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

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 Ca2+- and/or pH-sensitive fluorophores, transient exposures to 25-139.5 mM external K+ (K+(radical anion)) or 20 mu M veratridine in the presence of 2 mM Ca2+(radical anion) (extracellular pH (pH(radical anion)) constant at 7.35) caused reversible increases and decreases in intracellular free calcium concentration ([Ca2+](i)) and intracellular pH (pH,), respectively. In contrast, under external Ca2+-free conditions, the same stimuli failed to affect [Ca2+](i) but caused an increase in pH(i), the magnitude of which was related to the [K+](radical anion) applied and the change in membrane potential. Consistent with the properties of 9(H)(+)S in other cell types, the magnitude of the rise in pH, observed in the absence of external Ca2+ was not affected by the removal of external Na+ but was sensitive to external Zn2+ and temperature and was dependent on the measured transmembrane pH gradient (Delta pH(memb)). Increasing Delta pH(memb) by pretreatment with carbonylcyanide-p-trifluoromethoxyphenylhydrazone augmented both the high-[K+](radical anion)-evoked rise in pH(i) and the Zn2+-sensitive component of the rise in pH(i), suggestive of increased acid extrusion via a g(H)(+). The inhibitory effect of Zn2+ at a given Delta pH(memb) was further enhanced by increasing pH. from 7.35-7.8, consistent with a pH.-dependent inhibition of the putative g(H)(+) by Zn2+. 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 Zn2+ 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 Zn2+-sensitive proton conductive pathway is present in rat hippocampal neurons and contributes to H+ efflux under depolarizing conditions. (c) 2008 IBRO. Published by Elsevier Ltd. All rights reserved.