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.