UBC Theses and Dissertations
Regualtion of intracellular pH in cultured postnatal rat hippocampal neurons : the potential role of a voltage-gated proton conductance Cheng, Yen May
First studied in snail neurons, voltage-gated proton conductances (g[sub H+]) 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 g[subH+] 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 g[sub H+])in intracellular pH (pH[sub i]) regulation was examined in cultured postnatal rat hippocampal neurons by i) measuring the changes in [Ca²⁺]t and pH[sub i] evoked by membrane depolarization in neurons loaded with Ca²⁺ 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 HCO₃⁻/CO₂-free, HEPES-buffered conditions. Consistent with previous reports, under control conditions (3 mM K⁺[sub o], 2 mM Ca²⁺[sub o], , pH[sub o] 7.35, 37°C), exposure to 25 - 139.5 mM [K⁺[sub o]] caused reversible increases and decreases in [Ca²⁺]i and pH[sub i], respectively. Under 0 Ca²⁺[sub 0] conditions, the same stimuli failed to affect [Ca²⁺]i but caused increases in pH[sub i] that were dependent on [K⁺[sub o]] and, thus, membrane voltage. Consistent with the properties of g[sub H+]s in other cell types, the rise in pH[sub i] was sensitive to Zn²⁺and was dependent on the transmembrane pH gradient (ΔpH[sub memb]). Increasing ΔpH[sub memb] by treatment with the protonophore FCCP prior to high [K⁺[sub o]] exposure enhanced both the rise in pH[sub i] and the inhibitory effects of Zn²⁺, suggestive of increased acid extrusion via a g[sub H+]. Under 0 Ca²⁺ 0 , pH[sub o] 7.8 conditions, the inhibitory effects of Zn²⁺ at any given ΔpH[sub memb] were further enhanced, consistent with a pH[sub o]-dependent inhibition of the putative g[sub H+] by Zn²⁺. 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 microspectrofluorimetric studies, the currents were not sensitive to Zn²⁺ or temperature. Nonetheless, together these results suggest that a g[sub H+] may be present in rat hippocampal neurons and may contribute to H⁺ efflux under depolarizing conditions.