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Effects of intracellular magnesium on large conductance, calcium-activated potassium channels in rat… Zhang, Xian 1994

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EFFECTS OF INTRACELLULAR MAGNESIUM ON LARGE CONDUCTANCE, CALCIUM-ACTIVATED POTASSIUM CHANNELS IN RAT CEREBROVASCULAR SMOOTH MUSCLE CELLS by XIAN ZHANG M.D., Jiangxi Medical College, 1984 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Pharmacology and Therapeutics) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA April 1994 © Xian Zhang, 1994 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of pkjXYYnOJC^ ftWjM &- ~TUsLh£XJtofjuJ^<> & The University of British Columbia Vancouver, Canada Date •g£. n DE-6 (2/88) ii ABSTRACT Increasing the plasma concentration of Mg2+ above normal levels results in the dilation of cerebral arteries. Conversely, an abnormally low level of Mg2+ in plasma has been shown to increase cerebrovascular tone and to induce calcium-mediated vasospastic responses in cerebral vessels. It is known that large conductance, calcium-activated potassium channels (BK channels) play an important role in the regulation of myogenic tone in cerebrovascular smooth muscle cells (CVSMCs). In the vascular smooth muscle cells of systemic vessels, the properties of BK channels are themselves modulated by the intracellular concentration of free magnesium ions, [Mg2+]j. In CVSMCs, [Mg2+]j increases on elevation of the plasma concentration of magnesium ions. It therefore seemed possible that the vasodilatory effect of high plasma Mg2+ levels could result in part from direct, intracellular actions of the cation on BK channel function. The present project was undertaken to test this hypothesis. CVSMCs from the basilar, middle and posterior cerebral arteries of adult Wistar rats were dispersed using collagenase and trypsin and maintained in vitro for 48 hours prior to use. Recordings of single BK channel currents were made at room temperature (20 - 24 °C) from inside-out membrane patches excised from these cells, using a List EPC-7 patch clamp amplifier. Concentrations of Mg2+f higher than 1 mM reversibly reduced the amplitude of currents flowing through open BK channels. In this action, Mg2+f behaved as a fast blocker, reducing BK channel currents in a concentration and a voltage-dependent manner. The blocking effect of Mg2+j was well described by the Woodhull model, which postulates the physical occlusion i i i of the channel pore by a penetrating ion. However, the affinity and voltage-dependence of Mg2+j block were found to be dependent on the concentration of free intracellular calcium ions, [Ca2+]j, bathing the cytoplasmic face of membrane patches. Ca2+j may stabilize a conformation of the BK channel protein in which the Mg2+; binding sites are relocated closer to the inner membrane surface, reducing the voltage-dependency of Mg2+j block. In the presence of 1 |aM [Ca2+]j, Mg2+j enhanced the open probability (P0) of BK channels in a concentration-dependent manner, this effect being evident at the physiologically relevant concentration of 0.5 mM [Mg2+]f. Mg2+j shifted the Boltzmann curve relating P0 to membrane potential leftwards on the voltage axis, without any change in its slope. The enhancing effect of Mg2+j on P0 was, therefore, not itself a voltage-dependent process. These results suggest that the sites which Mg2+j must occupy to increase P0 are distinct from those which are involved in blocking current flow through the open channel. Quantitative considerations suggest that the blocking action of Mg2+f on BK channel currents is unlikely to play a significant role in modulating channel function under physiological conditions. However, physiological levels of Mg2+j would tonically facilitate the effect of Ca2+j on BK channel activation. The abnormally high or low levels of Mg2+j associated with hyper- or hypomagnesemia may also contribute to the dilation or contraction of cerebral vessels seen under these two conditions. iv TABLE OF CONTENTS ABSTRACT ii TABLE OF CONTENTS iv LIST OF FIGURES vii LIST OF TABLES ix ACKNOWLEDGEMENTS x Chapter 1. INTRODUCTION 1 1.1 The Biochemical And Physiological Roles Of Magnesium 1 1.2 Vasodilatory Action Of Mg2+ On Cerebrovascular Smooth Muscle 2 1.3 The Role of Mg2+-Ca2+ Interactions In The Regulation Of Contractility In Vascular Smooth Muscle 3 1.4 Measurements Of Mg2+j Concentration In Vascular Smooth Muscle Cells 4 1.5 The Role Of Ionic Currents In Regulation Of Vascular Smooth Muscle Tone 5 1.6 BK Channels Regulate Myogenic Tone In Cerebral Arteries 7 1.7 Mg2+j Directly Regulates BK Channels 8 1.8 Experimental Rationale 9 Chapter 2. EXPERIMENTAL PROCEDURES 11 2.1 Tissue Culture 11 2.2 Identification Of Dispersed CVSMCs 12 V 2.3 Electrophysiology 13 2.4 Solution Perfusing System 14 2.5 Experimental Solutions 15 2.6 Data Acquisition And Analysis 17 2.7 Statistics 18 Chapter 3. RESULTS 19 3.1 Identification Of BK Channels 19 3.1.1 BK channel conductance and reversal potential 19 3.1.2 [Ca2+]j dependence and blocking effect of [TEA+]j on BK channels 22 3.2 Effects Of Mg2+f On Single BK Channel Currents 22 3.2.1 Concentration-dependent block of BK channel currents by Mg2+j 22 3.2.2 The effect of [Ca2+]; on the Mg2+, block of BK channel currents at -60 mV . . 27 3.2.3 The dependence of Mg2+j block of BK channel currents on membrane potential 27 3.2.4 Application of the Woodhull model to the blocking effect of Mg2+} in BK channel currents 35 3.3 Effects Of Mg2+f On The Open Probability Of BK Channels 39 3.3.1 Mg2+j enhancement of BK channel open probability 39 3.3.2 Mg2+j enhancement of P0 is Ca2+S dependent 48 3.3.3 Effect of Mg2+j on the voltage-dependence of P0 in BK channel 48 Chapter 4. DISCUSSION 54 vi 4.1 Effects Of Mg2+j On BK Channel Currents 54 4.1.1 Mg2+j blocks BK channel currents in a concentration- and voltage-dependent manner 54 4.1.2 Mg2+j acts as a fast blocker of single BK channel currents 55 4.1.3 Possible mechanisms to account for the Mg2+; block of BK channel currents 56 4.2 Effects Of Mg2+j On The Activation Of BK Channels 59 4.2.1 Mg2+j increases the open probability of BK channels 59 4.2.2 Enhancement of BK channel activation by Mg2+j is not voltage-dependent . . 60 4.2.3 Physiological role of Mg2+f as a modulator of BK channel function in CVSMCs 61 4.3 Significance And Future Directions 66 BIBLIOGRAPHY 67 vii LIST OF FIGURES Figure Page 1 Current-voltage relationship of BK channels in the absence of Mg2+j 20 2 [Ca2+]j dependence and blocking effect of TEA+j on BK channels 23 3 Effects of 0.5, 1 and 4 mM [Mg2+]j on current flow in a single BK channel, as recorded at a membane potential of V = -60 mV 25 4 Concentration-dependent reduction by [Mg2+]j of current flow in single BK channels recorded at V = -60 mV 31 5 Effect of [Ca2+]j on Mg2+f block of single BK channel currents recorded at V = -60 mV 33 6 Effect of [Ca2+]j on the current-voltage relationship of single BK channel currents during Mg2+S block 36 7 The Woodhull model accounts for the concentration- and voltage-dependent Mg2+; block of BK channel currents at 100 |iM [Ca2+]f 40 8 The Woodhull model accounts for the voltage-dependent Mg2+j block of BK channel currents at 15 |iM [Ca2+]j 42 9 The block of BK channel currents by 2 mM [Mg2+]; fulfills a prediction of the Woodhull equation 44 10 Effects of 0.5 and 2 mM [Mg2+]j on the open probability of single BK channels recorded at V = -60 mV and [Ca2+]j = 1 [iM 46 V l l l 11 Effect of 2 mM [Mg2+]j on the activation of BK channels by membrane voltage in the presence of 15 \iM [Ca2+]j 52 12 A schematic diagram showing the possible contribution of Mg2+j to the relaxation of CVSMCs in the presence of abnormally high levels of plasma Mg2+ 64 ix LIST OF TABLES Table Page 1 Composition of solutions applied to the cytoplasmic face of inside-out membrane patches 16 2 Effect of various concentrations of [Mg2+]f on the current in single BK channel at V = -60 mV, designated as i.60 28 3 Average reduction in i^0 caused by different internal concentrations of Mg2+. 30 4 Effects of 2 mM [Mg2+]j on the open probability of BK channels recorded at V = -60 mV, in the presence of 1 and 15 \iM [Ca2+]; 49 5 Lack of effect of [Mg2+]j on the open probability of BK channels in the presence of 100 |xM [Ca2+]i 50 X ACKNOWLEDGEMENTS I would like to take this opportunity to thank my supervisor, Dr. Ernest Puil for his encouragement and support, and for his invaluable advice during the course of this project. I wish to express my sincere gratitude to my co-supervisor, Dr. David A. Mathers in Physiology for allowing me to work in his laboratory throughout the research project, and for his invaluable advice and his endless time on direction during the process of experiments and preparation of this thesis. In particular, thanks to Dr. Mathers and Dr. Puil for their providing me with a research assistantship throughout the course of this project. I would like to thank other members of my thesis committee, Dr. Michael J. A. Walker and Dr. B.R. Sastry for their valuable suggestions and constructive comments that made this project interesting and relevant. Thank-you as well to Dr. Steven Kehl and Dr. Peter Vaughan in Physiology for making time for my questions and for their encouragement. Thanks to Mr. John Sanker for his assistance in the preparation of figures for this thesis. Also thanks to Hong, Diane, Monica, Alison and Kathy for your technical assistance and friendship. To my parents, thanks isn't enough, your encouragement and support has always allowed me to take on new challenges. A special thank-you to my husband, xiaoming, for your understanding and support on everything, I couldn't have done it without you... 1 Chapter 1 INTRODUCTION 1.1 The Biochemical And Physiological Roles Of Magnesium Magnesium, Mg2+, is one of the most abundant ions in mammalian tissues, ranking fourth in concentration behind Ca2+, K+, and Na\ Tissue Mg2+ is distributed between three main compartments. Of the approximately 21-28 g of Mg2+ found in the body of a 70 Kg human, 60% resides in the mineral phase of hard tissues such as bone, and is therefore relatively nonexchangeable. Cells contain 38% of the total Mg2+ in the body. The remaining 2% Mg2+ is found in the extracellular compartment. Of this, about 35% is bound nonspecifically to proteins and the remainder exists in ionized form in whole-blood, serum and plasma (Vink et al. 1991; Altura 1991-1992). Over 300 enzymes in the body require Mg2+ for optimal function (Heaton 1980; Ebel and Gunther 1980). Of these, one of the most important classes in terms of energy metabolism is that of the transphosphorylase enzymes involved in the transfer of phosphate groups. These enzymes are found in virtually every metabolic pathway, including the glycolytic pathway, tricarboxylic acid cycle, and oxidative phosphorylation. All adenesine triphosphate (ATP)-consuming and ATP-producing reactions require Mg2+ for activation, with the Mg2+-nucleotide complex serving as the activated substrate. Included in this group of enzymes are Na+/K+ -ATPase, phosphofructokinase and creatine kinase. A further group of enzymes must bind directly to Mg2+ for activation. These include pyruvate kinase, enolase, and adenylate kinase. Mg2+ also regulates the permeability of the cell membrane to other ions, notably Ca2+ and 2 K+ (Bara and Guiet-Bura 1984; Altura and Altura 1985). Being an integral part of the cell membrane, Mg2+ regulates membrane fluidity and, perhaps indirectly, influences the activity of membrane-associated enzymes (Ebel and Gunther 1980). By regulating DNA synthesis and RNA aggregation, Mg2+ plays a vital role in controlling cell growth, reproduction and membrane structure (Rubin 1976; Gunther et al. 1984). As a consequence of its numerous biochemical functions, Mg2+ plays a pivotal role in the physiological control of neuronal activity (Chutkow 1990), cardiac excitability (Altura and Altura 1990; Shine 1979), neuromuscular transmission (Aikawa 1981), vasomotor tone, blood pressure and peripheral blood flow (Altura and Altura 1980, 1982, 1990; Altura et al. 1981). 1.2 Vasodilatory Action Of Mg2+ On Cerebrovascular Smooth Muscle The tone or contractile state of vascular smooth muscle results from spontaneous electrical activity in the muscle fibres, together with the influences of circulating and locally released vasoactive substances (Cook 1990). Mg2+ plays a critical role in the maintenance of vascular tone (Altura 1978). Cerebral vasospasm, stroke and premature atherosclerosis have been linked to chronic depletion of plasma Mg2+(Huang et al. 1990; Altura and Gupta 1992). Increasing the plasma Mg2+ concentration results in dilation of cerebral arterioles, an action that is probably mediated by Ca2+ channels (Altura and Altura 1982). Conversely, decreasing the Mg2+ content of plasma has been shown to increase cerebrovascular tone and even to induce Ca2+ mediated vasospastic responses (Altura et al. 1987). Further, the decrease in plasma levels of magnesium following traumatic brain injury may play a role in focal reductions in blood flow which accompany CNS trauma (Vink et al. 1987). Depletion of extracellular Mg2+ may 3 exacerbate posttraumatic vasospasm and result in more severe secondary hypoxic insults. A recent study has reported a deficiency in serum magnesium in patient with migraines (Mauskop et al. 1993). Despite these important clinical effects, the cellular mechanisms by which magnesium dilates blood vessels remain unclear. However, studies carried out on a variety of smooth muscle preparations indicate that Mg2+-Ca2+ interactions may play an important role in this process (Altura et al. 1982; Altura et al. 1986). 1.3 The Role Of Mg2+-Ca2+ Interactions In The Regulation Of Contractility In Vascular Smooth Muscle Although it is generally true that free intracellular Ca2+, Ca2+j is the key regulator of contractile state in vascular smooth muscle cells (VSMCs), the sources and sinks of this activator Ca2+ vary among smooth muscle tissues (Van Breemen et al. 1980; Fleckenstein 1983; Nabika et al. 1985). Even within a particular organ, different blood vessels may utilize different Ca2+-activating mechanisms for maintenance of myogenic and agonist-induced tone (Altura 1978; Bevan et al. 1985). Organic Ca2+ channel blockers, such as verapamil, nitrendipine, nisoldipine, and nimodipine, do not uniformly inhibit both myogenic and agonist-induced tone in intact arterioles and venules . In contrast, externally applied Mg2+ is able to inhibit myogenic, basal, and agonist-induced tone in all types of vascular smooth muscle (Turlapaty 1978; Iseri and French 1984; Altura et al. 1986). This capability seems to reflect the fact that Mg2+ can act on both voltage- and receptor-operated Ca2+ channels in VSMCs. The organic Ca2+ channel 4 blockers do not have this uniform capability. In consequence, Mg2+ possesses unique and potentially useful Ca2+ antagonistic properties, despite being three to five orders of magnitude less potent than the organic Ca2+ channel blockers (Altura et al. 1986). In addition to its effects at the VSMCs membrane, Mg2+ also affects Ca2+ dynamics at intracellular sites in vascular smooth muscle (Altura et al. 1981). Lowering of external concentration of Mg2+, [Mg2+]0 from 1.2 mM to 0.3 mM results in a 5.8 fold increment in the concentration of free intracellular Ca2+, [Ca2+]j, and induces contraction in aorta VSMCs. However, elevation of [Mg2+]0 to 4.8 mM only slightly reduces [Ca2+]j in these cells (Zhang et al. 1992). 1.4 Measurements Of Mg2+j Concentration In Vascular Smooth Muscle Cells A major obstacle to understanding the role of Mg2* in the regulation of vasomotor tone has been the difficulty of measuring the concentration of free intracellular magnesium, [Mg2+]j, for it is the free ion concentration and not the total Mg2+ concentration which is the physiologically important parameter (Grubbs 1987; McGuigan et al. 1991). Until recently, methods for the measurement of [Mg2+]f have been relatively complex and unreliable. These methods include calculations based on Mg2+-dependent enzyme equilibria, measurements on Mg2+ -ligands whose complexed and free forms exhibit different NMR spectra, Mg2+ selective microelectrodes and null point titration techniques (McGuigan et al. 1991). Using these techniques, [Mg2+]j has been measured at 0.1 mM in rat tail artery VSMCs (Palaty 1971), while a value of 0.4 mM has been obtained from porcine carotid artery cells (Dillon 1986). 5 Recently, Mag-fura-2 fluorescent probes have become available which are analogous to the well established fluorescent indicators for calcium (Raju et al. 1989). Using these probes, an estimate of [Mg2+]j = 0.31 mM has been reported for rat aortic VSMCs, when [Mg2+]0 was 0.80 mM (Schachter 1990). Using the same preparation, Zhang et al. (1992) reported that [Mg2+]j = 0.63 mM when the incubation media contained a physiologically appropriate [Mg2+]0 of 1.2 mM. However, when [Mg2+]0 was raised to 4.8 mM, [Mg2+]j increased to 1.63 mM within a period of 5 minutes. These results show that [Mg2+]; is strongly dependent on the external Mg2+ concentration in VSMCs. A recent study has shown that [Mg2+]j = 0.48 mM in dog cerebrovascular smooth muscle cells (CVSMCs), when [Mg2+]0 is at 1.2 mM levels (Altura et al. 1993), but comparable data for rat CVSMCs are not yet available. Although [Mg2+]j values up to 3 mM have been reported in other cell types (Corkey et al. 1986; McGuigan et al. 1991), all the studies presently available for VSMCs show that [Mg2+]( is less than 1 mM, and typically lies in the range of 0.3 - 0.6 mM, when [Mg2+]0 = 0.6 - 1.2 mM which is considered to be the physiologically normal range of this parameter (Altura 1992; Mudge and Weiner 1990). 1.5 The Role Of Ionic Currents In The Regulation Of Vascular Smooth Muscle Tone Vascular smooth muscle tone is suppressed by the removal of external Ca2+, by addition of Ca2+ channel blockers, and by membrane hyperpolarization (Osol et al. 1986; Harder et al. 1987; Klockner et al. 1989). The generation and maintenance of tension in VSMCs are dependent on processes which modulate intracellular levels of free Ca2+. Calcium ions enter 6 the cytoplasmic compartment of VSMCs in several ways, including transsarcolemmal entry through voltage-dependent Ca2+ channels, Na+-Ca2+ exchange, and receptor-operated Ca2+ channels (Nelson et al. 1990). The depolarizing phase of the action potential in VSMCs is mediated by an inward current, carried largely by Ca2+ ions which enter the cell through voltage-dependent Ca2+ channels (Harder 1983; Klockner et al. 1989). Depolarization and/or the increase in [Ca2+]j in turn trigger potassium selective outward currents, which serve to repolarize the cell membrane to its original potential. The general role of these K+ currents in the modulation of vascular excitability is well recognized. In VSMCs, K+ currents play a crucial role in the vascular response to endogenous and pharmacological vasodilators, by influencing the resting membrane potential, and the time course and amplitude of electrical changes superimposed on the membrane potential (Brayden et al. 1991). Increases in K+ conductance result in membrane hyperpolarization, with subsequent closure of voltage-dependent Ca2+ channels (Standen et al. 1989; Bulbring and Tomita 1987) and enhanced Ca2+ extrusion via the Na+-Ca2+ exchanger (Lauger 1987). hi both cases, the physiological response is a reduction in vascular smooth muscle tone. The known types of K+ channels in VSMCs include delayed rectifier K+ channels (Okabe et al. 1987; Bolton et al. 1986; Stockbridge et al. 1991; Bonnet et al. 1991), ATP-sensitive K+ (KATP) channels (Standen et al. 1989; Brayden et al. 1991), and Ca2+-activated K+ (KQ,) channels (Beech et al. 1987; Inoue et al. 1985, 1987; Benham et al. 1986; Wang and Mathers 1993). Of these, K^ channels have been the most widely studied (for review, see Tomita 1988; Latorre et al. 1989; Edwards and Weston 1990; Kolb 1990). Based on their single channel conductance, calcium sensitivity, voltage dependence, and pharmacological properties, 7 three main kinds of K^ channels have been described in VSMCs. These are designated as large, 150 - 250 pS conductance Kc, channels (BK channels), intermediate, 30 - 120 pS conductance K ,^ channels (IK channels), and small conductance (SK channels) K^ channels (Ruby 1988; Cook 1990). Differences in the mechanisms of activation of these channels may reflect their functional roles, with some channels maintaining resting tone, and others terminating contraction induced by vasoconstrictors. 1.6 BK Channels Regulate Myogenic Tone In Cerebral Arteries Isolated cerebral arteries exhibit a degree of active tone even in the absence of neural input and circulating hormones (Asano et al. 1987; Masuzawa et al. 1990; Tanoi et al. 1992; Suzuki et al. 1992). This spontaneous tension generation is referred to as myogenic tone. It has been proposed that myogenic tone is a key element in the autoregulation of cerebral blood flow. Elevation of intravascular pressure leads to membrane depolarization, enhanced Ca2+ influx and contraction of CVSMCs (Brayden and Nelson 1992). Myogenic tone is abolished by the removal external Ca2+ (Asano et al. 1987; Tanoi et al. 1992; Suzuki et al. 1992) or by the addition of Ca2+ channel blockers (Asano et al. 1987; Tanoi et al. 1992). It is believed that a negative feedback control of this process is provided by KQ, channels, which are opened by intracellular Ca2+ accumulation and by membrane depolarization (Brayden and Nelson 1992). In support of this view, charybdotoxin (CTX), a relatively specific blocker of BK channels, has been shown to increase myogenic tone in cerebral arteries. This effect is associated with membrane depolarization and Ca2+ influx via voltage-dependent calcium channels (Brayden and Nelson 1992; Asano et al. 1993). Since the BK channel is the only known K+ channel in 8 CVSMCs which is blocked by CTX, these results suggest that BK channels play an important role in the regulation of myogenic tone in cerebral vessels (Nelson et al. 1990; Brayden and Nelson 1992; Asano et al. 1993). In contrast, KATP channels do not appear to play an important role in the normal regulation of myogenic tone in cerebral arteries (Brayden and Nelson 1992; Asano 1993). 1.7 Mg2+j Directly Regulates BK Channels In patch clamp studies, Mg2+S has been found to block BK channel currents in variety of non-vascular preparations. In the presence of 10 |iM [Ca2+]j, 5 to 50 mM [Mg2+]j reduced inward and outward currents through the open BK channels of rat skeletal muscle in a concentration- and voltage-dependent manner (Ferguson and Magleby 1989; Ferguson 1991). In pancreatic islet fi cells, studies using 10 to 20 mM [Mg2+]j showed a block of outward current which increased with membrane depolarization (Tabcharani and Misler 1989). In addition to exerting a blocking effect on BK channel currents, Mg2^ has also been found to influence the gating of these channels. Millimolar concentrations of Mg2+S reduce the open probability of BK channels in the human erythrocyte membrane (Grygorczyk and Schwarz 1983). In contrast to this inhibitory effect, however, 1-10 mM [Mg2+]j has been shown to increase the open probability of BK channels in rat skeletal muscle. This effect involved an increased affinity of BK channels for Ca2+f, and an increase in the apparent cooperativity of Ca2+ activation, that is in the minimum number of Ca2+ ions needed to activate each channel (Golowasch et al. 1986; Oberhauser et al. 1988). Squire and Petersen (1987) have observed a concentration-dependent activation of BK channels in mouse salivary acinar cells by 1 |iM -9 1 mM [Mg2+]j, at a constant [Ca2+]s of 10 nM. In rat hippocampal neurons, McLarnon and Sawyer (1993) reported that Mg2+S potentiated BK channel activation when internal [Ca2+]j was 5 |iM. In VSMCs, 2 mM [Mg2+]j increased the open probability of BK channels in the presence of 0.1 and 0.3 p,M [Ca2+]j in pig coronary and pial vessels (Trieschmann and Isenberg 1989). These studies have also shown that Mg2+S is unable to activate BK channels in the absence of Ca2+j (Golowasch et al. 1986; Oberhauser et al. 1988; Tabcharani and Misler 1989; McLarnon and Sawyer 1993). 1.8 Experimental Rationale Evidence reviewed above shows that increasing the plasma concentration of Mg2+ above normal levels results in the dilation of cerebral arteries. Conversely, an abnormally low levels of Mg2+ in plasma has been shown to increase cerebrovascular tone and to induce calcium-mediated vasospastic responses in cerebral vessels. It is known that large conductance, calcium-activated K+ channels (BK channels) play an important role in the regulation of myogenic tone in cerebrovascular smooth muscle cells. In the vascular smooth muscle cells of systemic vessels, the function of BK channels is itself modulated by the intracellular concentration of free Mg2+j. In CVSMCs, [Mg2+]f increases on elevation of the plasma concentration of magnesium ions. It may therefore be hypothesized that the dilation of cerebral vessels by elevated plasma concentrations of Mg2+ could involve direct, intracellular effects of the cation on BK channel function, mediated at the cytoplasmic face of the CVSMC membrane. To test this hypothesis, the effects of physiologically relevant concentrations of Mg2+j on the function of BK channels in CVSMCs were examined using standard patch clamp 10 techniques. A low extracellular concentration of free calcium ions was employed in these studies, in order to suppress inward Ca2+ current on depolarization of isolated membrane patches. This study was restricted to the effects of Mg2+ at the intracellular membrane surface, since previous studies have shown that there are no direct effects on BK channel function if Mg2+ is applied to the extracellular membrane surface (Golowasch 1986; MacKinnon 1989; Oberhauser 1988). 11 Chapter 2 EXPERIMENTAL PROCEDURES 2.1 Tissue Culture Primary cell cultures were prepared from the middle, basilar, posterior communicating and posterior cerebral arteries of adult Wistar rats (Charles River, Montreal) weighing 250-300 g. After being exposed to C02 until unconscious, the rats were decapitated. Brains were removed under sterile conditions, and placed in a 60 mm culture dish containing Ca2+- and Mg2+-free Hank's Balanced Salt Solution (HBSS, Gibco Laboratories, Grand Island, NY) of the following composition (mM): 138 NaCl, 5 KC1, 0.3 KH2P04, 0.3 Na2HP04.7H20, 18 Dextrose, 4 NaHC03,15.7 HEPES with penicillin 100 U/ml and streptomycin 100 ng/ml (Sigma Chemical Company, St. Louis, MO), pH 7.4. Under the low power of a dissection microscope, the basilar, middle posterior communicating and posterior cerebral arteries and their first and second order side branches were removed. Arteries were placed in a 60 mm culture dish containing 10 ml potassium glutamate (KG) buffer solution of composition (mM): 140 glutamic acid monopotassium, 16 NaHCOj, 0.5 NaH2P04, 16.5 Dextrose and 25 HEPES, pH 7.4. Following incubation in KG solution at 37°C for 10 minutes, the vessels were minced with iridectomy scissors into 0.5 mm fragments. The fragment suspension was centrifuged for 1 minute, and the supernatant was discarded. The tissue pellet was resuspended in 3 ml of 0.1% trypsin (Type C, Sigma, dissolved in KG solution), and incubated at 37°C for 7 minutes . After being centrifuged for 1 minute, the supernatant was again discarded. The tissue was then resuspended and incubated 12 in 3 ml of KG solution containing 0.3% collagenase (Type 1A, Sigma) and 0.5% trypsin inhibitor (Sigma) at 37°C for 15 minutes. The cell suspension was centrifuged and the supernatant again decanted. Dispersed cells were resuspended in Minimum Essential Medium (Gibco, Grand Island, New York) containing 15% horse serum (heat-inactivated, Gibco) at 4°C in order to inhibit enzyme activity. The cells were then washed three times with 6 ml of KG solution. A final cell suspension was prepared in 1 ml of KG solution. 15 mm diameter sterile glass coverslips precoated with poly-D-lysine (Sigma) were placed in 35 mm tissue culture dishes. A 0.2 ml volume of cell suspension was plated onto each glass coverslip. Cells were allowed to settle for 45 minutes at room temperature (20-24°C). Each culture dish was then filled with 2 ml of maintenance solution containing (in mM): 130 NaCl, 5 KC1, 0.8 CaCl2, 1.3 MgCl2, 5 Glucose, 10 HEPES, penicillin (Sigma) 100 U/ml, and streptomycin (Sigma) 100 |ig/ml. Cultures were maintained at 4°C in a refrigerator for 48 hours prior to use. 2.2 Identification Of Dispersed CVSMCs Under a light microscope (Olympus CCK-1, X300 magnification), CVSMCs could usually be recognized by their oval shapes and by their length/breadth ratios in the range 1.5-2:1. On contact with a patch electrode, these cells adopted a spherical morphology, suggesting that they were capable of a contractile response. Cells were further identified by use of the Masson trichrome stain (Masson 1929; Spatz et al. 1983), in which the cytoplasm of CVSMCs was stained red. Cells on the coverslip were first fixed with 2.5% formalin in phosphate buffer solution (PBS) containing (in mM): 149 NaCl, 2 KH2P04, 4.2 NajHPO^ pH 7.4 for 10 13 minutes. Cell nuclei were then stained with 50% iron haematoxylin (1:1 in H20) for 5 minutes, followed by differentiation with 1 % acid alcohol for a few seconds. The cells were washed with distilled water, and treated with 2% Ponceau acid solution (dissolved in 1% acetic acid) for 3 minutes. The Ponceau acid stained the cytoplasm of smooth muscle cells red. Cells were differentiated with 1% phosphotungstic acid for 5 minutes. Under the light microscope, the nuclei of CVSMCs, endothelial cells and fibroblasts were stained black, the cytoplasm of CVSMCs was stained red, and the cytoplasm of other cell types present was stained blue. 2.3 Electrophysiology Single channel recordings were carried out at room temperature (20-24°C) using standard patch clamp techniques (Hamill et al. 1981). One culture dish containing CVSMCs was taken out of the refrigerator at the time of recording. The maintenance solution in the dish was discarded and replaced with 2 ml of a control bath saline appropriate to the experimental design. The culture dish was then mounted on the stage of an inverted, phase-contrast microscope (Olympus CK, Tokyo, X300 magnification). Patch electrodes were pulled from capillary tubes of borosilicate glass (1.5 mm OD X 0.75 mm ID, Frederik Haer Corp., Brunswick, ME) using a two-stage vertical puller (David Kopf 700). The outside diameter of electrode tips was 2-3 urn. Electrodes were coated with 3140 RTV sealant (Dow Corporation, Midland, Michigan). This reduced pipette bath capacitance by forming a hydrophobic surface and increasing the effective wall thickness (Hamill et al. 1981). Electrodes were fire-polished prior to use to produce a clean and smooth tip rim. This 14 facilitated the formation of a high resistance seal between the electrode and the cell membrane (Hamill et al. 1981). Electrode resistances were in the range of 10 - 20 MQ when filled with standard external solution. Recordings were made using the inside-out patch clamp configuration (Hamill et al. 1981). A low resistance seal was produced upon mechanical contact between the patch electrode and the cell membrane. With gentle suction, a cell-attached, gigohm seal was formed. To form an inside-out patch, the electrode was then withdrawn from the cell surface, creating a membrane vesicle at the electrode tip. Brief passage through the solution/air interface ruptured the outer membrane of this vesicle. The intracellular surface of the membrane patch was then exposed to the bath saline. 2.4 Solution Perfusing System A gravity-fed perfusion system was used to change the saline bathing the cytoplasmic face of the patch membrane. This system was composed of several 60 ml syringe reservoirs mounted above the stage of the microscope. Plastic taps were connected tightly to the end of each syringe, allowing for selection of the appropriate test solution. A short plastic tube ran from this assembly to an L-shaped glass tube held in a micromanipulator. Following patch isolation, the tip of the patch electrode was moved to within 0.5 mm of the outlet of the perfusion system, facilitating rapid exchange of bath solutions. A perfusion rate of 5 ml/min was maintained throughout the duration of recordings. Experiments using very low calcium salines to suppress activity of BK channels showed that solution changes were complete within 30 seconds of switching to a new saline. 15 2.5 Experimental Solutions Patch electrodes were filled with a standard external solution of composition (in mM): 140 KC1,1.48 CaCl2, 3 EGTA, 10 HEPES, pH 7.4. The free calcium concentration in this solution was 50 nM, as calculated using the programme Max Chelator, obtained from Stanford University, California. The cytoplasmic face of the membrane patch was normally exposed to a control internal solution containing (in mM): 140 KC1, 0.1 CaCl2, 10 HEPES, pH 7.4. The free calcium concentration of this composition was 100 |J.M. To examine the effects of Mg2+j in this high free calcium condition, 0.5,1,2,4, or 8 mM MgCl2 was added to the control internal solution. In a second group of recordings, the cytoplasmic face of the membrane patch was bathed in a control saline of composition (in mM): 140 KC1, 2.86 CaCl2, 3 EGTA, 10 HEPES, pH 7.4. The free calcium concentration in this saline was calculated at 1 |iM. In a third group, the control solution facing the cytoplasmic face of membrane patch contained 140 KC1, 0.015 CaCl2, 10 HEPES, pH 7.4. In this group, the free calcium concentration was 15 |j,M. To test the effects of Mg2+f in the presence of 1 or 15 |J,M free [Ca2+]j, 0.5 or 2 mM [Mg2+]j was added to these salines. Table 1 lists the internal solutions used in this study. To demonstrate the Ca2+ sensitivity of BK channels, the cytoplasmic membrane face of patches was exposed to a very low free calcium solution of the following composition (in mM): 140 KC1, 0.27 CaCl2, 3 EGTA, 10 HEPES, pH 7.4 with a calculated free calcium of 5 nM. In some experiments, 1 mM tetraethylammonium (TEA+, Sigma, chloride salt) was applied to the cytoplasmic membrane face. TEA+ has been reported to block BK channels from the intracellular face of the CVSMC membrane (Wang and Mathers 1991). 16 Table 1. Composition of solutions applied to the cytoplasmic face of inside-out membrane patches in this study. Solutions [Ca2+L 100 vM [Ca2+] ; 15 /iM [Can 1 AiM [Ca2+] ; [Mg2+]i Control 0.5 mM [Mg2+] ; 1 mM [Mg2+] ; 2 mM [Mg2+]j 4 mM [Mg2+]; 8 mM [Mg2+]j Control 2 mM [Mg2+]j Control 0.5 mM [Mg2+]i 2 mM [Mg2+]i Composition (in mM) KCl CaCl2 Free MgCl2 Free EGTA HEPES [Ca2+] ; [Mg2+] ; 140 0.1 0.1 0 0 0 10 140 0.1 0.1 0.5 0.5 0 10 140 0.1 0.1 1 1 0 10 140 0.1 0.1 2 2 0 10 140 0.1 0.1 4 4 0 10 140 0.1 0.1 8 8 0 10 140 0.015 0.015 0 0 0 10 140 0.015 0.015 2 2 0 10 140 2.87 0.001 0 0 3 10 140 2.86 0.001 0.51 0.5 3 10 140 2.84 0.001 2.03 2 3 10 17 2.6 Data Acquisition And Analysis Single channel currents and membrane patch voltages were recorded using a List EPC-7 amplifier (Medical Systems Corp., New Jersey). Signals were filtered at a bandwidth of DC-2 kHz (8-pole, Bessel), stored using a Sony video cassette recorder, and displayed on a rectilinear pen recorder (DC-100 Hz, Gould, Model 220, Ohio). Data analysis proceeded off-line using RECORDER and TAC programs (Instrutech Corporation, New York) running on an Atari Mega 4 computer (Atari, Sunnyvale, California). The current signals were subjected to analogue-to-digital (A-D) conversion at 8 kHz. The threshold for single BK channel current detection was set at 50% of the mean single BK channel current. Frequency distributions of single channel current amplitudes were constructed by measuring about 1000 openings in each data set. The mean amplitude of single channel currents was obtained as the midpoint of a Gaussian curve fitted by eye to these distributions. Reversal potentials for single channel currents were determined as the zero current intercepts of fit lines applied to current-voltage plots by linear regression. Currents and voltages were denoted with respect to the cytoplasmic face of membrane patches. The probability, P0 of finding a single BK channel in the open state during a recording of total duration, Ttot was calculated from the relation P0 = ( T, + T2 + ...+TN ) / NTtot Here, N is the total number of functional BK channels in the patch. Tx, T2, ...TN are the times when at least 1, 2, ...N channels were open (Mayer et al. 1990). N was estimated by depolarizing the membrane patches to +60 mV in the presence of 100 nM [Ca2+]j. 18 2.7 Statistics Data presented in this thesis were expressed as mean ± standard error of the mean (S.E.M.). "n" represents the number of patches studied. Statistical analysis was carried out using the SYSTAT 4.1 software package. When more than two experimental groups were being compared, analysis of variance between experimental groups (ANOVA) was performed. For paired data, significance was determined using a paired t-test. Data sets for which P < 0.05 were considered to be significantly different. 19 Chapter 3 RESULTS 3.1 Identification Of BK Channels Isolated inside-out membrane patches excised from CVSMCs displayed a variety of single channel currents when exposed to symmetrical 140 mM K+ solutions. Large conductance, Ca2+ -activated K+ (BK) channels were observed in 68% (48/71) of inside-out patches excised from CVSMCs. BK channels were identified by their large conductance (>150 pS), dependence of their open probability on [Ca2+]j and by their sensitivity to block by internally applied TEA+, as reported in previous studies on these cells (Wang and Mathers 1991, 1993). In some patches, channels with conductances in the range of 10 - 90 pS were also observed. These were readily distinguished from BK channels by their significantly smaller conductance and their insensitivity to the changes in [Ca2+]j. No other calcium-dependent ion channels of large conductance were present in these patches. 3.1.1 BK channel conductance and reversal potential In patches exposed to symmetrical 140 mM K+ solutions, 100 |iM [Ca2+]j and 0 mM [Mg2+]j, the current-voltage (I-V) relationship of BK channels was linear over the voltage range -80 to +80 mV, with a reversal potential of 0 mV (Fig. 1). The range of conductance values encountered in different patches was from 150 to 255 pS, and the mean single channel conductance obtained was 192 ± 10 pS (n=9). 20 Fig. 1 Current-voltage relationship of BK channels in the absence of Mg2+, Single BK channel currents were recorded in inside-out patches of CVSMC membrane voltage-clamped to membrane potentials, V in the range -80 to +80 mV. Patches were exposed to symmetrical 140 mM K+ solutions, and [Ca2+]j and [Ca2+]0 were 100 |iM and 50 nM, respectively. The straight line was fitted to the data by least squares regression and corresponded to a mean conductance of 192 ± 10 pS (n=9). Currents reversed at -1 mV. Symbols indicate mean ± SEM. In this and in all subsequent figures showing standard errors, this parameter is omitted if its value is less than the size of the symbol. 21 i (pA) 20 16 12 8 0-Outward 1 0 0 - 8 0 - 6 0 - 4 0 - 2 0 , - 4 0 20 40 60 80 100 V (mV) Inward - 1 6 20 L 22 3.1.2 [Ca2+], dependence and blocking effect of [TEA+], on BK channels To test the effect of [Ca2+]j on the open probability (P0) of BK channels, recordings were made when [Ca2+]j was decreased from 100 \xM to 5 nM. The membrane potential was voltage-clamped at -60 mV. Fig. 2A shows single channel currents recorded from a membrane patch containing one active BK channel. In this patch, the channel remained open most of the time in the presence of 100 |iM [Ca2+]j (P„ = 0.73). After decreasing [Ca2+]f to 5 nM, the open probability of the channel was greatly reduced to 0.0002. This effect reversed on return to the solution containing 100 p.M [Ca2+]j (P0 = 0.69). These observations show that the open probability of the BK channels was strongly enhanced by an increase in [Ca2+]j. Internal application of TEA+ is known to block BK channel currents in CVSMCs with a Kd of 0.83 mM (Wang and Mathers 1993). At the end of some recordings, 1 mM TEA+ was applied to the cytoplasmic face of the patch to verify its blocking effect on BK channel currents. As shown in Fig. 2B, 1 mM [TEA+]j reduced the single channel current by more than 50%, as expected from previous studies (Wang and Mathers 1993). 3.2 Effects Of Mg2+, On Single BK Channel Currents 3.2.1 Concentration-dependent block of BK channel currents by Mg2*, The effect of Mg2+j on BK channel currents was initially studied in patches bathed in symmetrical 140 mM K+ solutions with [Ca2+]( =100 |iM. Under these conditions, adding Mg2+j at 1 mM or a higher concentration reversibly reduced (blocked) the amplitude of BK channel currents recorded at a membrane potential of -60 mV, designated as i^,. Fig. 3 shows the effects of 0.5, 1 and 4 mM [Mg2+]f on i.60 as measured in a single BK channel from one 23 Fig. 2 [Ca2+], dependence and blocking effect of [TEA*], on BK channels A. The effect of changing [Ca2+]j from 100 \xM to 5 nM on the activity of a single BK channel. This patch was exposed to symmetrical 140 mM K+ solutions and the membrane potential was voltage-clamped at -60 mV. "0" denotes the closed state and "1" denotes the open state of the BK channel. The channel remained open most of the time (P„ = 0.73) in the presence of 100 \xM [Ca2+]j. After decreasing [Ca2+]j to 5 nM, the open probability fell to P0 - 0.0002. This effect reversed on return to the solution containing 100 (J.M [Ca2+]j (P0 = 0.69). Bandwidth of recording DC-2 kHz. B. The effect of 1 mM [TEA+]j on the current flow in a single BK channel voltage-clamped at a membrane potential of -60 mV, with [Ca2+]j = 100 |iM. TEA+ was applied to the cytoplasmic membrane face of the patch. "1" denotes the channel open level and "0" denotes the channel closed level. Bandwidth of recording was DC-2 kHz. 24 A I 2+-1 0 0 UK [ c a £ ] j O ^Jutwui^HH'ji^WiKOII^ frjUHWiwi^ihn^ 2+ i 5 nM [ C a £ T ] j 2+-, 1 0 0 uK [ C a ' ] , . < o 100ms B ° --^H^f n \>t(4^tf\rtfi--0 mM [TEA*],-o --. i 4 (l" 1 mM [TEA + ] j < a o ""> 10ms 25 Fig. 3 Effects of 0.5, 1 and 4 mM [Mg2*], on current flow in a single BK channel, as recorded at a membrane potential of V = -60 mV These traces illustrate the concentration-dependent blocking effect of Mg2+j on current flow in a single BK channel recorded at a membrane potential of V = -60 mV, designated as i^,. This patch was exposed to symmetrical 140 mM K+ solutions. The control bath solution contained 100 |J.M [Ca2+]j and 0 mM [Mg2+]j. The recording pipette contained 50 nM free [Ca2+]0. "0" represents closed channel current level and "1" represents open channel current level. Bandwidth of recordings was DC-2 kHz. The control value of i^0 was -9.5 pA. Adding 0.5 mM [Mg2+]j to the bath solution did not alter i^,. However, a further increase in [Mg2+]j to 1 and 4 mM reduced i^0 from -9.5 to -8.1 (a 15% reduction) and to -5.1 pA (a 47% reduction of the original current), respectively. These effects reversed on washout of magnesium ions. 26 O o Control 0.5 mM [Mg2+]; O 1 mM [Mg2+]; O 1 4 mM [Mg2+]; 0 Wash < Q O O 100ms 27 membrane patch. In this patch, 1 mM [Mg2+]f reduced i^from -9.5 to -8.1 pA (a 15% reduction). A further increase in [Mg2+]j to 4 mM reduced i^, to -5 pA (a 47% reduction of the original current). However, there was no significant reduction of i^, in the presence of 0.5 mM [Mg2+]j. Data from several experiments of this type are shown in Table 2. The mean percentage reduction in i^, caused by various concentrations of Mg2+f are summarized in Table 3. This table shows that application of 0.5, 1, 2, 4 or 8 mM [Mg2+]f decreased i^, by 0.6% (n=6, P>0.05, ANOVA), 17% (n=7, P<0.01, ANOVA), 31% (n=8, P<0.01, ANOVA), 46% (n=8, P<0.01, ANOVA) and 64% (n=8, P<0.01, ANOVA), respectively. Fig. 4 plots the percentage reduction in i^ „ against the various concentrations of Mg2+i5 and demonstrates that Mg2+; reduced BK channel currents in a concentration-dependent manner. 3.2.2 The effect of [Ca2*], on the Mg2+, block of BK channel currents at -60 mV Since the gating of BK channels is regulated by [Ca2+]j, it seemed possible that changes in [Ca2+]j could alter the blocking effect of Mg2+j on BK channel currents. This possibility was tested by studying patches exposed to 1, 15 or 100 |iM [Ca2+]i and voltage-clamped at V = -60 mV (Fig. 5). 2 mM [Mg2+]j reduced i^, from -11.5 pA to -7.8 pA (a 31% reduction) at 100 |iM [Ca2+]f (n=8, P<0.01, ANOVA), but this concentration of Mg2+, did not alter i^, in patches exposed to 1 or 15 |iM [Ca2+]j (n=6, P>0.05, ANOVA). 3.2.3 The dependence of Mg2*, block of BK channel currents on membrane potential Fig. 5 showed that low [Ca2+]j solution protected BK channel currents from Mg2+j block at a membrane potential of -60 mV. It was of interest to determine whether this was also the 28 Table 2. Effect of various concentrations of internal magnesium ions, Mg2+j on the current in single BK channels at V = -60 mV, designated as i^,. In all of these experiments, [Ca2+]j = 100 [iM, [Ca2+]0 = 50 nM, [K+]j = [K+]0 = 140 mM. The percent reduction in i^, was calculated by comparing the value of the Mg2+j blocked current with the value of the control current in the same patch. "—" indicates that washout data were unattainable because of loss of seal integrity during the recording. 29 Solutions 0.5 mM [Mg2t], 1 mM [Mg2+]j 2 mM [Mg2t]( 4 mM [Mg2l 8 mM [Mg2t]j Patch No. 1 2 3 4 5 6 1 2 3 4 5 6 7 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 Control (pA) -13.8 -10.2 -12.3 -12.9 -12.7 -9.4 -15.1 -12.5 -11.0 -11.4 -11.9 -12.9 -9.5 -12.1 -9.3 -10.3 -13.6 -11.6 -12.3 -13.3 -9.3 -10.3 -11.9 -14.2 -13.8 -9.4 -9.0 -9.0 -11.9 -12.4 -12.1 -11.5 -11.1 -12.7 -9.2 -9.1 -11.0 1-60 Mg2+, (pA) -13.5 -10.4 -11.8 -13.4 -12.6 -9.2 -12.5 -10.6 -8.9 -8.9 -9.0 -9.8 -8.1 -8.1 -6.3 -5.2 -8.5 -7.6 -9.9 -9.2 -7.4 -6.5 -6.3 -8.0 -7.4 -4.0 -5.2 -5.1 -6.3 -2.4 -3.2 -4.7 -4.0 -4.6 -4.6 -5.1 -3.0 Wash(pA) -13.4 -10.5 -12.3 -12.9 -11.5 -9.3 -15.3 -13.6 -11.0 -9.8 -11.6 -13.3 -9.3 -12.1 -9.2 -9.8 — -10.7 -12.5 -13.3 -9.4 -12.8 — -13.4 — -10.0 — -9.5 -11.5 — -12.1 -10.4 — -11.5 -10.1 -8.6 -8.4 Percent reduction ini.«, 2% -1.6% 4% -4% 1% 2% 17% 15% 19% 21% 24% 24% 15% 33% 32% 41% 38% 35% 20% 30% 20% 37% 47% 44% 46% 57% 43% 44% 47% 81% 73% 59% 64% 64% 51% 44% 73% 30 Table 3. Average reductions in i ,^ caused by different concentrations of Mg2+i. "n" represents the number of patches from which the mean value was obtained. "S" indicates that the data obtained in the presence of Mg2+j were significantly different from control values (P<0.01, ANOVA), while "NS" indicates that the data, with Mg2+; present, were not significantly different from control values (P>0.05, ANOVA test). At 100 /xM [Ca2+]j and -60 mV membrane potential, 1, 2, 4, and 8 mM [Mg2+]; reduced the current by 17%, 31%, 46% and 64%, respectively, while 0.5 mM [Mg2+]i had no significant effect on [Mg2+]; 0.5 mM (n=6) 1 mM (n=7) 2mM (n=8) 4 mM (n=8) 8 mM (n=8) Current at -60 mV, Control -11.9 ±0.70 -12.0 ±0.66 -11.5 ±0.59 -11.9 ±0.74 -11.1 ±0.48 Mg2+; -11.8 ±0.70 -9.8 ±0.56 -7.8 ±0.54 -6.1 ±0.46 -4.0 ±0.34 i-6o (PA) Wash -11.7 ±0.63 -12.0 ±0.82 -11.0 ±0.62 -11.4 ±0.76 -10.2 ±0.61 Percent reduction 0.6 ±1.17 17.3 ±1.46 31.3 ±2.72 45.6 ±1.98 63.6 ±4.32 ANOVA test NS S s s s 3 1 Fig. 4 Concentration-dependent reduction by Mg2+| of current flow in single BK channels recorded at V = -60 mV, i^ , This figure shows the percent reduction in i^, caused by addition of 0.5,1,2,4 or 8 mM [Mg2+]j to the bath solution. [Ca2+]j = 100 |iM, [Ca2+]0 = 50 nM. Each data point represents the mean value calculated from 6-8 patches. The smooth curve was fitted to the data by least squares non-linear regression. 32 7 8 9 10 2+ Concentrat ion of Mg . (mM) 33 Fig. 5 Effect of [Ca2+], on Mg2+, block of single BK channel currents recorded at V = -60 mV, i_«9 The effects of 2 mM [Mg2+]j on i^, recorded from patches exposed to 1,15 or 100 \xM [Ca2+]j are compared in this figure. At 1 or 15 |iM [Ca2+]t there was no significant difference in i ^ between the control and 2 mM [Mg2+]; data groups (n=6, P > 0.05, ANOVA). However, at 100 |iM [Ca2+]j, 2 mM [Mg2+]j reduced i^, from -11.5 pA to -7.8 pA (n=8, P < 0.01, ANOVA, asterisk). 34 i_Rn (PA) "SO -60 Control P < 0.01 2-K Z 3 2 mM [Mg ] 15 -- 1 0 - 5 ^ 'A i 1 /JM [Ca 2 + ] , 15 /xM [Ca 2 + ] . 100 /xM [Ca 2 + ] . (n=6) (n = 6) (n = 8) 35 case at positive membrane potentials. The voltage-dependence of Mg2+; block was therefore studied in patches exposed to either low [Ca2+]j (15 nM) or high [Ca2+]j (100 |iM). In the absence of Mg2+i5 the I-V relationship of BK channels in symmetrical 140 mM K+ solutions was linear over the voltage range -80 to +80 mV. Fig. 6A shows the effect of 1 mM (n=4) and 2 mM [Mg2+]f (n=5) on the I-V relationship of BK channels, in patches exposed to [Ca2+]j = 100 |iM. Under these conditions, Mg2+f reduced both inward and outward currents in BK channels, without altering the reversal potential. The reduction in BK channel currents increased on raising [Mg2+]( from 1 mM to 2 mM (P<0.05, ANOVA). In addition, the I-V relationship of BK channels in the presence of Mg2+j became non-linear, since outward currents were reduced to greater degree than were inward currents. This indicated that the blocking effect of Mg2+j was voltage-dependent. Fig. 6B shows the effect of 2 mM [Mg2+]j on the I-V relationship of BK channels exposed to the solution containing [Ca2+]j = 15 |iM (n=6). Under these conditions, Mg2+S had negligible effect on inward currents, but produced a sharper voltage-dependent reduction of outward currents than the same concentration of Mg2+f did in the presence of 100 nM [Ca2+]j (P<0.01, ANOVA). Again, the reversal potential for BK channel currents was unchanged by Mg2+j. The data shown in Fig. 6 provide evidence for the dependency of Mg2+S block on [Ca2+](, since altering the latter parameter also changed the voltage-dependence of Mg2+j block. 32.4 Application of the Woodhull model to the blocking effect of Mg2*, on BK channel currents The data presented above show that Mg2+i reduced BK channel currents in a concentration-36 Fig. 6 Effect of [Ca2*], on the current-voltage relationship of single BK channel currents during Mg2*! block. A. The I-V relationship of single BK channels studied in patches exposed to symmetrical 140 mM K+ solutions, in the presence of 100 |iM [Ca2+]i- The control data (circles) were fitted by least squares regression to a linear function over the voltage range -80 to +80 mV. I-V curves obtained after addition of 1 mM (triangles, n=4) and 2 mM (squares, n=5) [Mg2+]j were fitted by least squares non-linear regression. All I-V plots showed a reversal potential of -1 mV. B. The I-V relationship of single BK channels studied in patches exposed to symmetrical 140 mM K+ solutions, in the presence of 15 |iM [Ca2+]f. Control data (closed circles) were fitted by least squares regression to a linear function over the voltage range -80 to +80 mV. The I-V curve obtained after addition of 2 mM [Mg2+]j (open circles, n=6) was fitted by least squares non-linear regression. The reversal potential of these plots was 0 mV. 37 1 8 0 Y"/ 1 - 6 0 ' ' uL' i (pA) 1 1 - 4 0 - 2 0 J X ^ 20 16 12 8 4 ft / ( - 4 - 8 - 1 2 - 1 6 - 2 0 -1 20 • T • i i i 40 60 80 V (mV) Control 1 mM [Mg ]. 2 mM [Mg + ] ; B 1 8 0 1 - 6 0 i (pA) i i - 4 0 - 2 0 20 16 12 8 4 - 4 - 8 - 1 2 - 1 6 - 2 0 -~ 1 20 • o ^•^ o -^_J3- —"O"^ 1 1 1 40 60 80 V (mV) Control 2 mM [Mg2+]. 38 and voltage-dependent manner. It was of interest to determine whether the Mg2+; block could be accounted for by the WoodhuU model, first developed to describe the blocking effect of hydrogen ions (FT) on sodium currents (WoodhuU 1973). The WoodhuU model has been used to analyse the block of BK channels by charged species in several preparations (Blatz and Magleby 1984; Yellen 1984; Wang and Mathers 1993). The model makes the following assumptions: (a) The rates of binding and unbinding of the blocking ion at its active site in the channel vary as exponential functions of membrane potential, as predicted by Eyring rate theory; (b) when the blocking ion occupies the blocking site, ions normally permeant in the channel cannot pass through, or their rate of permeation becomes very small; (c) other ions do not interfere with the binding of the blocking ion to the blocking site; (d) all channels being considered are open, and the current carried by blocking ions is negligible; (f) the system is in a steady state of block, that is the time constants for channel opening are long compared to the time the blocking ion takes to reach a steady-state concentration in the channel. The WoodhuU model then yields the relationship io / ib = 1 + ( [B] / Kd (0)) exp ( zbWF/RT) where i0 / ib is the ratio of single channel currents in the absence (io) and in the presence (ib) of blocker; [B] is the concentration of the blocking ion; Kd (0) is the dissociation constant of the blocking ion at 0 mV; z is the valence of the blocking ion; V is the membrane potential; 5 is the fraction of the membrane field felt by the blocking ion at its binding site, sometimes called the "effective valence" (WoodhuU 1973; Yellen 1984); and F, R, and Tare the Faraday constant, the gas constant, and the absolute temperature, respectively. RT / F is 25.4 mV at 22 °C. A plot of In (i0 / ib-l) versus V should therefore yield a straight line of slope 6F/RT. 39 The value of 8 provides a measure of how steeply voltage-dependent the blocking process is. In the present study, the Woodhull model was found to account for both the concentration-and the voltage-dependence of Mg2+j block. At [Ca2+]j =100 p.M, the best fits to the data were obtained using Kd = 6.4 mM, 8 = 0.10 at 1 mM [Mg2+]j, and Kd = 6.6 mM, 8 = 0.08 at 2 mM [Mg2+]j (Fig. 7). These results suggest that, in the presence of high [Ca2+];, the magnesium ion traverses only a small part of the membrane field to reach its binding sites. At 15 nM [Ca2+]j, however, the best fit to the data was obtained using Kd = 10.7 mM and 8 =0.30 for the block by 2 mM [Mg2+]j (Fig. 8). The high value of 8 indicates that Mg2+( ions must traverse further through the membrane field to block BK channel currents, under conditions of low [Ca2+]j. Fig. 9 shows a plot of In (i0 / ib - 1) versus V for block by 2 mM [Mg2+]j, in the presence of 15 or 100 |iM [Ca2+]j. The adequate straight line fits to these data accord with the linear relationship predicted from the Woodhull model of channel block. 3.3 Effects Of Mg2+j On The Open Probability Of BK Channels 3.3.1 Mg2*, enhances BK channel activation in a concentration-dependent manner In addition to its blocking effect on BK channel currents, Mg2+j was also found to increase the open probability, P0 of these channels, under certain conditions. Fig. 10 shows representative BK channel currents recorded in a patch exposed to 0 (control), 0.5 and 2 mM [Mg2+]j at V = -60 mV and [Ca2+]j = 1 |J,M. On exposure to 0.5 mM [Mg2+]j, P0 increased from 0.0039 to 0.0067 (a 1.7 fold change, n=6, P<0.01, ANOVA). Exposure to 2 mM [Mg2+]f caused P0 to further increase to 0.017 (a 4.4 fold increase over the original P„, n=6, P<0.01, ANOVA). These results show that Mg2+f enhanced the open probability of BK channels in a 40 Fig. 7 The Woodhull model accounts for concentration- and voltage-dependent Mg2+; block of BK currents at 100 nM [Ca2+]L Patches were exposed to symmetrical 140 mM K+ solutions and 100 |iM [Ca2+]j. The pipette solution contained 50 nM [Ca2+]0. The membrane was voltage-clamped to potentials in the range -80 to +80 mV. The control I-V plot (0 mM [Mg2+]j) was fitted using least squares regression by the indicated straight line. The curved lines plot best fits using the Woodhull model to data for 1 and 2 mM [Mg2+]j. The fit parameters for equation ib / i0 = 1 + ( [B] / Kd (0) ) exp ( z6VF / RT ) were: at 1 mM [Mg2+]i} Kd (0) = 6.4 mM, 8 = 0.10; at 2 mM [Mg2+]h Kd (0) = 6.6 mM, 5 = 0.08. 41 i (pA) 20 1 mM [Mg + ] . K, = 6.4 mM Q 6 = 0.10 2 mM [Mg2 +] . K^ = 6.6 mM a 15 -10 5 -Y O / V 0.08 L A. _L _L -0 1 0 0 - 8 0 - 6 0 - 4 0 - 2 0 ^ 0 - 5 J L _L 20 4 0 60 80 100 V ( m V ) - 1 0 - 1 5 - 2 0 L 0 mM [Mg2 +] . 2+-O 1 mM [Mg ]. 2+. 2 mM [Mg ]. 42 Fig. 8 The Woodhull model accounts for the voltage-dependent Mg2+, block of BK currents at 15 \xM [Ca2+], Patches were exposed to symmetrical 140 mM K+ solutions and 15 |iM [Ca2+]j. The pipette solution contained 50 nM [Ca2+]0. The membrane was voltage-clamped to potentials in the range -80 to +80 mV. The control I-V plot was fitted using least squares regression by the indicated straight line. The curved line plots the best fit using the Woodhull model for data obtained in the presence of 2 mM [Mg2+]j The fit parameters for equation ib / i0 = 1 + ( [B] / Kd (0) ) exp (zSVF / RT) were : Kd (0) = 10.7 mM, 8 = 0.30. 43 i (pA) - 1 0 0 - 8 0 - 6 0 - 4 0 - 2 0 ^ C 20 40 60 80 100 V (mV) • Control o 2 mM [Mg ]. K. = 10.7 mM d 6 - 0.30 - 2 0 44 Fig. 9 The block of BK channel currents by 2 mM flVIg2*], fulfills a prediction of the Woodhull equation. Patches were exposed to symmetrical 140 mM K+ solutions and voltage-clamped to potentials in the range of -80 to +80 mV. Data for block by 2 mM [Mg2+]j in the presence of 15 |iM (closed circles) and 100 |iM [Ca2+]j (open circles) were ploted as In (i(0) / i ^ - 1 ) versus membrane potential, V. Here i(0) was the BK channel current seen in the absence of Mg2+j and i ^ was the current seen in the presence of the divalent cation. The straight lines, fitted by least squares regression, correspond to the values of Kd = 10.7 mM, 8 = 0.30 at 15 |iM [Ca2+]f, and Kd = 6.6 mM, 8 = 0.08 at 100 |xM [Ca2+]j. 45 2 1 0 - 1 - 2 - 3 - 4 - 5 - 6 ---• I o • 100 nM [Ca2+]. 15 • i r 2 + , luM [Ca ]. O __^—-^/> • / ^ i i i i / • ^ * — " O i i i i 1 0 0 - 8 0 - 6 0 - 4 0 - 2 0 0 20 40 60 80 100 V (mV) 46 Fig. 10 Effects of 0.5 and 2 mM [Mg2*], on the open probability of single BK channels recorded at V = -60 mV and [Ca2+], = 1 |iM This figure shows representative single BK channel currents, recorded in one patch exposed to symmetrical 140 mM K+ solutions, and voltage-clamped at V = -60 mV, in the presence of 1 [iM [Ca2+]j. During the control period, Pc of 0.0039 was calculated. Adding 0.5 mM [Mg2+]j to the bath solution increased P0 to 0.0067. Exposure to 2 mM [Mg2+]j caused P0 to increase to 0.017. These effects of [Mg2+]j reversed on washout of the magnesium ions (P0 = 0.0042). These P0 estimates were calculated from data segments containing 500 to 1000 openings. The dash lines indicate the average open channel currents in this patch, calculated as the mean of 500 to 1000 openings. The mean values were -13.6 pA (control), -13.5 pA (0.5 mM [Mg2+]j, -13.3 pA (2 mM [Mg2+]j) and -13.3 pA (wash). Note that the mean amplitude of these currents was unchanged by Mg2+j. "0" represents closed channel current level and "1" represents open channel current level. Bandwidth of recording DC-2 kHz. 47 o C o n t r o l o 0 . 5 mM [Mg^j j o 2 mM [Mg2*],-o Wash < Q. O O 100ms 48 concentration-dependent manner. No changes in the mean amplitude of BK channel currents was seen during these recordings. P„ returned to control values on washout of Mg2+f (Fig. 10). 3.3.2 Mg2*, enhancement of P0 is Ca2+f dependent The effect of [Ca2+]j on Mg2+j enhancement of P0 was studied in patches voltage- clamped to -60 mV and exposed to 1, 15 or 100 |J.M [Ca2+]j. As shown in Table 4, in the presence of 1 and 15 |iM [Ca2+]j, the mean control values of P0 without Mg2+f were 0.0039 and 0.0064, respectively. Addition of 2 mM [Mg2+]j increased P0 from 0.0039 to 0.017 (a 4.4 fold change) in 1 nM [Ca2+]j (n=6, P < 0.01, ANOVA), and from 0.0064 to 0.043 (a 6.7 fold increase) in 15 |iM [Ca2+]j (n=6, P < 0.01, ANOVA). The increase in P0 caused by 2 mM [Mg2+]; in the presence of 15 |iM [Ca2+]j was higher than that produced by 2 mM [Mg2+]j in the presence of 1 |iM [Ca2+]j (P < 0.05, ANOVA). However, as shown in Table 5, concentrations of Mg2+j ranging from 0.5 to 8 mM did not alter P0 in patches exposed to 100 |iM [Ca2+]j and voltage-clamped to V - -60 mV (n=6-8, P>0.05, ANOVA). 3.3.3 Effect of Mg2+, on the voltage-dependence of P0 in BK channels To quantify the effects of Mg2+j on the activation of BK channels by membrane voltage, the dependence of P0 on membrane potential was studied in the absence and presence of Mg2+;. These experiments were carried out using [Ca2+]j =15 |iM. In both the absence and presence of Mg2+j, the relationship between P0 and V was well described by the Boltzmann equation p^n+expc-zsrcv-vj)]-1 where AT is a slope factor, describing the steepness of the voltage dependence, and V0 is the 49 Table 4. Effects of 2 mM IMg2^ on the open probability of BK channels recorded at membrane potential of -60 mV, in the presence of 1 and 15 \xM [Ca2^. * P0 was significantly increased compared to control (P<0.01, ANOVA). ** P0 was significantly increased compared to control (P<0.01, ANOVA). t The mean increase in P0 was higher for the 15 |iM [Ca2+]i data group than in the 1 |J.M [Ca2+]i data group (P<0.01, ANOVA). Patch No. 1 2 3 4 5 6 Control 0.0030 0.0066 0.0021 0.0034 0.0039 0.0044 Open 1 \xM [Ca2+]i 2mM [Mg2+]i 0.017 0.016 0.007 0.018 0.017 0.023 Times increase inP0 5.7 2.4 3.3 5.3 4.4 5.2 Probability, P0 Control 0.0061 0.0043 0.0080 0.0052 0.0110 0.0030 15 \iM [Ca2+]j 2mM [Mg2+]; 0.038 0.040 0.052 0.029 0.061 0.020 Times increase inP0 6.2 9.4 6.5 5.6 5.5 6.7 mean ±s.e.m. 0.0039 0.017* 4.4f ±0.0006 ±0.0022 ±0.53 0.0064 0.043** 6.7t ±0.0012 ±0.0062 ±0.58 50 Table 5. Lack of effect of [Mg2*], on the open probability of BK channels in the presence of 100 |iM [Ca2+],. Patches were voltage-clamped at -60 mV and exposed to symmetrical 140 mM K+ solutions, with [Ca2+]j =100 nM. The pipette solution contained 50 nM [Ca2+]0. "n" represents the number of patches from which the mean value was determined. "NS" indicates that data obtained with Mg2+j present were not significantly different from control data ( P > 0.05, ANOVA). Open probability, P0 [Mg^iCmM) 0.5 (n=6) 1 (n=7) 2 (n=8) 4 (n=8) 8 (n=8) Control 0.68 ±0.11 0.56 ±0.10 0.57 ±0.11 0.69 ±0.08 0.64 ±0.09 Mg2+i 0.77 ±0.08 0.57±0.12 0.61 ±0.14 0.67 ±0.11 0.60 ±0.11 Wash 0.62 ±0.10 0.58 ±0.11 0.58 ±0.14 0.65 ±0.13 0.63 ±0.10 ANOVA NS NS NS NS NS 5 1 voltage at which P0 = 0.5. As shown in Fig. 11,2 mM [Mg2+]j increased P0 by shifting the activation curve of BK channels leftward by 29 mV along the voltage axis, without any change in the value of K. Therefore, the ability of Mg2+f to enhance P0 in BK channels was not itself a voltage-dependent process. 52 Fig. 11 Effect of 2 mM [Mg2*], on the activation of BK channels by membrane voltage in the presence of 15 |j,M [Ca2+], Patches were exposed to symmetrical 140 mM K+ solutions, and [Ca2+]i = 15 |iM, [Ca2+]0 = 50 nM. In both the absence and the presence of 2 mM [Mg2+]i5 the relation between open probability, P0 and voltage, V was well described by the Boltzmann equation: P0 = [ 1 + exp (-K ( V - V 0 ) ) ] _1, where K is a constant, and V0 is the potential at which P„ = 0.5. For the control data (open circles), the best fit curve was obtained using K = 0.06 mV'1 and V0 = +20 mV. In the presence of 2 mM [Mg2+]( (closed circles), K = 0.06 mV1 and V0 = -9 mV provided the best fit to the data. 53 2 mM [Mg2 +]. K = 0.06 mV V = - 9 mV o - 1 0 0 - 8 0 - 6 0 -40 - 2 0 0 20 40 60 80 100 V (mV) 54 Chapter 4 DISCUSSION 4.1 Effects Of Mg2+j On BK Channel Currents 4.1.1 Mg2*, blocks BK channel currents in a concentration- and voltage-dependent manner It was found in this study that Mg2+j reduced the amplitude of single BK channel currents in cultured rat cerebrovascular smooth muscle cells (CVSMCs). The reduction in BK channel currents caused by magnesium ions can be referred to as Mg2+j block, without implying a specific mechanism. The present results showed that this Mg2+j block was reversible and occurred in a concentration-dependent manner. When recorded at [Ca2+]j = 100 p,M, the Mg2+j block was present at negative potentials and increased in magnitude as the membrane voltage was made more positive. However, when [Ca2+]j was reduced to 15 |iM, Mg2+; had no effect on the amplitude of inward currents, but block was evident for outward currents. Magnesium ions have been reported to act as blocking particles in various types of ion channels. Depending upon the ion channel being considered, Mg2+ can enter the pore from either the external or the intracellular membrane surface. Mg2+ block of ion flux can occur by interaction with high affinity sites (with a Kd in the range of 10"6 to 10"5 M) on the extracellular side of the pore, as in the case of N-methyl-D-aspartate (NMDA) receptor channels (Mayer and Westbrook 1987; Johnson and Ascher 1990), or at the cytoplasmic side of a channel, as in the case of inwardly-rectifying potassium channels (Vandenberg 1987; Matsuda 1988; Horie and Irisawa 1989). Mg2+ block can also occur through interaction with 55 low affinity sites (Kd values in the order of 10 "3 M), as exemplified by blocking sites in voltage-dependent sodium channels (Albitz et al. 1990; Pusch et al. 1989), Ca2+ channels (Lansman et al. 1986; Wu and Lipsius 1990), and ATP-sensitive K+ channels (Horie et al. 1987). The data in this study show that Mg2+j block of BK channels involved an interaction with low affinity sites, since millimolar levels of Mg2+f were required to produce a 50% reduction in BK channel current amplitude. 4.1.2 Mg2+, acts as a fast blocker of single BK channel currents In general, channel blockers produce a variety of changes in the appearance of single channel currents, an observation that may be explained by supposing that individual blockers obstruct channel currents for different lengths of time, relative to the bandwidth of the recording system. On this basis, three classes of channel blockers can be distinguished (Yellen 1984; Hille 1992). Slow blockers produce clearly resolved interruptions in single channel currents, so that each blocking event looks like a closing of the channel. An intermediate class of blockers (flicker blockers) produces rapid fluctuations in current during a channel opening. These are the result of interruptions long enough to detect, but too brief to resolve as full channel closures. Fast blockers produce frequent, extremely brief interruptions of the channel current which can only be detected as an apparent reduction in the mean level of open channel current. Otherwise, the channels appear to be gating normally. This study showed that the Mg2+j block of BK channel currents was graded, and that there was no obvious increase in open channel noise or change in the open probability of BK channels in the presence of effective concentrations of Mg2+f. The Mg2+j block manifested itself 56 simply as a reduction in the mean open channel current. The blocking and unblocking events must therefore be much too fast to detect at a bandwidth of DC-2 kHz. Hence, this process can be considered to be a fast block, as defined by Yellen (1984). 4.1.3 Possible mechanisms to account for the Mg2*, block of BK channel currents Block of current flow in ion channels could occur by several mechanisms, including physical blockade of the channel pore by a penetrating ion (WoodhuU 1973; Yellen 1984), allosteric interactions affecting the ion conduction pathway (Armstrong 1971; Armstrong and Hille 1972), the screening of negative surface charges (Dani 1986; Imoto et al. 1988; MacKinnon and Miller 1989), and competitive interaction between the blocker and permeant ions in the channel vestibule (Ferguson 1991). The WoodhuU model used in this study is an example of the first of these classes of mechanisms. This model has been found to describe the block of BK channels by H+ (WoodhuU 1973), Na+ (Yellen 1984), TEA+ (Blatz and Magleby 1984; Wang and Mathers 1993), and other quaternary ammonium ions (Villarroel et al. 1988). In the present study, the WoodhuU model was found to account for the concentration- and voltage-dependent Mg2+S block of BK channel currents obtained in the presence of 100 |iM [Ca2+]j. As expected from the model, similar Kd and 8 values were obtained for the block by both 1 mM and 2 mM Mg2+j. However, when [Ca2+]f was lowered to 15 \iM, the observed block caused by 2 mM [Mg2+]j could not be well fitted by the same parameters as were employed for the data obtained at [Ca2+]j = 100 \xM. These changes in the values of Kd and 8 suggest that Ca2+j alters the extent to which Mg2+f ions must penetrate the membrane field, 57 in order to reach their binding sites. In addition, Ca2+j also changed the affinity of these binding sites for magnesium ions. These phenomena are not predicted by simple application of the Woodhull model. A possible explanation for this discrepancy is that increasing [Ca2+]f stabilizes a new conformation of the BK channel protein, in which the Mg2+j binding sites are relocated closer to the inner membrane face, thereby reducing the voltage-dependency of channel block. Evidence that high internal calcium concentrations do indeed alter the conformation of BK channels has been obtained from study of BK channel kinetics. It has been shown that high levels of [Ca2+]j stabilize the channel in the open state and favour the occurrence of long-duration channel openings (Wang and Mathers 1993). An entirely distinct hypothesis for channel block would propose that the blocking species binds to a site on the channel protein and allosterically alters the conformation of this protein. This binding site need not itself reside in the membrane electric field, and may perhaps not even be in the pore. In cases in which the binding site is outside the membrane field, this field is presumed to act directly on the channel macromolecule, altering the affinity or availability of the target site for the blocking species. Binding of blocker to this site would cause the pore to block itself in an unspecified manner. For example, binding of the blocker could stabilize closed conformational states of the channel. This mechanism would again produce a voltage-dependent block (Armstrong 1971; Armstrong and Hille 1972). An allosteric model of Mg2+j block cannot be rigorously excluded by this study. However, allosteric models imply that the blocking species should preferentially interact with some conformations of the channel protein, thereby altering the kinetic behaviour of the channel, and likely also changing the channel open probability (Demo and Yellen 1992; Hille 1992). 58 However, the present study showed that Mg2+S could reduce single BK channel currents under conditions in which P0 was unaltered. A more rigorous assessment of the allosteric model will require kinetic analysis of BK channels in the presence of blocking concentrations of Mg2+j. A third mechanism which could account for Mg2+j block of single BK channel currents is one in which Mg2+j screens negative surface charges at the cytoplasmic membrane surface, rather than binding to a specific target site. In this model, channel proteins are seen as possessing a small net negative charge in their internal and external vestibules, which attracts a layer of cations to these regions (Hille et al. 1975; Guy 1984; Finer-Moore and Stroud 1984). The influence of blocking cations on current flow in channel proteins is attributed to their ability to screen these fixed negative charges on the inner and outer aspects of the channel protein (Dani 1986; Villarroel and Eisenman 1987; 1989). Imoto et al. (1988) have found that eliminating negative charges from the intracellular vestibule of acetylcholine receptor channels decreased the blocking effect of Mg2+j on currents in these channels. It might therefore be possible that Mg2+( reduces BK channel currents by screening negative charges at the intracellular channel vestibule, thereby decreasing the concentration of K+ available for channel permeation. The voltage-dependent nature of Mg2+f block would result from the increased entry of Mg2+ into the intracellular vestibule at positive membrane potentials. If screening of membrane surface charges is involved in the Mg2+S block of BK channel currents, then other divalent cations may be expected to exert similar blocking effects, and act with similar affinities. Evidence in support of this view has been reported in the case of BK channels of rat skeletal muscle (MacKinnon et al. 1989). However, Ferguson (1991) has reported that the Kd's for BK channel block by Mg2+j, Ca2+j and other divalent cations did in 59 fact vary by some two to threefold. However, this result need not necessarily exclude the surface charge screening model, since it is possible that the accessibility of surface charges to divalent cations is sterically limited to smaller species, rendering large cations less effective as channel blockers (Dani 1986). In the present study, the blocking effect of 2 mM [Mg2+]f in the presence of 15 \iM [Ca2+]f was some threefold more voltage-dependent than that measured in [Ca2+]j = 100 |iM. It seems unlikely that a 4% change in the total concentration of divalent cations could strongly alter the voltage-dependency of Mg2+j block, solely by the screening of negative surface charges. The fourth mechanism which may play a role in the Mg2+j block of BK channel currents is a competitive interaction between Mg2+j and K+. Decreasing the intracellular concentration of K+ enhances the block of BK channel currents by Mg2+f in rat skeletal muscle cells (Ferguson 1991). This effect could arise if Mg2+j competitively displaced K+ from the intracellular vestibule of the channel (Toyoshima and Urwin 1988; Ferguson 1991). The present study was performed using a physiologically appropriate concentration of internal K+. It is possible that a greater blocking effect of Mg2+f would have been observed using solutions containing a low concentration of internal K+. 4.2. Effects Of Mg2+j On The Activation Of BK Channels 4.2.1 Mg2*, increases the open probability of BK channels Although Mg2+j cannot by itself activate BK channels, it has been shown to increase the open probability, P0 of BK channels in the presence of Ca2+j, in a variety of preparations (Golowasch 1986; Oberhauser et al. 1988; Tabcharani and Misler 1989; McLarnon and Sawyer 60 1993). In the present study, Mg2+f was found to increase the open probability of BK channels in a concentration-dependent manner, in the presence of 1 \xM [Ca2+]j. Furthermore, in the presence of 15 |iM [Ca2+]j, the increase in P0 caused by 2 mM [Mg2+]j was greater than that seen in the presence of 1 |xM [Ca2+]j. This suggests that Mg2+j enhanced the apparent cooperativity for calcium activation of the BK channel, that is it raised the number of calcium ions required to activate each BK channel. Golowasch et al. (1986) have proposed that Mg2+S causes an allosteric conformational change which reveals additional Ca2+j binding sites in the BK channels of skeletal muscle cells. A similar process may also occur in the present preparation. However, when a high concentration of Ca2+j (100 jiM) was employed, there was no significant increase in P0 in the presence of [Mg2+]j. 4.2.2 Enhancement of BK channel activation by Mg2+, is not voltage-dependent In the present study, plots of P0 versus membrane potential for BK channels were well described by the Boltzmann relationship, using the value of slope factor, K = 0.06 mV"1 when [Ca2+]j = 15 |iM. This corresponds to an e-fold increase in P0 for a depolarization of 17 mV, in good agreement with previous results in this preparation (Wang and Mathers 1993). Benham et al. (1986) have reported that BK channels of mesenteric artery smooth muscle cells exhibit a somewhat lower voltage-dependency (an e-fold increase for a 30 mV depolarization). It was found that addition of 2 mM [Mg2+]j did not alter the value of K required for good fits of the data to the Boltzmann relationship. Apparently, Mg2+j did not alter the steepness of voltage-dependence of BK channel activation. Similar results have been obtained in previous studies on BK channels in other tissues (Golowasch et al. 1986; Oberhauser et al. 1988; 61 Tabcharam 1989). The value of K can be related to z, the gating charge which moves in the membrane field on the opening of single channel, by the expression 1 / K = kT/ ze, where k is the Boltzmann constant, T is the absolute temperature and e is the elementary charge (Hille 1992). Since kT/ e = 25.4 mV at 22 °C, this implies that z = 1.5. Probably, therefore, two elementary charges must move in the membrane field during the opening of a single BK channel in the CVSMCs membrane. This process was not altered by the presence of Mg2+j. In the presence of Mg2+j, a parallel, leftward shift was seen in the Boltzmann curves describing BK channel activation. This showed that the effect of Mg2+; on BK channel activation was itself not a voltage-dependent process, in contrast to the blocking effect of the divalent cation on single channel current, described previously. These results suggest that the sites which Mg2+S must occupy in order to increase P0 are distinct from those which are involved in blocking current flow through the open BK channel. 4.2.3 Physiological role of Mg2+, as a modulator of BK channel function in CVSMCs In the present study, 0.5 mM [Mg2+]( produced no detectable block of BK channel currents when studied at a membrane potential of -60 mV, and when using [Ca2+]j = 1 |iM. However, under the same conditions, 0.5 mM [Mg2+]j significantly increased the open probability of BK channels in CVSMCs. The question therefore arises as to the physiological relevance of these results for understanding the vasodilatory action of Mg2+ on cerebral vessels. Measurements obtained from CVSMCs have shown that [Mg2+]j is near 0.5 mM (Altura et al. 1993). Relaxed vascular smooth muscle cells typically exhibit [Ca2+]j levels in the range of 40 - 120 nM (Nabika et al. 1985; Wang et al. 1990; Wang and Mathers 1993), but may 62 show [Ca2+]j values approaching 10 nM during maximal contraction (Kuriyama et al. 1982). Therefore, the 0.5 mM [Mg2+]j and 1 ^M [Ca2+]j levels used in the present study could be considered to be within the normal physiological range. The mean resting membrane potential of CVSMCs has been reported to be -63 mV in rat middle cerebral arteries (Hirst et al. 1986), -55 mV in cat basilar arteries (Harder 1981), and -50 mV in guinea-pig basilar arteries (Fujiwara et al. 1982). At the peak of the action potential in rat CVSMCs, the membrane potential is unlikely to become more positive than about 10 mV (Hirst et al. 1986). Hence, effects of 0.5 [Mg2+]j on BK channel currents recorded at negative membrane potentials and at 1 [iM [Ca2+]j should be of physiological relevance. The present study showed that 0.5 - 2 mM [Mg2+]; did not detectably block current flow in BK channels at a membrane potential of -60 mV with [Ca2+]j = 1 |iM. In addition, no detectable block of BK channel currents was observed when patches were exposed to 15 \iM [Ca2+]j and 2 mM [Mg2+]j over the physiologically relevant range of membrane potentials (-60 mV to +10 mV). In this potential range, Mg2+; block was evident only in the presence of 100 HM [Ca2+]j. These results suggest that, under physiological conditions, the blocking action of Mg2+j on BK channel currents is of negligible importance in the regulation of channel function. Indeed, even under extreme pathological conditions, in which [Ca2+]j is raised to abnormal levels in the presence of membrane depolarization, the percentage reduction of BK channel currents caused by 1 mM [Mg2+]j is likely to remain less than 20%. In contrast to these results, 0.5 mM [Mg2+]j significantly increased the open probability of BK channels studied using [Ca2+]j = 1 |iM at membrane potentials within the physiological range. It seems likely, therefore that, under the physiological conditions, intracellular Mg2+ 63 serves to tonically facilitate BK channel activation in CVSMCs. Variations in [Mg2+]j associated with fluctuations in [Mg2+]0 could exert small but potentially significant effects on the open probability of BK channels in CVSMCs. This could in turn influence the negative feedback control of myogenic tone afforded by BK channel openings, which must be finely calibrated if excessive or insufficient repolarizing K+ currents are to be avoided. Fig. 12 proposes a scheme by which Mg2+S may contribute to the vasodilation of cerebral vessels seen in the presence of abnormally high concentrations of plasma Mg2+. Here, an increase in [Mg2+]0 reduces the inward Ca2+ current through voltage-dependent calcium channels (VDCs), and decreases the concentration of intracellular free Ca2+. This in turn decreases the Ca2+-dependent activation of contractile proteins and lowers the tonic level of BK channel activation in the cell. The increase in [Mg2+]0 also leads to an increment in [Mg2+]i, which enhances the open probability of BK channels at the available level of [Ca2+]j . This action compensates for the decreased levels of intracellular free Ca2+, further predisposing the cell to undergo relaxation. In the presence of abnormally low levels of plasma Mg2+, the associated fall in [Mg2+]j is expected to lower the Ca2+ -sensitivity of BK channels, and this effect acts synergistically with a concomitant rise in [Ca2+]j, predisposing the cell towards contraction. Under conditions of hyper- or hypomagnesemia, therefore, the facilitatory effect of [Mg2+]j on BK channel function may contribute to the development of abnormal dilation and contraction in cerebral vessels. 64 Fig. 12 A schematic diagram showing the possible contribution of Mg2+i to the relaxation of CVSMCs in the presence of abnormally high levels of plasma Mg2* [Ca2+]0 and [Ca2+]j represent the extracellular and intracellular concentrations of free Ca2+, respectively. [Mg2+]0 and [Mg2+]( represent the extracellular and intracellular concentrations of free Mg2+, respectively. VDCs indicates the voltage-dependent calcium channel, while SR denotes the sarcoplasmic reticulum. The symbol t indicates a rise in the concentration of the ion indicated, while i denotes a fall in ion concentration. The symbol •—www—* represents the contractile proteins of the CVSMC cytoplasm. The symbol © or 6 indicates activation or inhibition, respectively. See the text for further explanation. 65 66 4.3 Significance And Future Directions In view of the clinical importance of cerebrovascular diseases, and of the ability of Mg2+ to dilate cerebral vessels and prevent cerebral vasospasm, it is important that the mechanisms of Mg2+ action on cerebrovascular smooth muscle be well understood. The present study has contributed to this goal by describing a tonic, facilitatory effect of internal Mg2+ on the function of BK channels, which may play a role in setting the tone of cerebral arteries. Further experiments will be required to understand the nature of the interaction between Mg2+j and the BK channel protein. The assessment of possible allosteric models for Mg2+S action will require detailed analysis of the effects of Mg2+j on the kinetics of BK channels. 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