T T X 95 Fig. 3.9. The effect of Z n 2 + on high [K +] 0- induced rises in pH- in neurons with a normal ApHmemb. Experiments were performed under HC0 37C02-free, HEPES-buffered conditions, in the absence of C a 2 + 0 . A, in neurons with a resting pHj of 7.04, a 5 min exposure to 75 mM [ K + ] 0 caused an increase in pH- of 0.27 p H units. Following the recovery of pHj to resting levels, a second depolarization in the presence of 250 uM Z n 2 + evoked a smaller rise in pHj of 0.20 p H units. The trace is the averaged response of 12 neurons on a single coverslip. B, in a similar experiment to (A), exposure to 75 mM [ K + ] 0 from a resting pHj 7.30 induced a small rise in pH-of 0.12 p H units under control conditions. Upon the return to resting conditions, pH- recovered to a lower value of p H 7.22. At this point, a second depolarization in the presence of 250 uM Z n 2 + caused a rise in pHj of 0.15 p H units. The trace is the averaged response of 15 neurons on a single coverslip. Together, these results suggest that the rise in pHi and, possibly, the inhibitory effect of Z n 2 + , observed under depolarizing conditions in the absence of external C a 2 + is dependent on the resting pHj (and/or ApHmemb) immediately prior to the stimulus. 5 min 97 Fig. 3.10. The effect of Z n 2 + on high [K + ] 0 - induced increases in pH- in neurons with a ApHmemb increased by pre-treatment with F C C P . Experiments were performed under HC0 3 " /C02-free, HEPES-buffered conditions, in the absence of external Ca 2 + . A, in neurons with a resting p H of -7.15, 1 uM FCCP caused a rapid fall in pHi to -6.55. pHj then began to slowly recover and, when it reached -6.70 ( A p H m e m b = 0.65), a 5 min exposure to 75 mM [K + ] 0 induced a rise in pH, of 0.47 p H units. The trace is the averaged response of 17 neurons on a single coverslip and is representative of 11 experiments. B, in neurons with a similar FCCP-induced ApHmemb of = 0.55, exposure to 75 mM [K ] 0 in the presence of 250 uM Zn elicited a smaller increase in pHj of 0.37 pH units. The trace is the averaged response of 13 neurons and is representative of 16 experiments. Thus, in neurons with an increased ApHmemb, Z n 2 + has a significant (P < 0.0001) inhibitory effect on the magnitude of the rise in pHj observed under depolarizing, 0 Ca 0 conditions. 7.4 O C a 2+ 98 1 uM F C C P 75 mM [K + ] n x C L 7.2 4 7.0 6.8 6.6 6.4 5 min B x Q. 6.4 J 5 min 99 Fig. 3.11. The rise in pHj observed under depolarizing conditions in the absence of external C a 2 + , and its inhibition by Z n 2 + , are dependent on the A p H m e m b . A, plot of the magnitude of the rise in pHj (ApH,) observed in response to a 5 min exposure to 75 mM [K + ] 0 under various conditions. Under control and FCCP-pretreated conditions in the absence of Z n 2 + , ApHj increases by 0.64 pH units for every unitary increase in the value of ApHmemb immediately prior to the depolarization (P < 0.0001; r = 0.87). In the presence of Z n 2 + (250 pM Z n 2 + and 1 pM 2+ FCCP + 250 pM Zn z ), the relationship falls to a 0.43 pH unit increase in ApHj for every unitary increase in ApHmemb (P < 0.0001; r = 0.89). The regression lines describing the relationships between ApHj and ApHmemb under control and Zn2+-containing conditions are significantly different (P < 0.01). B, plot of the initial rate of increase in pH, in response to a 5 min exposure to 75 mM [K + ] 0 under various conditions. Similar to the magnitude of the rise in pHj, the initial rate of increase was also dependent on the value of ApH m e m b immediately prior to the depolarization, both under control conditions (P < 0.05, r = 0.49) and in the presence of Z n 2 + (P < 0.01, r = 0.62). Again, the regression lines describing the two sets of data were significantly different (P < 0.01). 100 A _ O Control 0.0 0.2 0.4 0.6 0.8 1.0 A P H m e m b (PHo-pH;) B X Q. Q. T5 0.007 0.006 0.005 H 0.004 0.003 0.002 0.001 0.000 O Control A 1 jiM F C C P • 250 uM Z n 2 + A 1 uM F C C P + 250 uM Zn A 2+ 0.0 0.2 0.4 0.6 0.8 1.0 ApH memb 101 Fig. 3.12. The effects of removing extracellular Z n 2 + with the membrane impermeant chelator, D T P A . Experiments were performed under HC0 37C02-free, HEPES-buffered conditions in the absence of external Ca 2 + . A, in two different neuronal populations with a similar A p H m e m b , 250 u.M Zn did not inhibit the rise in pHj observed in response to a 5 min exposure to 75 mM [K + ] 0 when the Z n 2 + chelator DTPA was also present. The control trace is the averaged response of 8 neurons on a single coverslip. The trace of pHj in the presence of 1.5 mM DTPA and 250 uM Zn is the averaged response of 8 sister neurons on a single coverslip and is representative of 3 experiments. B, application of DTPA following depolarization in the presence of Zn causes a rapid increase in the rate of rise of pH-,. DTPA was added to the perfusion medium 1.5 min into the 5 min exposure to 75 mM [K +] 0; Z n 2 + was removed from the perfusate at the same time. With the addition of DTPA, the rate of rise of pHi increased from 0.0012 pH units/s to 0.0024 pH units/s. The trace is the averaged response of 7 neurons on a single coverslip and is representative of 4 experiments. 250 uM Zn 1.5 m M D T P A 103 Fig. 3.13. The effects of increased p H 0 on the rise in pHi and the inhibitory effects of Zn 2 + . Experiments were performed under HC0 37C0 2-free, HEPES-buffered conditions in the absence of external Ca 2 + . A, in neurons with a resting pHj of -7.25 at pH 0 7.35, perfusion with a pH 0 7.8 solution caused a rise in p H j to -7.50. A 5 min exposure to 75 mM [ K + ] 0 under these conditions evoked a rise in p H i of 0.26 pH units. This is not significantly different from the rise in pHj observed under pH 0 7.35 conditions in neurons with a similar ApHmemb. B, plots of the magnitudes of the rises in pHj (A pH) observed in response to a 5 min exposure to 75 mM [ K + ] 0 in the absence of external C a 2 + under the conditions shown on the figure. The magnitude of the rise in p H j observed at pH 0 7.80 is not significantly different from that observed under pH 0 7.35 conditions when the ApHmemb is taken into account (plot includes all control data generated at pH 0 7.35, including those from neurons pre-treated with FCCP; n = 48) and the two sets of data are shown fitted to the same solid line (r = 0.81; P O.0001). Under pH 0 7.80 conditions, the magnitude of ApHj in the presence of 250 pM Z n 2 + is also dependent on ApH m e m b , rising 0.47 pH units per unitary increase in ApHm emb (r = 0.11\ P < 0.001). C, bar chart comparing the magnitudes of ApHj under the various conditions shown in selected neuronal populations with a ApHmemb between 0.25 and 0.35 pH units. At pH 0 7.80, Z n 2 + exhibits an additional inhibitory effect on the rise in p H , compared to pH 0 7.35. *, indicates a statistically significant difference (P <0.05) between rise in pHj measured in the presence of Z n 2 + and under control pH0-matched conditions. 0, indicates a statistically significant difference (P <0.001) between the rises in pHj observed in the presence of Z n 2 + at pH 0 7.80 and pH 0 7.35. n values shown within the bars correspond to the number of neuronal populations (i.e. coverslips) examined; error bars represent S.E.M. 104 0.0 0.2 0.4 0.6 0.8 A P H m e m b (PH 0 - P H ) O pH 0 7.35 A pH 0 7.80 • pH 0 7.80, 250 uM Zn 1.0 2+ 0.3 n 0.2 x C L < 0.1 0.0 X 10 X 8 4> • pH 0 7.35 • pH o 7.35, 250 uM Z n 2 + L\S pH 0 7.80 pH o 7.80, 250 uM Z n 2 + 105 Fig. 3.14. Effects of reducing temperature on the internal alkalinization evoked by high -t- 2+ [K ] 0 under 0 C a 0 conditions. Experiments were performed under HC0 37C02-free, HEPES-buffered conditions. A, under control conditions at 37°C, a 5 min exposure to 75 mM [K + ] 0 evoked an increase in pHj of 0.14 pH units, from a resting pH- of 7.26. At room temperature (RT, ~22°C), the same depolarizing stimulus evoked a smaller rise in pHj (0.03 pH units) in a neuron with a similar resting pHj. Each trace is the response of an individual neuron from experiments performed on the same day with a single batch of cultures. B and C, bar charts comparing the magnitudes of the rise (ApHj) and initial rate of rise (dpHJdf) of pHj observed at 37°C and at room temperature, in selected neurons with a ApH m e m b of 0.05 - 0.15 pH units. *, indicates a statistically significant difference between measurements made under control and RT conditions (P <0.001 and 0.05 for ApHi and dp¥L\/dt, respectively), n values in bars correspond to the number of individual neurons examined; error bars represent s.E.M. 0.15 0.10 0.05 0.00 7 37°C 0.0016 - | 0.0014 -0.0012 -w 'c 0.0010 -X 0.0008 -x~ 0.0006 -Q. T 3 0.0004 -0.0002 -0.0000 - 7 — r 37°C * T I RT 107 Fig. 3.15. Depolarizing voltage steps evoke outward currents that slowly run-up. Cultured neurons were patch-clamped at room temperature in the conventional whole-cell configuration, using TMAMeS03-based bath and pipette solutions. pH 0 and pH, were 7.5 and 6.0, respectively. A, 10 min after establishing the whole-cell configuration, depolarizing voltage steps from Vh = -20 mV evoked small, non-inactivating outward currents. Thereafter, each subsequent family of voltage steps applied at 10 min intervals elicited outward currents of increasing amplitude; 110 min after establishing the whole-cell configuration, the currents had increased by approximately five-fold. Both families of leak-subtracted currents were obtained from the same neuron and are representative of 22 experiments. B, I-V curves for the current families shown in (A). With current run-up, the I-Vrelationship became steeper and the activation threshold moved from +20 to 0 mV. C, time-course of current run-up during depolarization to +40 mV. While run-up is initially rapid, current size typically stabilizes by 90 min after establishing the whole-cell configuration, at which time test manoeuvres could be applied. Each data point represents at least 3 neurons; error bars represent s .E .M. 108 10 min 110 min +60 mV -20 mV-B -40 -20 0 20 40 60 -100 -vm (mV) • 10 min a— 110 min 200 150 H < C L 100 A 50 H 80 i i 1 1 1 1 20 40 60 80 100 120 Time (min) 109 Fig. 3.16. The outward current is likely not carried by CI or M g 2 + ions. Outward currents were recorded at room temperature from cultured neurons patch-clamped in the conventional whole-cell configuration with TMAMeS0 3-based bath and pipette solutions (pH 0 7.5, pHj 6.0). A, I-V curves for currents recorded under control conditions (8 mM [Cl] 0; Control and Wash) and in the presence of 20 mM [CI ]0. Increasing [CI ] 0 failed to exert a significant effect on the magnitudes of the currents. Each data point represents measurements made in at least 2 neurons; error bars represent S . E . M . (when not present they are contained within the symbol area). B, I-V curves for currents recorded under control conditions (4 mM [Mg 2 +] 0; Control and Wash) and in the presence of 20 mM [Mg 2 +] 0. Increasing [Mg 2 +] 0 did not have a significant effect on the magnitudes of the outward currents. Each data point represents results from at least 3 neurons; error bars represent S . E . M . (where not present they are contained within the symbol). 110 -40 < CL 400 300 200 100 -20 -100 - • — Control - v - 20 mM [Cr]n W a s h e -40 350 300 250 - • — Control -v— 20 mM [Mg 2 + ] 0 • W a s h -20 -50 J 20 40 vm (mV) 60 80 I l l Fig. 3.17. Tail currents and mean reversal potentials (Vrey) of outward currents measured at a range of transmembrane pH gradients (ApHm e mb). Cells were patch-clamped in the whole-cell configuration with TMAMeS03-based bath and pipette solutions. Pipette solution p H was fixed at 6.0; A p H m e m b was varied by perfusing neurons with bath solutions of p H 6.5, 7.0, 7.5 and 8.0. A, in a neuron with a A p H m e m b of 1.5 p H units, following a 4 s activating voltage step from F n = -20 mV to +60 mV, 20 mV voltage steps to various potentials evoked families of tail currents. The reversal potential was estimated by plotting the average amplitude of the currents measured between 1 - 1.5 ms after the beginning of the test pulses against voltage. Dashed line represents the zero current level. Activating current has been truncated for clarity. B, mean reversal potentials measured at various A p H m e m b S . F r e v varies with the A p H m e m b with a slope of 9.8 mV/pH unit (r = 0.97, P < 0.05). Dashed line represents the EH calculated by the Nernst equation (slope 58 mV/pH unit), n-values are 2 for ApHm emb = 0.5 and 2 p H units; 7 for ApHmemb =1.0 and 1.5 p H units. Error bars represent S . E . M . 112 113 Fig. 3.18. The time course of activation is best fit by a double exponential function. Outward currents were recorded from neurons patch-clamped at room temperature in the whole-cell configuration, using TMAMeS03-based bath and pipette solutions. The same outward current at +60 mV as shown in Fig. 3.15,4 (right panel) is depicted here fitted to, A, the sum of a single exponential and a linearly rising component and, B, a double exponential function (see Materials and Methods, Section 2.3.4). Panels on the left show the rising phase of the current against time; fitted functions are shown in green. Panels on the right are plots of residuals vs. time for each function, r-values for fits in (A) and (B) were 0.98 and 0.99, respectively. From the plots of residuals it can be seen that the double exponential function provides a better description of the entire current trace, particularly in the early rising phase. 115 Fig. 3.19. Time constants of activation and deactivation of the outward currents. A, activation time constants for the initial fast rising phase (xact(i)) and secondary, slow rising phase (jact(2)) of the outward currents were obtained by fitting current traces to double exponential functions and are plotted as a function of voltage. At more positive potentials, the value of xact(i) became smaller, indicative of a faster initial rising phase. In contrast, the value of xact(2) increased at more positive potentials, in agreement with the more pronounced slowly rising phase of currents recorded at higher voltages. Each data point represents measurements obtained from at least 3 neurons; error bars represent S . E . M . B, deactivation time constants (xtaii) were obtained by fitting tail currents such as those shown in Fig. 3.17.4 to single exponential functions and are plotted as a function of voltage. At more negative potentials the value of xtaii decreased, indicative of faster deactivation at these voltages. Each data point represents measurements obtained from at least 3 neurons; error bars represent S . E . M . • ° - Tact(1) Tact(2) 1 \ -1 1 1 1 1 0 20 40 60 80 Voltage (mV) 1 1 1 1 1 1 70 -60 -50 -40 -30 -20 -10 Voltage (mV) 117 Fig. 3.20. Lack of effect of Zn on the outward currents. Outward currents were recorded in response to depolarizing voltage-steps from neurons patch-clamped at room temperature in the whole-cell configuration with TMAMeS03-based bath and pipette solutions. pH 0 and pHj were 7.5 and 6.0, respectively. A, consecutively recorded families of outward currents recorded from a single neuron after completion of run-up, under control conditions and in the presence of 250 uM Zn . The presence of Zn did not affect either the magnitude or the time course of the outward currents. B, I-V curves for currents recorded under control conditions and in the presence of Zn 2 + . Z n 2 + had no effect on the magnitude of the outward currents or activation threshold. Each data point represents measurements obtained from at least 3 neurons (except for Z n 2 + at +60 mV; n = 1); error bars represent S . E . M . (where not present they are contained within the symbol area). 118 -100 VM (M V) 119 Fig. 3.21. L a c k of effect of increasing temperature on the outward currents. Currents were recorded from neurons patch clamped in the whole-cell configuration, using TMAMeS03-based bath and pipette solutions of pH 7.5 and 6.0 (temperature corrected), respectively. A, outward currents recorded 10 min after establishment of the whole-cell configuration at room temperature (Control) and at 30°C. B, I-V relationships of currents recorded 10 min after establishment of the whole-cell configuration at room temperature and at 30°C. There is no difference in the magnitude of the currents recorded under each condition. Each data point represents measurements obtained from at least 7 neurons; error bars represent s.E.M., when absent they are contained within the symbol. Control 140 120 -\ 100 80 H A 60 - I — • Control -v — 30°C -20 (I -20 -20 40 60 80 121 4. D I S C U S S I O N This thesis presents the results of the first formal investigation into the potential contribution of a voltage-gated proton conductance to pHj regulation in mammalian central neurons. Although previous studies have documented voltage-dependent, Zn2+-sensitive pHj changes in rat hippocampal neurons, leading to the suggestion of gH+ activity in this cell type (e.g. Diarra et al., 1999; Sheldon and Church, 2002), they did not examine whether the pHj changes shared other properties common to gH+s, nor did they look for an associated H + current. With a specific focus on the potential role of a gH+, the present study characterized the pHj response to depolarizing conditions and attempted to record associated H+-selective currents that would confirm the presence of a gH+ in cultured rat hippocampal neurons. Accordingly, this discussion will not only examine the mechanisms regulating the intracellular proton environment in hippocampal neurons under depolarizing conditions, but will also address the possible reasons underlying the difficulties of recording H + currents in this cell type. 4.1. The pHj response to depolarization is dependent on the presence of external C a 4.1.1. The role of a Ca2+, H+-ATPase Under physiological conditions, in the presence of C a 2 + 0 , depolarization-evoked changes in pHj have been described in a variety of neuronal and non-neuronal cell types and attributed to a range of mechanisms, such as simple changes in the driving force for passive transmembrane H + movement (e.g. Austin and Wray, 1993; Sanchez-Armass et al., 1994); increased glycolysis and lactate production (e.g. Zhan et al., 1998); and H + influx via Na v channels or Na+-dependent C a 2 + release from mitochondria and the subsequent displacement by C a 2 + ions of protons from shared binding sites (e.g. Meech and Thomas, 1980; Ou-Yang et al., 1995; Meyer et al., 2000). 2+ In the present study, the pHj response to depolarization in the presence of external Ca was 122 shown to be dependent on C a 2 + influx; the temporal relationship between the increase in [Ca2+]-and fall in pH- is consistent with the secondary activation of an acid-loading mechanism by the rise in [Ca2+]- (see Trapp et al., 1996b). Indeed, the sensitivity of the [Ca2+], and pH- transients to eosin B suggests that a PMCA is responsible for at least part of the fall in pH-. This was unsurprising, given the fact that the PMCA has previously been implicated in Ca2+-dependent pHj shifts in a number of tissue preparations, including snail neurons (Schwiening et al., 1993), cultured rat cerebellar granule cells (Wu et al., 1999), red blood cells (Gatto and Milanick, 1993) and, importantly, rat hippocampal CA1 neurons (Smith et al., 1994; Trapp et al., 1996b). The finding that the [Ca ]j and pH- changes induced by depolarization in the presence of Ca o were not completely inhibited by the PMCA inhibitor eosin B indicates that other mechanisms (e.g. passive binding of calcium to intracellular buffers in exchange for protons and mitochondrial uptake of Ca (Meech and Thomas, 1980; Werth and Thayer, 1994)) must also contribute to the fall in pHj. It is evident, however, that in order to observe the potential H + -extruding activity of a gH+, the acid-loading activities of the PMCA and any other C a 2 + -dependent pathways must be circumvented. 4.1.2. The rise in pHj observed in the absence of external Ca2+ is not due to known rat hippocampal neuron EC efflux pathways Although they did not examine the underlying mechanism(s), Smith et al. (1994) found that the 94-application of glutamate receptor agonists to CA1 neurons in the presence of Ca 0 evoked an extracellular alkalinization (consistent with the activation of a PMCA; see above) whereas, in the absence of C a 2 + 0 , an extracellular acidification was observed. The latter finding is consistent with the present results that, under 0 C a 2 + 0 conditions, a depolarization-evoked intracellular alkalinization was unmasked by the elimination of C a 2 + influx and the associated fall in pH-. 123 Examination of the potential role of the acid-extruding NHE in the rise in pH; is complicated by the lack of a selective pharmacological inhibitor of NHE in rat hippocampal neurons (Raley-Susman et al., 1991; Schwiening and Boron, 1994; Baxter and Church, 1996). Nevertheless, the observation that the rise in pHj was not affected by the removal of external Na + , as well as previous reports that NHE activity is reduced in Ca -free solutions such as those used here (Tornquist and Tashjian, 1991; Murao et al., 2005), indicates that NHE does not make a major contribution to the rise in pHi observed under depolarizing conditions in the absence of external C a 2 + . In addition to NHE activity, the contributions of H + efflux via non-inactivating, TTX-sensitive Na v channels and/or K+-dependent H + transport pathways to the high-[K+]0 evoked rise in pHi were also shown to be negligible. Although neither of these pathways have to date been reported to play major roles in pHj regulation in rat hippocampal neurons, an examination of their potential contributions was necessary in this study due to the experimental conditions used. This is particularly true of non-inactivating Na v channels, as persistent Na + currents can be potentiated by increased external K + (Somjen and Muller, 2000). The lack of effect of T T X on the rise in pHj, and the similarity of the rise in pHj evoked by veratridine (at [K + ] 0 = 3 mM; .[Na+] 0 =138 mM) to that evoked by high [K + ] 0 (and, thus, reduced [Na+]0), therefore suggest that the alkalinization was not an artefact of the experimental conditions. Lastly, all experiments in the present study were performed under nominally H C 0 3 /C02-free, HEPES-buffered conditions, such that the HC03~-dependent pHj regulating mechanisms present in rat hippocampal neurons (see Schwiening and Boron, 1994; Baxter and Church, 1996) should not be contributing to the observed changes. There have been, however, reports that depolarization can evoke rises in pHj in glial cells under similar buffering conditions via an electrogenic Na + /HC0 3 " cotransporter that exhibits a very high affinity for HC0 3 " 124 (Brookes and Turner, 1994; O'Connor et al., 1994; Boussouf et al., 1997). Although Na + /HC0 3 " cotransporters have recently been shown to be expressed in rat hippocampal neurons (Schmitt et al., 2000; Cooper et al., 2005), their potential contribution to the depolarization-evoked rises in pHj observed under 0 C a 2 + 0 conditions in the present study were not assessed and it is therefore possible that the pHj changes may have been mediated by inward Na + /HC0 3 " cotransport with a 1:2 or 1:3 stoichiometry (see Fig. 1.1). However, because the removal of external Na + had no effect on the depolarization-evoked internal alkalinization, a contribution from Na + /HC0 3 " cotransport to the rise in pHj appears unlikely. 4.2. A gu+ likely contributes to voltage-dependent H + efflux from rat hippocampal neurons While the increase in pHj evoked by depolarization under 0 C a 2 + 0 conditions is not due to acid extrusion by previously described pHj regulating mechanisms in rat hippocampal neurons, it does bear the hallmarks of being mediated by a gH+. The possible contribution of a gH+ to the depolarization-evoked intracellular alkalinization is suggested by a number of lines of evidence. First, consistent with the voltage-dependence of gH+ gating (for a review, see DeCoursey, 2003), the magnitude of the rise in pHj observed under depolarizing, 0 C a 2 + 0 conditions was dependent on [K + ] 0 and, hence, membrane voltage. Arguing against the possibility that the rise in pH, is due to a gH+ is the fact that H + efflux was observed even when the electrochemical gradient for H + movement was not apparently outwardly directed (e.g. when [K + ] 0 = 50 mM, Vm = -16 mV; EH = -12.3 mV when pHi = 7.15, pH 0 = 7.35, 37°C). However, this finding is tempered by the fact that the local pH in the vicinity of the putative gH+ may be much lower than measured in the bulk cytoplasm; as noted by DeCoursey and Cherny (1994b), the gH+ may be activated in situations not predictable from bulk pH measurements made with cytoplasmically 125 located fluorescent dyes. The voltage-dependence of the internal alkalinization observed in the present study is consistent with previous findings that the recovery of pHj from internal acid loads in rat hippocampal neurons is enhanced under conditions of elevated [K + ] 0 (Sheldon and Church, 2002) and that a Zn2+-sensitive rise in pHj is observed in hippocampal neurons in response to anoxia (Diarra et al , 1999; Sheldon and Church, 2002), during which time there is a marked membrane depolarization (e.g. Tanaka et al., 1997). Second, the rise in pHj evoked by depolarization under 0 C a 2 + 0 conditions was also dependent on the A p H m e m b , consistent with the pH-dependent gating of gH+s described in other cell types (e.g. Kapus et al., 1993a; Cherny et al., 1995). It could be argued that the increasing magnitude of the alkalinization at greater ApHm embS might simply reflect a greater outwardly-directed H + gradient rather than increased gH+ activation (see Section 4.4). However, the fact that the degree of inhibition of H + efflux by Z n 2 + also increases with A p H m e m b (as evidenced by the greater level of separation of the regression lines at larger A p H m e m b values, shown in Fig. 3.11), is consistent with an increasing contribution of a Zn2+-sensitive gH+ to the H + efflux. The pH-dependent gating of gH+s allows them to effectively respond to internal acid loads by shifting t^hreshold, the most negative voltage at which a detectable H + current is activated, towards more negative potentials (Kapus et al., 1993a; Cherny et al., 1995). This may be particularly important in rat hippocampal neurons, which experience large falls in pH- in response to anoxia, during which time the major acid-extruding mechanism, NHE, becomes inhibited (Sheldon and Church, 2004). Indeed, as noted above, a Zn -sensitive rise in pHi during anoxic depolarization in these neurons may be due to H + efflux via a gH+ (Diarra et al., 1999; Sheldon and Church, 2002). Third, as already mentioned in this Discussion, the depolarization-evoked rise in pH-observed in the absence of external C a 2 + was sensitive to inhibition by Z n 2 + , consistent with the 126 properties of all gH+s described to date (for reviews see DeCoursey and Cherny, 1994; Eder and DeCoursey, 2001; DeCoursey, 2003). In agreement with previous studies (Peral and Ilundain, 1995; Cherny and DeCoursey, 1999), the site of action of Z n 2 + appeared to be extracellular. Furthermore, the inhibitory actions of Z n 2 + on the rise in pHj were, independent of the effects of changing A p H m e r n b , sensitive to pH 0 . It has been postulated that this may reflect competitive binding of Z n 2 + and H + to an external site on the surface of H + channels (Cherny and DeCoursey, 1999). The inhibitory effects of Zn may have important implications in pHj regulation and cell survival in the face of anoxic insults. The concentrations of extracellular Z n 2 + reached during cerebral ischemia are sufficient to inhibit gH+s (Koh et al., 1996; Choi and Koh, 1998; Lee et al., 1999; Wei et al., 2004) and block of gH+s (in addition to increases in [Zn2+]j) may contribute to the damaging effects of the cation by preventing the alleviation of the internal acidosis that occurs during anoxia and ischemia. Indeed, not only does inhibition of the putative gH+ by Z n 2 + augment the detrimental fall in pHj that occurs during anoxia (Diarra et al., 1999; Sheldon and Church, 2002) but, because gH+s couple to NHE (DeCoursey and Cherny, 1994a; Demaurex et al., 1995), inhibition of gH+s may promote the activation of the acid extruding NHE that occurs upon reoxygenation, thereby enhancing injurious Na + influx at this time (Sheldon et al., 2004b). Lastly, the marked reduction in the rate of rise of pHi when temperature was reduced from 37°C to ~22°C is in agreement with the remarkable sensitivity to temperature of gH+ gating kinetics (for a review, see DeCoursey, 2003). That the absolute magnitude of the depolarization-evoked rise in pHj is also reduced at 22°C could be due to the effects of temperature on the rate of gH+ activation, as well as on the open-channel conductance, although this property of gH+s has been somewhat less well defined (see DeCoursey, 2003) and it is impossible in the present study 127 to distinguish between the two factors. Nevertheless, the overall sensitivity of the depolarization-evoked alkalinization to temperature provides further support for the hypothesis that it is mediated by a gH+. 4.3. Estimation of current density from H + fluxes From the initial rates of increase in pHj observed upon depolarization under Ca2 + 0-free conditions in the presence and absence of Z n 2 + (Fig. 3.115), it is possible to calculate the initial Zn2+-sensitive H + flux (JH) from a single neuron that is presumably due to H + efflux through a gH+. J H was calculated as [dpH,] - B r d P H i ^  . dt , r \ Control { dt , Zinc (Equation 4.1) where (dpHj/d()controi and (dpHj/dr)zinc are the rates of change of pHi in the absence and presence of Z n 2 + , respectively, and B\ is the (pHj-dependent) intracellular buffering capacity. In this form, H + efflux is a positive quantity. Using values of (dpHjAk)c0ntroi and (dpHj/dOzinc taken from the regression lines in Fig. 3.115, and a B\ (20 mM/pH unit) taken from Fig. 3 in Bevensee et al. (1996), the J H caused by exposure to 75 mM [K + ] 0 (equivalent to depolarization to -7 mV; see Fig. 3.7) in neurons with a ApHmemb ° f 0-6 ( p H 6.75) was calculated as 0.028 mM/s. For simplicity, if one assumes a spherical neuronal diameter of 20 pm, this implies a H + current density at -7 mV of 9 fA/pm (for a detailed description of this calculation, see Appendix). This value is within the range of 3-16 fA/pm 2 calculated from Zn2+-sensitive rates of pHj recovery from internal acid loads in rat alveolar epithelial cells, which are known to possess a gH+ (Murphy et al., 2005). Assuming a specific membrane capacitance of 1.0 pF/cm 2 (Hille, 2001), the current density can be converted to a normalized H + current of 0.9 pA/pF. Although there are no comparable values from mammalian neurons, this value is at the lower limit of the 128 normalized H + currents measured in a variety of cell types over a wide pH range (pH- 5.5-7.3; see DeCoursey, 2003). Therefore, it is at least feasible that the Zn2+-sensitive changes in pHj observed upon depolarization in the absence of C a 2 + 0 in rat hippocampal neurons reflect H + efflux via a gH+. In addition, the calculations suggest that in rat hippocampal neurons, either the Zn2+-sensitive efflux observed in response to depolarization only requires the activation of a small fraction of the available pool of putative voltage-gated H + channels, or the total gH+ is small compared to other cells (see Section 4.7). 4.4. Mediators of Zn 2 +- insensitive, voltage-dependent H + efflux under 0 C a 2 + 0 conditions While the above considerations are consistent with the possibility that a gH+ underlies the Z n 2 + -sensitive, voltage-dependent H + efflux observed in the absence of external Ca 2 + , the question remains as to the basis of the Zn -insensitive rise in pH, also observed. As already noted above, the contributions of several previously described acid-extrusion pathways in rat hippocampal neurons were ruled out. In addition, although H + efflux via voltage-gated K + channels was not considered in the present study, to date there have been no reports of H + currents through K + channels (see DeCoursey, 2003). Aside from H + efflux via proteins such as carriers, channels and pumps, however, additional pathways exist for H + permeation through the plasma membrane, including transient water wires, weak acid/base shuttles, and/or phospholipid "flip-flop" (for reviews, see Lukacs et al., 1993; DeCoursey, 2003). Without attempting to define the mechanism(s) behind the Zn2+-insensitive H + efflux observed here under depolarizing, 0 C a 2 + 0 conditions, it is important to note that the change in Vm evoked by exposure to high [K + ] 0 is enough to create an outwardly directed electrochemical gradient for H + movement across the plasma membrane, as has been reported in highly H+-permeable rat mesenteric vascular smooth muscle cells (Austin and Wray, 1993) and rat brain synaptosomes (Sanchez-Armass et al., 129 1994). In rat hippocampal neurons with a resting pHi of 7.15 pH units (pH 0 = 7.35; 37°C), EH is -12 mV; thus, under resting conditions (i.e. 3 mM [K +] 0 , Vm = -62 mV), En is positive to Vm and the electrochemical gradient for protons is inwardly directed. Conversely, exposure to 75 mM [K + ] 0 brings Vm to -7 mV, past Eu, and the efflux of protons is favoured. To summarize the findings of the microspectrofluorimetric studies, voltage-dependent, Z n 2 + -sensitive increases in pHj are unmasked in rat hippocampal neurons under depolarizing conditions in the absence of external Ca 2 + . These changes in pHj have characteristics that strongly support the hypothesis that they are mediated by H + efflux via a gH+. As such, depolarization should also evoke outward H + currents that share the same characteristics as the pH; changes (i.e. sensitivity to voltage, Z n 2 + , temperature). 4.5. The depolarization-evoked outward current is carried in part by protons Although the bath and pipette solutions in this study were designed to minimize the concentration of permeant ions, the possibility existed that the remaining ions in the solutions could contribute to the outward currents recorded from hippocampal neurons. The finding, however, that the currents were not significantly affected by changes in the concentrations of M g 2 + or Cl ions, or by complete replacement of T M A + with Cs + , suggests that these ions contribute little, if at all, to the outward currents observed. That the outward currents recorded in this study are carried, at least in part, by protons is suggested by the fact that the VKV varies with ApHm emb- While the low slope of 10 mV/pH unit change in ApHm emb may be indicative of the contribution of other ions to the currents observed, it is more likely due to imperfect control over pHj and thus, ApHm emb, a problem that often plagues studies of H + currents. Indeed, in the present study, measurements of bulk cytosolic pHi 130 in patch-clamped neurons were always higher than the pipette pH. Considering the fact that p H microdomains have been observed within neurons (Schwiening and Willoughby, 2002; Willoughby and Schwiening, 2002), it is possible that the p H near the intracellular face of the plasma membrane may deviate even further from the assumed (i.e. pipette) pHj, resulting in an even greater overestimation of the A p H m e m b - The difficulties of attempting to control pHj using internal perfusion are well known. In snail neurons 90 - 120 um in diameter, effective control of pHi (measured with H+-sensitive microelectrodes) was only achieved using highly buffered solutions (100 mM) applied via a suction pipette one-third the cell diameter (Byerly and Moody, 1986). Even under these conditions, the slope of the relationship between Vrey and ApHm emb in these cells, which are known to exhibit H + currents, was only 21 mV/pH unit change in ApHm emb (Byerly et al., 1984), markedly lower than the theoretical 58 mV/pH unit change in A p H m e m b (see Section 1.4.1.3). Given that the hippocampal neurons in the present study had cell bodies 12-20 urn in diameter and were internally perfused with a moderately buffered solution (50 mM MES) via a patch pipette <1 urn in diameter, the small change of F r e v with (an assumed) ApHmemb does not seem unfeasible for H + currents. As noted by DeCoursey (2003), it is important to realize that the difference between F r e v and the equilibrium potential for H + may not be an error; rather it may provide an accurate reflection of pHj. 4.6. Comparison of outward currents with H + currents from other cell types 4.6.1. Current run-up H + current run-up has been described in snail neurons and rat alveolar epithelial cells (Byerly et al., 1984; Cherny et al., 1995). The time course in these cells, however, was in the order of minutes, not hours, as seen in rat hippocampal neurons in the present study. In rat alveolar epithelial cells, run-up of H + currents was attributed to the removal of the indirect inhibition of 131 H + currents conferred by NHE activity as the bath solution was changed to a Na+-free saline (Cherny et al., 1995). NHE acts to inhibit gH+s by extruding protons and dissipating the ApHmemb (Cherny et al., 1994b; Demaurex et al., 1995); block of NHE activity by the removal of N a + 0 helps to maintain a low pHj and large ApHmemb, which in turn lowers the t^hreshold of gH+s. In snail neurons, H + current run-up was thought to be due to improvement of the control of pHj over time (Byerly et al., 1984). The run-up of the outward presumptive H + currents in hippocampal neurons may be due to one or both of these phenomena, particularly the latter, in light of the poor control of pHj in these cells (see above, Section 4.5). 4.6.2. Gating kinetics Gating kinetics are the greatest source of variability among the H + currents recorded in different cell types, both in terms of the time course of activation, which may or may not follow an exponential function, and in the rates of opening and closing, which can vary over three orders of magnitude (for reviews, see DeCoursey, 1998, 2003). Several methods have been used to describe the time course of activation of H + currents, including measurements of time to half-peak current (Byerly et al., 1984; Kapus et al., 1993a), measurements of maximum rate-of-rise of H + currents (DeCoursey and Cherny, 1994a; Cherny et al., 1995), and fits to a single exponential after a delay (DeCoursey and Cherny, 1998; DeCoursey et al , 2001). In this study, I adopted the practice of fitting the activation time course of the outward current to a double exponential and, although the fit was not always perfect, it provided a defined and clearly understandable reflection of the activation kinetics (see also DeCoursey, 1998; DeCoursey et al., 2001). Although not quantified, the activation of H + currents in human skeletal muscle also appears to follow a double exponential time course (see Fig. AA in Bernheim et al , 1993). The activation time constants obtained in this study are compared with those obtained 132 for H + currents in other cell types in Table 4.1. Due to the wide range of experimental conditions used in studies of H + currents, an absolute numerical comparison of activation time constants is not feasible. Thus, Table 4.1 gives values of x a c t in terms of fast (a few milliseconds), medium (a few hundred milliseconds) and slow (seconds). The values of x a c t ( i ) and x a ct(2) obtained in hippocampal neurons are comparable to the values of x a c t seen in amphibian oocytes and mammalian non-neuronal cells, respectively, and appear to be similar to those of H + currents recorded from human skeletal muscle (estimated by eye from Fig. 4A in Bernheim et al., 1993). Thus, the activation time constants of the outward, presumed H + currents recorded in rat hippocampal neurons are within the wide range of values described in the various cells that are known to possess H + currents. Additionally, although the time course of the current in hippocampal neurons is much slower than that of H + currents in invertebrate neurons, it is interesting to note that the fast rising component of the hippocampal outward current is an order of magnitude faster than the time course of H + currents in non-excitable mammalian cells; perhaps there exists some specialization of gH+s in mammalian excitable cells such as neurons and skeletal muscle. The time constant of tail current decay in rat hippocampal neurons is also compared to that of H + currents from other cells in Table 4.1. It is important to note, however, that values of Xt an have not been systematically reported in many studies of H + currents and, thus, the comparisons are made with estimates made by DeCoursey (1998, 2003) from data in published figures. Again, the values of x t a i i observed in hippocampal neurons in the present study are within the range of values described in other mammalian cell types. 4.6.3. Sensitivity to Zn2+ and changes in temperature In contrast to the properties of gH+-mediated H + currents described to date, the outward currents 133 observed in rat hippocampal neurons in the present study were neither inhibited by Z n 2 + nor enhanced by increases in temperature. As sensitivity to Z n 2 + is considered a defining property of H + currents, these results suggest that the outward currents observed here are not, in fact, H + currents mediated by a gH+. Considering the dependence of VKV on ApH m e mb, the coincidence of the time constants with those of H + currents in other cell types and, particularly, the results of the microspectrofluorimetric studies presented in this thesis, all of which are suggestive of the presence of a gu+ in rat hippocampal neurons, the insensitivity of the outward currents to the non-selective treatments of Z n 2 + and changing temperature are not sufficient to refute the possibility that this cell type indeed possesses a gu+. It is possible that these apparently contradictory results are due to some of the technical difficulties associated with recording H + currents in small mammalian neurons (see Section 4.7). Additional experiments will be required to substantiate or refute the lack of effect of Zn and temperature on the outward currents recorded from hippocampal neurons. 4.7. T e c h n i c a l cons ide ra t i ons a n d fu ture d i r ec t ions The conditions used for the isolation of H + currents vary among experimental preparations, and optimal pipette and bath solutions for recording H + currents have not yet been completely established (DeCoursey and Cherny, 1994b). As already noted, under the conditions used in this study, the control over pHi in whole-cell patch-clamped hippocampal neurons was likely quite poor, as reflected in measurements of the Vrev of the outward current. Additionally, although unlikely, the slight dependence of Vm on pH, may reflect a contribution to the current of other, undefined charged species in the experimental solutions (e.g. the highly abundant H + buffers). If so, depending on the magnitude of this undefined current, a H + current may be obscured, particularly if ApH m e mb and, thus, H + currents, are small. The effects of Z n 2 + and temperature 134 under such conditions could also be masked. As noted above (see Section 4.3), there also lies the possibility that the total gn+ in rat hippocampal neurons may be small compared to non-neuronal cells. This would also result in small FT1" currents that could be difficult to resolve in the presence of contaminating currents. An additional reason that the gH+-mediated component of the outward currents might be too small to detect may lie in the long duration of the whole-cell patch-clamp experiments (due to current run-up) and the dialysis of intracellular factors that may be required for or enhance H + currents in rat hippocampal neurons. Although cytosolic factors are not, as a rule, considered essential for H + flux through a gH+, there have been reports in mammalian, non-neuronal cells that the activities of gH+s may require or be regulated by second messengers. Specifically, these are PKC (Nanda and Grinstein, 1991; Calonge and Ilundain, 1996; Gekle et al., 1997); phospholipase A 2 (PLA 2; Kapus et al., 1993b; Susztak et al., 1997; Lowenthal and Levy, 1999); and A A (DeCoursey and Cherny 1993; Kapus et al., 1994; Gordienko et al , 1996; Cherny et al., 2001). Indeed, cytosolic PLA2-generated A A has been shown to be an absolute requirement for stimulation of H + efflux through the NADPH-associated proton channel (Lowenthal and Levy, 1999; Mankelow et al., 2003). Therefore, in future experiments it may be desirable to assess the effects of PKC stimulation with 1,2-dioctanoyl-rac-glycerol (DOG) or PMA (Calonge and Ilundain, 1996; DeCoursey et al., 2001), and/or the presence of P L A 2 or A A , on the outward current in hippocampal neurons. Should there be an enhancement of the outward current, as would be expected of H currents in other cell types, the effects of Zn and temperature could then be re-assessed. Alternatively, experiments could be performed using the perforated-patch configuration (which would be required in studies using PMA; see DeCoursey et al., 2001) to preserve second messenger pathways. The problems associated with pHi control are augmented under these conditions, due to the restricted diffusion between the pipette and cytosol; however, 135 H + currents have been studied using the permeabilized-patch approach in human eosinophils, where control over pHj was achieved with a standing N H 4 + gradient (DeCoursey et al., 2001). As an alternative to the direct measurement of H + currents, the presence of a gH+ could be studied with simultaneous measurements of pHj and membrane current made from whole-cell voltage-clamped neurons internally dialysed with solutions of low buffering capacity (e.g. 10 mM). In the first description of H + currents in snail neurons (Thomas and Meech, 1982), changes in clamp current and pHj in response to injections of HC1 were assessed under voltage-clamp and attributed to a gH+. The advantage of this approach is that manoeuvres that influence gH+s should affect in parallel both the pHj and current changes. Although I have not yet been able to simultaneously record p H and current, preliminary measurements of pHj in voltage-clamped hippocampal neurons loaded with BCECF via the patch pipette indicate that pHj increases with depolarization (Fig. 4.1). 4.8. Conclusions and functional implications This study examined the changes in pH* evoked by depolarization and the possibility that these changes are mediated in part by a gH+. Microspectrofluorimetric measurements of pH, and [Ca2+]i in the presence of external C a 2 + under depolarizing conditions support previous findings that depolarization causes an intracellular acidification and that this is due to the activation of a plasma membrane C a 2 + , H+-ATPase consequent upon C a 2 + influx. Removal of external C a 2 + to study the pH, response to depolarization independently of changes in [Ca2+]j uncovered a voltage-dependent intracellular alkalinization. The" rise in pHj was sensitive to changes in ApHmemb, exposure to Z n 2 + and reductions in temperature in a manner entirely consistent with the properties of gH+s described to date in other cell types. Thus, based on the results of the 136 microspectrofluorimetric experiments, it is likely that rat hippocampal neurons possess a gH+ and, under appropriate conditions, these cells should also exhibit voltage-dependent, outwardly directed H + currents. Indeed, using solutions designed to isolate H + currents, outward currents exhibiting a time course within the range described for H + currents in other cell types and a slight dependence on the electrochemical gradient for protons were observed. However, contradictory to the properties of gH+s in other cell types, the currents were not sensitive to Z n 2 + or changes in temperature. Although this may suggest that the currents are not carried by protons via a hippocampal neuronal gH+, technical concerns may limit the validity of these electrophysiological measurements, which should be considered preliminary and, in light of the microspectrofluorimetric studies, insufficient to refute the hypothesis that a gH+ is present in rat hippocampal neurons. Further experimentation, perhaps involving simultaneous microspectrofluorimetric and electrophysiological recordings, will be required to further substantiate or disprove the possibility that a gH+ is present in rat hippocampal neurons. Should the existence of a hippocampal gH+ eventually be confirmed, it would have important implications in our understanding of pHj regulation in these neurons in a variety of situations. First, as previously suggested (Diarra et al., 1999; Sheldon and Church, 2002), the gH+ may act as an important acid extrusion mechanism under anoxic conditions, during which the fall in ATP results in the inhibition of the other major H + efflux pathways, such as NHE (Sheldon and Church, 2004). As noted in Section 4.2, this may provide an additional explanation for the detrimental effects of Z n 2 + on neuronal survival. Additionally, due to the established coupling of gH+s and NHE activity (DeCoursey and Cherny, 1994a; Demaurex et al., 1995), the contribution of a gH+ to the alleviation of anoxia-induced internal acid loads could reduce the contribution of NHE to acid extrusion following the anoxic period and thereby lessen 137 the potentially detrimental increase in intracellular Na + via this pathway during this time (Sheldon et al., 2004b). Under more physiological conditions, a gH+ could help mediate H + extrusion in response to action potentials and metabolic activity, similar to the scenario envisioned by Thomas and colleagues whereby gH+s allow for the rapid recovery of p H following membrane depolarization and the activation of a PMCA (Meech and Thomas, 1987; Schwiening et al., 1993). In snail neurons, the activation of gH+s is rapid enough that significant numbers of H + channels may open during each action potential. In hippocampal neurons, the outward currents observed in this study activated slowly but, similar to gu+s in skeletal myotubes (Bernheim et al., 1993), the fast component of activation may allow sufficient channel opening during one or a train of action potentials. Lastly, a gH+ may be important for H + extrusion under conditions of low external Ca 2 + , under which hippocampal neurons exhibit spontaneous burst firing (Jefferys and Haas, 1982; Taylor and Dudek, 1982). Despite a lack of C a 2 + influx and associated activation of an acid-loading PMCA, pHj in rat hippocampal neurons falls in response to spontaneous activity brought about by exposure to nominally Ca2+-free media (Xiong et al., 2000). Thus, under these conditions a gu+ may be activated in response to the intracellular acidification and membrane depolarization and contribute to the recovery of pHj. 138 Table 4.1. Comparison of outward currents recorded in rat hippocampal neurons with varieties of H + currents from other cell types ff" current type Hippocampal n o e p x (oxidase neuron (neuron) (oocyte) (epithelial) (phagocyte) related) Gated by T a c , (at +60 mV) V, ( A p H m e m b ) V, A p H m e m D 1. Medium 2. Slow Fast V, ApHmemb Medium V, ApHmemb V, A p H m e m b Slow Slow V, pH 0 , pHj, AA?, N A D P H oxidase activity? Slow Tui (at -40 mV) Cells expressing Slow Fast Snail neuron Medium Medium Frog and newt oocytes Rat alveolar epithelium, frog proximal tubule Slow Microglia, neutrophils, eosinophils, mast cells, macrophages, basophils, HL-60, THP-l , C H O , PLB cells Very slow Eosinophils, neutrophils, PLB cells Abbreviations: V, voltage; ApH m emb, transmembrane pH gradient (pH 0 - pHj); A A , arachidonic acid; xact, activation time constant; x t aii, tail current time constant. The categories of x are arbitrary, because time courses did not always follow a single exponential (hippocampal outward currents were fit to a double exponential and thus have two x values), depend on pHj, and x values from various studies were not all obtained at the same pHj. The categories of xa c t are based on criteria established by DeCoursey (1998), where 'fast' means a time constant of a few milliseconds, 'medium' means a few hundred milliseconds, 'slow' means seconds. A similar approach is used in the description of xtaii, where 'fast' means a few milliseconds, 'medium' means under a hundred milliseconds, 'slow' is a few hundred milliseconds and 'very slow' means seconds. In rat hippocampal neurons, due to the double exponential time course of activation, two activation time constants are given. Table adapted from DeCoursey (2003). 139 Fig. 4.1. Depolarization causes increases in pH- in cells with low internal buffering capacity. Preliminary measurements of pHj in cells voltage-clamped in the whole-cell configuration and loaded with 100 uM BCECF via the patch pipette. Internal and external solutions were the same TMAMeS03-based solutions used in all voltage-clamp studies, except for the reduced internal buffer (10 mM MES; iso-osmotically replaced with TMAMeSOs). The experiment was performed at room temperature, pHj 6.0, pH 0 7.5. The record begins immediately after establishment of the whole-cell configuration. After stabilization of pHj, a 1 min depolarizing voltage step to +60 mV from F n = -20 mV elicited a rapid increase in pHj that recovered upon return to resting conditions. 141 5. R E F E R E N C E S Austin C, Wray S (1993) Changes of intracellular pH in rat mesenteric vascular smooth muscle with high-K+ depolarization. J Physiol 469:1-10. Banfi B, Maturana A, Jaconi S, Arnaudeau S, Laforge T, Sinha B, Ligeti E, Demaurex N, Krause K H (2000) A mammalian H + channel generated through alternative splicing of the N A D P H oxidase homolog NOH-1. Science 287:138-142. Banfi B, Schrenzel J, Niisse O, Lew DP, Ligeti E, Krause K H , Demaurex N (1999) A novel H + conductance in eosinophils: unique characteristics and absence in chronic granulomatous disease. J Exp Med 190:183-194. Barish M E , Baud C (1984) A voltage-gated hydrogen ion current in the oocyte membrane of the axolotl, Ambystoma. J Physiol 352:243-263. Bassnett S, Reinisch L, Beebe DC (1990) Intracellular pH measurement using single excitation-dual emission fluorescence ratios. Am J Physiol 258:C171-C178. Baxter K A (1995) Regulation of intracellular pH in cultured fetal rat hippocampal pyramidal neurons. M . Sc. thesis. Vancouver, BC: Department of Anatomy, University of British Columbia. Baxter K A , Church J (1996) Characterization of acid extrusion mechanisms in cultured fetal rat hippocampal neurones. J Physiol 493:457-470. Bennett M V L , Zukin RS (2004) Electrical coupling and neuronal synchronization in the mammalian brain. Neuron 41:495-511. Bernheim L, Krause R M , Baroffio A, Hamann M , Kaelin A, Bader CR (1993) A voltage-dependent proton current in cultured human skeletal muscle myotubes. J Physiol 470:313-333. Bevensee MO, Bashi E, Boron WF (1999) Effect of trace levels of nigericin on intracellular pH and acid-base transport in rat renal mesangial cells. J Membr Biol 169:131-139. Bevensee MO, Boron WF (1998) pH regulation in mammalian neurons. In: pH and brain function (Kaila K, Ransom BR, eds), pp 211-231. New York: Wiley-Liss, Inc. Bevensee MO, Cummins TR, Haddad GG, Boron WF, Boyarsky G (1996) pH regulation in single CAI neurons acutely isolated from the hippocampi of immature and mature rats. J Physiol 494:315-328. Bevensee MO, Schwiening CJ, Boron WF (1995) Use of BCECF and propidium iodide to assess membrane integrity of acutely isolated CAI neurons from rat hippocampus. J Neurosci Meth 58:61-75. 142 Blank PS, Silverman HS, Chung OY, Hogue BA, Stern MD, Hansford RG, Lakatta EG, Capogrossi M C (1992) Cytosolic pH measurements in single cardiac myocytes using carboxy-seminaphthorhodafluor-1. Am J Physiol 263:H276-H284. Bonanno JA (1991) K + - H + exchange, a fundamental cell acidifier in corneal epithelium. Am J Physiol 260:C618-C625. Boron WF (2004) Regulation of intracellular pH. Adv Physiol Educ 28:160-179. Boussouf A, Lambert RC, Gaillard S (1997) Voltage-dependent Na + -HC0 3 " cotransporter and N a + / H + exchanger are involved in intracellular pH regulation of cultured mature rat cerebellar oligodendrocytes. Glia 19:74-84. Bouyer P, Bradley SR, Zhao J, Wang W, Richerson GB, Boron WF (2004) Effect of extracellular acid-base disturbances on the intracellular pH of neurones cultured from rat medullary raphe or hippocampus. J Physiol 559:85-101. Boyarsky G, Hanssen C, Clyne L A (1996) Inadequacy of high K+/nigericin for calibrating BCECF. I. Estimating steady-state intracellular pH. Am J Physiol 271 :C1131-C1145. Brett C L , Kelly T, Sheldon C, Church J (2002) Regulation of Cl"-HC03~ exchangers by cAMP-dependent protein kinase in adult rat hippocampal CA1 neurons. J Physiol 545:837-853. Bright GR, Fisher GW, Rogowska J, Taylor DL (1989) Fluorescence ratio imaging microscopy. Methods Cell Biol 30:157-192. Bright GR, Fisher GW, Rogowska J, Taylor DL (1987) Fluorescence ratio imaging microscopy: temporal and spatial measurements of cytoplasmic pH. J Cell Biol 104:1019-1033. Bromberg Y, Pick E (1983) Unsaturated fatty acids as second messengers of superoxide generation by macrophages. Cell Immunol 79:240-252. Brookes N, Turner RJ (1994) K+-induced alkalinization in mouse cerebral astrocytes mediated by reversal of electrogenic Na + -HC0 3 " cotransport. Am J Physiol 267:C1633-C1640. Buckler KJ, Vaughan-Jones RD (1998) Effects of mitochondrial uncouplers on intracellular calcium, pH and membrane potential in rat carotid body type I cells. J Physiol 513:819-833. Buckler KJ, Vaughan-Jones RD (1990) Application of a new pH-sensitive fluoroprobe (carboxy-SNARF-1) for intracellular pH measurement in small, isolated cells. Pfliigers Arch 417:234-239. Byerly L, Meech R, Moody WJ (1984) Rapidly activating hydrogen ion currents in perfused neurones of the snail, Lymnaea stagnalis. J Physiol 351:199-216. Byerly L, Moody WJ (1986) Membrane currents of internally perfused neurones of the snail, Lymnaea stagnalis, at low intracellular pH. J Physiol 376:477-491. 143 Byerly L, Suen Y (1989) Characterization of proton currents in neurones of the snail, Lymnaea stagnalis. J Physiol 413:75-89. Calonge M L , Ilundain A A (1996) PKC activators stimulate H + conductance in chicken enterocytes. Pfliigers Arch 431:594-598. Chen Y, Cann MJ, Litvin TN, Iourgenko V, Sinclair ML, Levin LR, Buck J (2000) Soluble adenylyl cyclase as an evolutionarily conserved bicarbonate sensor. Science 289:625-628. Cheng C, Reynolds IJ (1998) Calcium-sensitive fluorescent dyes can report increases in intracellular free zinc concentration in cultured forebrain neurons. J Neurochem 71:2401-2410. Cherny V V , DeCoursey T E (1999) pH-dependent inhibition of voltage-gated H + currents in rat alveolar epithelial cells by Z n 2 + and other divalent cations. J Gen Physiol 114:819-838. Cherny V V , Henderson L M , Xu W, Thomas L L , DeCoursey TE (2001) Activation of NADPH oxidase-related proton and electron currents in human eosinophils by arachidonic acid. J Physiol 535:783-794. Cherny V V , Markin VS, DeCoursey T E (1995) The voltage-activated hydrogen ion conductance in rat alveolar epithelial cells is determined by the pH gradient. J Gen Physiol 105:861-896. Cherny V V , Murphy R, Sokolov V, Levis RA, DeCoursey T E (2003) Properties of single voltage-gated proton channels in human eosinophils estimated by noise analysis and direct measurement. J Gen Physiol 121:615-628. Chesler M (2003) Regulation and modulation of pH in the brain. Physiol Rev 83:1183-1221. Chesler M (1990) The regulation and modulation of pH in the nervous system. Prog Neurobiol 34:401-427. Chesler M , Kaila K (1992) Modulation of pH by neuronal activity. Trends Neurosci 15:396-402. Choi DW, Koh JY (1998) Zinc and brain injury. Annu Rev Neurosci 21:347-375. Church J, Baimbridge K G (1991) Exposure to high-pH medium increases the incidence and extent of dye coupling between rat hippocampal CAI pyramidal neurons in vitro. J Neurosci 11:3289-3295. Church J, Baxter K A , McLarnon JG (1998) pH modulation of C a 2 + responses and a C a 2 + -dependent K + channel in cultured rat hippocampal neurones. J Physiol 511:119-132. Colvin RA, Davis N, Nipper RW, Carter PA (2000) Zinc transport in the brain: routes of zinc influx and efflux in neurons. J Nutr 130:1484S-1487S. 144 Cooper DS, Saxena NC, Yang HS, Lee HJ, Moring A G , Lee A, Choi I (2005) Molecular and functional characterization of the electroneutral Na/HC03 cotransporter NBCnl in rat hippocampal neurons. J Biol Chem 280:17823-17830. Cross AR, Jones O T G (1991) Enzymic mechanisms of superoxide production. Biochem Biophys Acta 1057:281-298. Daniel H , Kottra G (2004) The proton oligopeptide cotransporter family SLC15 in physiology and pharmacology. Pfliigers Arch 447:610-618. DeCoursey T E (2003) Voltage-gated proton channels and other proton transfer pathways. Physiol Rev 83:475-579. DeCoursey T E (1998) Four varieties of voltage-gated proton channels. Front Biosci 3:d477-d482. DeCoursey T E (1991) Hydrogen ion currents in rat alveolar epithelial cells. Biophys J 60:1243-1253. DeCoursey TE, Cherny V V (1998) Temperature dependence of voltage-gated H + currents in human neutrophils, rat alveolar epithelial cells, and mammalian phagocytes. J Gen Physiol 112:503-522. DeCoursey T E , Cherny V V (1994a) Na + -H + antiport detected through hydrogen ion currents in rat alveolar epithelial cells and human neutrophils. J Gen Physiol 103:755-785. DeCoursey TE, Cherny V V (1994b) Voltage-activated hydrogen ion currents. J Membrane Biol 141:203-223. DeCoursey T E , Cherny V V (1993) Potential, pH, and arachidonate gate hydrogen ion currents in human neutrophils. Biophys J 65:1590-1598. DeCoursey T E , Cherny V V , DeCoursey A G , Xu W, Thomas L L (2001) Interactions between NADPH oxidase-related proton and electron currents in human eosinophils. J Physiol 535:767-781. DeCoursey T E , Cherny V V , Zhou W, Thomas L L (2000) Simultaneous activation of NADPH oxidase-related proton and electron currents in human neutrophils. Proc Natl Acad Sci USA 97:6885-6889. Demaurex N, Grinstein S, Jaconi M , Schlegel W, Lew DP, Krause K H (1993) Proton currents in human granulocytes: regulation by membrane potential and intracellular pH. J Physiol 466:329-344. Demaurex N, Orlowski J, Brisseau G, Woodside, Grinstein S (1995) The mammalian Na + /H + antiporters NHE-1, NHE-2, and NHE-3 are electroneutral and voltage independent, but can couple to an H + conductance. J Gen Physiol 106:85-111. 145 Diarra A, Sheldon C, Brett CL, Baimbridge K G , Church J (1999) Anoxia-evoked intracellular pH and C a 2 + concentration changes in cultured postnatal rat hippocampal neurons. Neuroscience 93:1003-1016. Dipolo R, Beauge L (1982) The effect of pH on C a 2 + extrusion mechanisms in dialyzed squid axons. Biochim Biophys Acta 688:237-245. Doering A E , Lederer WJ (1993) The mechanism by which cytoplasmic protons inhibit the sodium-calcium exchanger in guinea-pig heart cells. J Physiol 466:481-499. Eder C, DeCoursey T E (2001) Voltage-gated proton channels in microglia. Prog Neurobiol 64:277-305. Eder C, Fischer HG, Hadding U, Heinemann U (1995) Properties of voltage-gated currents of microglia developed using macrophage colony-stimulating factor. Pfliigers Arch 430:526-533. Frankenhaeuser B, Hodgkin A L (1957) The action of calcium on the electrical properties of squid axons. J Physiol 137:218-244. French CR, Sah P, Buckett KJ, Gage PW (1990) A voltage-dependent persistent sodium current in mammalian hippocampal neurons. J Gen Physiol 95:1139-1157. Fushimi K, Verkman AS (1991) Low viscosity in aqueous domain of cell cytoplasm measured by picosecond polarization microfluorimetry. J Cell Biol 112:719-725. Gatto C, Milanick M A (1993) Inhibition of the red blood cell calcium pump by eosin and other fluorescein analogues. Am J Physiol 264:C1577-C1586. Gekle M , Silbernagl S, Oberleithner H (1997) The mineralocorticoid aldosterone activates a proton conductance in cultured kidney cells. Am J Physiol 273:C1673-C1678. Glantz SA (2002) Primer of Biostatistics. New York: McGraw-Hill Companies, Inc. Gordienko DV, Tare M , Parveen S, Fenech CJ, Robinson C, Bolton TB (1996) Voltage-activated proton current in eosinophils from human blood. J Physiol 496:299-316. Gottfried JA, Chesler M (1994) Endogenous H + modulation of NMD A receptor-mediated EPSCs revealed by carbonic anhydrase in rat hippocampus. J Physiol 478:373-378. Graber M , Pastoriza-Munoz E (1993) Regulation of cell pH by K + / H + antiport in renal epithelial cells. Am J Physiol 265:F773-F783. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pfliigers Arch 391:85-100. 146 Henderson L M , Chappell JB, Jones OTG (1988) Internal pH changes associated with the activity of N A D P H oxidase of human neutrophils. Further evidence for the presence of an H + conducting channel. Biochem J 251:563-567. Henderson L M , Moule SK, Chappell JB (1993) The immediate activator of the NADPH oxidase is arachidonate not phosphorylation. Eur J Biochem 211:157-162. Hille B (2001) Ion channels of excitable membranes. Sunderland, MA: Sinauer. Hoyt KR, Reynolds IJ (1998) Alkalinization prolongs recovery from glutamate-induced increases in intracellular C a 2 + concentration by enhancing C a 2 + efflux through the mitochondrial Na + /Ca 2 + exchanger in cultured rat forebrain neurons. J Neurochem 71:1051-1058. Ikuma M , Binder HJ, Geibel J (1998) Role of apical H-K exchange and basolateral K channel in the regulation of intracellular pH in rat distal colon crypt cells. J Membr Biol 166:205-212. Jefferys JGR, Haas HL (1982) Synchronized bursting of CAI hippocampal pyramidal cells in the absence of synaptic transmission. Nature 300:448-450. Kaila K, Ransom B (1998) Concept of pH and its importance in neurobiology. In: pH and brain function. (Kaila K, Ransom B, eds), pp 3-10. New York: Wiley-Liss, Inc. Kanai Y, Hediger M A (2004) The glutamate/neutral amino acid transporter family SLC1: molecular, physiological and pharmacological aspects. Pflugers Arch 447:469-479. Kapus A, Romanek R, Grinstein S (1994) Arachidonic acid stimulates the plasma membrane H + conductance of macrophages. J Biol Chem 269:4736-4745. Kapus A, Romanek R, Qu A Y , Rotstein OD, Grinstein S (1993a) A pH-sensitive and voltage-dependent proton conductance in the plasma membrane of macrophages. J Gen Physiol 102:729-760. Kapus A, Susztak K, Ligeti E (1993b) Regulation of the electrogenic H + channel in the plasma membrane of neutrophils: possible role of phospholipase A 2 , internal and external protons. Biochem J 292:445-450. Kelly T, Church J (2004) pH modulation of currents that contribute to the medium and slow afterhyperpolarizations in rat CAI pyramidal neurones. J Physiol 554:449-466. Kerchner GA, Canzoniero L M T , Yu SP, Ling C, Choi DW (2000) Z n 2 + current is mediated by voltage-gated C a 2 + channels and enhanced by extracellular acidity in mouse cortical neurons. J Physiol 528:39-52. Khodorov B, Pinelis V, Vergun O, Storozhevykh T, Fajuk D, Vinskaya N, Arsenjeva E, Khaspekov L, Lyzin A, Isaev N, AndreevaN, Victorov I (1995) Dramatic effects of external alkalinity on neuronal calcium recovery following a short-duration glutamate challenge: the role of the plasma membrane C a 2 + / H + pump. FEBS Lett 371:249-252. 147 Khodorov B, Valkina O, Turovetsky V (1994) Mechanisms of stimulus-evoked intracellular acidification in frog nerve fibres. FEBS Lett 341:125-127. Koh JY, Suh SW, Gwag BJ, He Y Y , Hsu CY, Choi DW (1996) The role of zinc in selective neuronal death after transient global cerebral ischemia. Science 272:1013-1016. Krishtal OA, Ospichuk Y V , Shelest TN, Smirnoff SV (1987) Rapid extracellular pH transients related to synaptic transmission in rat hippocampal slices. Brain Res 436:352-356. Kuno M , Kawawaki J, Nakamura F (1997) A highly temperature-sensitive proton current in mouse bone marrow-derived mast cells. J Gen Physiol 109:731-740. Lee JM, Zipfel GJ, Choi DW (1999) The changing landscape of ischemic brain injury mechanisms. Nature 399 (6738 Suppl):A7-A14. Lipton P (1999) Ischemic cell death in brain neurons. Physiol Rev 79:1431-1568. Liu J, Diwu Z, Leung W (2001) Synthesis and photophysical properties of new fluorinated benzo[c]xanthene dyes as intracellular pH indicators. Bioorg Med Chem Lett 11:2903-2905. Lowenthal A, Levy R (1999) Essential requirement of cytosolic phopholipase A 2 for activation of the H + channel in phagocyte-like cells. J Biol Chem 274:21603-21608. Lukacs GL, Kapus A, Nanda A, Romanek R, Grinstein S (1993) Proton conductance of the plasma membrane: properties, regulation, and functional role. Am J Physiol 265:C3-C14. Mackenzie B, Hediger M A (2004) SLC11 family of H+-coupled metal-ion transporters NRAMP1 and DMT1. Pfliigers Arch 447:571-579. Mahaut-Smith MP (1989) Separation of hydrogen ion currents in intact molluscan neurones. J Exp Biol 145:439-454. Mankelo TJ, Pessach E, Levy R, Henderson L M (2003) The requirement of cytosolic phospholipaase A 2 for the PMA activation of proton efflux through the N-terminal 230-amino-acid fragment of gp91p h o x. Biochem J 374:315-319. Martinez-Zaguilan R, Martinez G M , Lattanzio F, Gillies RJ (1991) Simultaneous measurement of intracellular pH and C a 2 + using the fluorescence of SNARF-1 and fura-2. Am J Physiol 260:C297-C307. Meech RW, Thomas RC (1987) Voltage-dependent intracellular pH in Helix aspersa neurones. J Physiol 390:433-452. Meech RW, Thomas RC (1980) Effect of measured calcium chloride injections on the membrane potential and internal pH of snail neurones. J Physiol 298:111-129. 148 Meyer T M , Munsch T, Pape HC (2000) Activity-related changes in intracellular pH in rat thalamic relay neurons. NeuroReport 11:33-37. Morihata H, Kawawaki J, Sakai H, Sawada M , Tsutada T, Kuno M (2000) Temporal fluctuations of voltage-gated proton currents in rat spinal microglia via pH-dependent and -independent mechanisms. Neurosci Res 38:265-271. Mozhayeva GN, Naumov AP (1983) The permeability of sodium channels to hydrogen ions in nerve fibres. Pfliigers Arch 396:163-173. Mullins LJ, Tiffert T, Vassort G, Whittembury J (1983) Effects of internal sodium and hydrogen ions and of external calcium ions and membrane potential on calcium entry in squid axons. J Physiol 338:295-319. Murao H, Shimizu A, Hosoi K, Iwagaki A, Min K Y , Kishima G, Hanafusa T, Kubota T, Kato M , Yoshida H, Nakahari T (2005) Cell shrinkage evoked by Ca2+-free solution in rat alveolar type II cells: C a 2 + regulation of Na + -H + exchange. Exp Physiol 90:203-213. Murphy R, Cherny V V , Morgan D, DeCoursey T E (2005) Voltage-gated proton channels help regulate pHj in rat alveolar epithelium. Am J Physiol 288:L398-L408. Nagle JF, Morowitz HJ (1978) Molecular mechanisms for proton transport in membranes. Proc Natl Acad Sci USA 75:298-302. Nanda A, Grinstein S (1991) Protein kinase C activates an H + (equivalent) conductance in the plasma membrane of human neutrophils. Proc Natl Acad Sci USA 88:10816-10820. 2+ Nowicky A V , Duchen MR (1998) Changes in [Ca ]j and membrane currents during impaired mitochondrial metabolism in dissociated rat hippocampal neurons. J Physiol 507:131-145. O'Connor ER, Sontheimer H, Ransom BR (1994) Rat hippocampal astrocytes exhibit electrogenic sodium-bicarbonate co-transport. J Neurophys 72:2580-2589. Ou-Yang Y, Kristian T, Kristianova V, Mellergard P, Siesjo BK (1995) The influence of calcium transients on intracellular pH in cortical neurons in primary culture. Brain Res 676:307-313. Ou-Yang Y, Mellergard P, Siesjo BK (1993) Regulation of intracellular pH in single rat cortical neurons in vitro: A microspectrofluorometric study. J Cereb Blood Flow Metab 13:827-840. Paalasmaa P, Kaila K (1996) Role of voltage-gated calcium channels in the generation of activity-induced extracellular pH transients in the rat hippocampal slice. J Neurophysiol 75:2354-2360. Park KS, Jo I, Pak Y K , Bae SW, Rhim H, Suh SK, Park SJ, Zhu M H , So I, Kim K W (2002) FCCP depolarizes plasma membrane potential by activating proton and Na + currents in bovine aortic endothelial cells. Pfliigers Arch 433:344-352. 149 Peral MJ, Ilundain A A (1995) Proton conductance and intracellular pH recovery from an acid load in chicken enterocytes. J Physiol 484:165-172. Prince DA, Connors BW (1986) Mechanisms of interictal epileptogenesis. Adv Neurol 44:275-299. Pusch M , Ludewig U, Jentsch TJ (1997) Temperature dependence of fast and slow gating relaxations of C1C-0 chloride channels. J Gen Physiol 109:105-116. Raley-Susman K M , Cragoe EJ, Sapolsky RM, Kopito RR (1991) Regulation of intracellular pH in cultured hippocampal neurones by an amiloride-insensitive Na + /H + exchanger. J Biol Chem 266:2739-2745. Ramnath RR, Strange K, Rosenberg PA (1992) Neuronal injury evoked by depolarizing agents in rat cortical cultures. Neuroscience 51:931-939. Richmond PH, Vaughan-Jones RD (1997) Assessment of evidence for K + - H + exchange in isolated type-1 cells of neonatal rat carotid body. Pflugers Arch 434:429-437. Roos A, Boron WF (1981) Intracellular pH. Physiol Rev 61:296-434. Rose CR, Ransom BR (1997) Regulation of intracellular sodium in cultured rat hippocampal neurones. J Physiol 499:573-587. Ross G M , Shamovsky IL, Woo SB, Post Jl, Vrkljan PN Lawrance G, Sole M , Dostaler SM, Neet K E , Riopelle RJ (2001) The binding of zinc and copper ions to nerve growth factor is differentially affected by pH: implications for cerebral acidosis. J Neurochem 74:515-523. Sanchez-Armass S, Martinez-Zaguilan R, Martinez G M , Gillies RJ (1994) Regulation of pH in rat brain synaptosomes. I. Role of sodium, bicarbonate, and potassium. J Neurophysiol 71:2236-2248. Schmitt B M , Berger UV, Douglas RM, Bevensee MO, Hediger M A , Haddad GG, Boron WF (2000) N a / H C 0 3 cotransporters in rat brain: expression in glia, neurons, and choroid plexus. J Neurosci 20:6839-6848. Schrenzel J, Lew DP, Krause K H (1996) Proton currents in human eosinophils. Am J Physiol 271:C1861-C1871. Schwiening CJ, Boron WF (1994) Regulation of intracellular pH in pyramidal neurones from the rat hippocampus by Na+-dependent Cl "HC03" exchange. J Physiol 475:59-67. Schwiening CJ, Kennedy HJ, Thomas RC (1993) Calcium-hydrogen exchange by the plasma membrane Ca-ATPase of voltage-clamped snail neurons. Proc R Soc Lond B 253:285-289. Schwiening CJ, Willoughby D (2002) Depolarization-induced pH microdomains and their relationship to calcium transients in isolated snail neurones. J Physiol 538:371-382. 150 Seksek O, Bolard J (1996) Nuclear pH gradient in mammalian cells revealed by laser microspectrofluorimetry. J Cell Sci 109:257-262. Seksek O, Henry-Toulme N, Sureau F, Bolard J (1991) SNARF-1 as an intracellular pH indicator in laser microspectrofluorometry: a critical assessment. Anal Biochem 193:49-54. Sensi SL, Canzoniero L M T , Yu SP, Ying HS, Koh JY, Kerchner GA, Choi DW (1997) Measurement of intracellular free zinc in living cortical neurons: routes of entry. J Neurosci 17:9554-9564. Sheldon C, Cheng Y M , Church J (2004a) Concurrent measurements of the free cytosolic concentrations of H + and Na + ions with fluorescent indicators. Pfliigers Arch 449:307-318. Sheldon C, Church J (2004) Reduced contribution from Na + /H + exchange to acid extrusion during anoxia in adult rat hippocampal CA1 neurons. J Neurochem 88:594-603. Sheldon C, Church J (2002) Intracellular pH response to anoxia in acutely dissociated adult rat hippocampal CA1 neurons. J Neurophysiol 87:2209-2224. Sheldon C, Diarra A, Cheng Y M , Church, J (2004b) Sodium influx pathways during and after anoxia in rat hippocampal neurons. J Neurosci 24:11057-11069. Siesjo BK, Katsura K, Kristian T (1996) Acidosis-related damage. Adv Neurol 71:209-236. Silver RA, Whitaker M , Bolsover SR (1992) Intracellular ion imaging using fluorescent dyes: artefacts and limits to resolution. Pfliigers Arch 420:595-602. Smith GA, Brett C L , Church J (1998) Effects of noradrenaline on intracellular pH in acutely dissociated adult rat hippocampal CA1 neurons. J Physiol 512:487-505. Smith SE, Gottfried JA, Chen JCT, Chesler M (1994) Calcium dependence of glutamate receptor-evoked alkaline shifts in hippocampus. NeuroReport 5:2441-2445. Spray DC, Scemes E (1998) Effects of intracellular pH (and Ca ) on gap junction channels. In: pH and brain function (Kaila K, Ransom BR, eds), pp 477-490. New York: Wiley-Liss Inc. Somjen GG, Miiller M (2000) Potassium-induced enhancement of persistent inward current in hippocampal neurons in isolation and in tissue slices. Brain Res 885:102-110. Storm JF (1990) Potassium currents in hippocampal pyramidal cells. Prog Brain Res 83:161-187. Straubinger R M , Papahadjopoulos D, Hong K (1990) Endocytosis and intracellular fate of liposomes using pyranine as a probe. Biochemistry 29:4929-4939. Susztak K, Mocsai A, Ligeti E, Kapus A (1997) Electrogenic H + pathway contributes to stimulus-induced changes of internal pH and membrane potential in intact neutrophils: role of cytoplasmic phospholipase A 2 . Biochem J 325:501-510. 151 Takahashi M , Billups B, Rossi D, Sarantis M , Hamann M , Attwell D (1997) The role of glutamate transporters in glutamate homeostasis in the brain. J Exp Biol 200:401-409. Tanaka E, Yamamoto S, Kudo Y, Mihara S, Higashi H (1997) Mechanisms underlying the rapid depolarization produced by deprivation of oxygen and glucose in rat hippocampal CA1 neurons in vitro. J Neurophysiol 78:891-902. Taylor CP (1993) Na + currents that fail to inactivate. Trends Neurosci 16:455-60. Taylor CP, Dudek FE (1982) Synchronous neuronal afterdischarges in rat hippocampal slices without active chemical synapses. Science 218:810-812. Thomas JA, Buchsbaum RN, Zimniak A, Racker E (1979) Intracellular pH measurements in Ehrlich ascites tumour cells utilising spectroscopic probes generated in situ. Biochemistry 18:2210-2218. Thomas RC, Meech RW (1982) Hydrogen ion currents and intracellular pH in depolarized voltage-clamped snail neurones. Nature 299:826-828. Thomas RC, Schwiening CJ (1998) Intracellular pH regulation in invertebrate neurons. In: pH and brain function (Kaila K, Ransom BR, eds), pp 195-210. New York: Wiley-Liss, Inc. Tombaugh GC (1994) Mild acidosis delays hypoxic spreading depression and improves neuronal recovery in hippocampal slices. J Neurosci 14:5635-5643. Tombaugh GC, Somjen G G (1998) pH modulation of voltage-gated ion channels. In: pH and brain function (Kaila K, Ransom BR, eds) pp 395-416. New York: Wiley-Liss, Inc. Tornquist K, Tashjian A H Jr (1991) Importance of transients in cytosolic free calcium concentrations on activation of Na + /H + exchange in GH4C1 pituitary cells. Endocrinology 128:242-50. Trapp S, Luckermann M , Brooks PA, Ballanyi K (1996a) Acidosis of rat dorsal vagal neurons in situ during spontaneous and evoked activity. J Physiol 496:695-710. Trapp S, Luckermann M , Kaila K, Ballanyi K (1996b) Acidosis of hippocampal neurones mediated by a plasmalemmal C a 2 + / H + pump. NeuroReport 7:2000-2004. Traynelis SF (1998) pH modulation of ligand-gated ion channels. In: pH and brain function (Kaila K, Ransom BR, eds), pp 417-446. New York: Wiley-Liss, Inc. Tsien R Y (1989) Fluorescent probes of cell signalling. Ann Rev Neurosci 12:227-253. Valkina ON, Vergun OV, Turovetsky VB, Khodorov, BI (1995) Changes of cytoplasmic pH in frog nerve fibers during K+-induced membrane depolarization. FEBS Lett 361:145-148. 152 Valkina ON, Vergun OV, Turovetsky VB, Khodorov, BI (1993) Effects of repetitive stimulation, veratridine and ouabain on cytoplasmic pH in frog nerve fibres: role of internal Na + . FEBS Lett 334:83-85. Vignes M , Blanc E, Guiramand J, Gonzalez E, Sassetti I, Recasens M (1996) A modulation of glutamate-induced phosphoinositide breakdown by intracellular pH changes. Neuropharmacology 35:1595-1604. Voipio J, Kaila K (1993) Interstitial Pco2 and pH in rat hippocampal slices measured by means of a novel fast C02/H(+)-sensitive microelectrode based on a PVC-gelled membrane. Pfliigers Arch 423:193-201. Wang GH, Randall RD, Thayer SA (1994) Glutamate-induced intracellular acidification of cultured hippocampal neurons demonstrates altered energy metabolism resulting from C a 2 + loads. J Neurophysiol 72:2563-2569. Wei G, Hough CJ, Sarvey JM (2004) Characterization of extracellular accumulation of Z n 2 + during ischemia and reperfusion of hippocampus slices in rat. Neuroscience 125:867-877. Werth JL, Thayer SA (1994) Mitochondria buffer physiological calcium loads in cultured rat dorsal root ganglion neurons. J Neurosci 14:348-356. Wilding TJ, Cheng B, Roos A (1992) pH regulation in adult rat carotid body glomus cells. J Gen Physiol 100:593-608. Willoughby D, Schwiening CJ (2002) Electrically evoked dendritic pH transients in rat cerebellar Purkinje cells. J Physiol 544:487-499. Willoughby D, Thomas RC, Schwiening CJ (1998) Comparison of simultaneous pH measurements made with 8-hydroxypyrene-l,3,6-trisulphonic acid (HPTS) and pH-sensitive microelectrodes in snail neurons. Pfliigers Arch 436:615-622. Wu M L , Chen JH, Chen WH, Chen YJ, Chu K C (1999) Novel role of the Ca 2 +-ATPase in NMDA-induced intracellular acidification. Am J Physiol 277:C717-C727. Xiong ZQ, Saggau P, Stringer JL (2000) Activity-dependent intracellular acidification correlates with the duration of seizure activity. J Neurosci 20:1290-1296. Yanaka A, Carter KJ, Goddard PJ, Heissenberg M C , Silen W (1991) H +-K +-ATPase contributes to regulation of pH; in frog oxynticopeptic cells. Am J Physiol 26LG781-G789. Yao H, Gu X Q , Haddad G G (2003) The role of HC03"-dependent mechanisms in pH; regulation during 0 2 deprivation. Neuroscience 117:29-35. Yao H, Haddad G G (2004) Calcium and pH homeostasis in neurons during hypoxia and ischemia. Cell Calcium 36:247-255. 153 Zhan RZ, Fujiwara N, Tanaka E, Shimoji K (1998) Intracellular acidification induced by membrane depolarization in rat hippocampal slices: roles of intracellular C a 2 + and glycolysis. Brain Res 780:86-94. 154 6. A P P E N D I X From equation 4.1 (pg 127), the Zn2+-sensitive H + flux from a single neuron with a pH- of 6.75 (pH 0 7.35, 37°C) in response to depolarization with 75 mM [K + ] 0 (Vm = -7 mV) was calculated to be 0.028 mM/s. Assuming that this H + flux from a spherical neuron (i.e. no processes) with a radius of 10 um, the volume of the cell is equal to 4188 um3 (4.19 x 10"9 cm3; volume of a sphere = 47ir3), with a surface area of 1257 jam2 (surface area = 47rr2). Thus, the number of protons crossing the membrane per unit time can be calculated as follows: Number of protons = JH x Volume x TV where N is Avogadro's number. The number the proton extrusion rate was determined to be 70650000 FlVs, which is equivalent to an outward current of 11.3 pA. Dividing this current by the assumed cell surface area gives a current density of 9 fA/um . From the current density, the channel density can be calculated using estimates of the single channel conductance reported in other studies. Assuming a single channel conductance of 40 fS at pH, 6.75 (see Cherny et al., 2003), the channel density is estimated to be 32 channels/um2. 

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