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 (gH+) 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+o) or 20 microM veratridine in the presence of 2 mM Ca2+o (extracellular pH (pHo) constant at 7.35) caused reversible increases and decreases in intracellular free calcium concentration ([Ca2+]i) and intracellular pH (pHi), respectively. In contrast, under external Ca2+-free conditions, the same stimuli failed to affect [Ca2+]i but caused an increase in pHi, the magnitude of which was related to the [K+]o applied and the change in membrane potential. Consistent with the properties of gH+s in other cell types, the magnitude of the rise in pHi 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 (DeltapHmemb). Increasing DeltapH(memb) by pretreatment with carbonylcyanide-p-trifluoromethoxyphenylhydrazone augmented both the high-[K+]o-evoked rise in pHi and the Zn2+-sensitive component of the rise in pHi, suggestive of increased acid extrusion via a gH+. The inhibitory effect of Zn2+ at a given DeltapHmemb was further enhanced by increasing pHo from 7.35-7.8, consistent with a pHo-dependent inhibition of the putative gH+ 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.