Na+-H+ antiport detected through hydrogen ion currents in rat alveolar epithelial cells and human neutrophils

Abstract

Voltage-activated H+-selective currents were studied in cultured adult rat alveolar epithelial cells and in human neutrophils using the whole-cell configuration of the patch-clamp technique. The H+ conductance, g(H), although highly selective for protons, was modulated by monovalent cations. In Na+ and to a smaller extent in Li+ solutions, H+ currents were depressed substantially and the voltage dependence of activation of the g(H) shifted to more positive potentials, when compared with the 'inert' cation tetramethylammonium (TMA+). The reversal potential of the g(H), V(rev), was more positive in Na+ solutions than in inert ion solutions. Amiloride at 100 μM inhibited H+ currents in the presence of all cations studied except Li+ and Na+, in which it increased H+ currents and shifted their voltage- dependence and V(rev) to more negative potentials. The more specific Na+- H+ exchange inhibitor dimethylamiloride (DMA) at 10 μM similarly reversed most of the suppression of the g(H) by Na+ and Li+. Neither 500 μM amiloride nor 200 μM DMA added internally via the pipette solution were effective. Distinct inhibition of the g(H) was observed with 1% [Na+](o), indicating a mechanism with high sensitivity. Finally, the effects of Na+ and their reversal by amiloride were large when the proton gradient was outward (pH(o)//pH(i) 7//5.5), smaller when the proton gradient was abolished (pH 7//7), and absent when the proton gradient was inward (pH 6//7). We propose that the effects of Na+ and Li+ are due to their transport by the Na+-H+ antiporter, which is present in both cell types studied. Electrically silent H+ efflux through the antiporter would increase pH(i) and possibly decrease local pH(o), both of which modulate the g(H) in a similar manner: reducing the H+ currents at a given potential and shifting their voltage-dependence to more positive potentials. A simple diffusion model suggests that Na+-H+ antiport could deplete intracellular protonated buffer to the extent observed. Evidently the Na+-H+ antiporter functions in perfused cells, and its operation results in pH changes which can be detected using the g(H) as a physiological sensor. Thus, the properties of the g(H) can be exploited to study Na+-H+ antiport in single cells under controlled conditions.