Potential contribution of a voltage-activated proton conductance to acid extrusion from rat hippocampal neurons

Abstract We examined the potential contribution of a voltage-gated proton conductance ( g H + ) to acid extrusion from cultured postnatal rat hippocampal neurons. In neurons loaded with Ca 2+ - and/or pH-sensitive fluorophores, transient exposures to 25–139.5 mM external K + (K + o ) or 20 μM veratridine in the presence of 2 mM Ca 2+ o (extracellular pH (pH o ) constant at 7.35) caused reversible increases and decreases in intracellular free calcium concentration ([Ca 2+ ] i ) and intracellular pH (pH i ), respectively. In contrast, under external Ca 2+ -free conditions, the same stimuli failed to affect [Ca 2+ ] i but caused an increase in pH i , the magnitude of which was related to the [K + ] o applied and the change in membrane potential. Consistent with the properties of g H + s in other cell types, the magnitude of the rise in pH i observed in the absence of external Ca 2+ was not affected by the removal of external Na + but was sensitive to external Zn 2+ and temperature and was dependent on the measured transmembrane pH gradient (ΔpH memb ). Increasing ΔpH memb by pretreatment with carbonylcyanide- p -trifluoromethoxyphenylhydrazone augmented both the high-[K + ] o -evoked rise in pH i and the Zn 2+ -sensitive component of the rise in pH i , suggestive of increased acid extrusion via a g H + . The inhibitory effect of Zn 2+ at a given ΔpH memb was further enhanced by increasing pH o from 7.35–7.8, consistent with a pH o -dependent inhibition of the putative g H + by Zn 2+ . Under conditions designed to isolate H + currents, a voltage-dependent outward current was recorded from whole-cell patch-clamped neurons. Although the outward current appeared to show some selectivity for protons, it was not sensitive to Zn 2+ or temperature and the H + -selective component could not be separated from a larger conductance of unknown selectivity. Nonetheless, taken together, the results suggest that a Zn 2+ -sensitive proton conductive pathway is present in rat hippocampal neurons and contributes to H + efflux under depolarizing conditions.