Effects of buffer concentration on voltage-gated H+ currents: does diffusion limit the conductance?

The single-channel proton conductance of the voltage-gated H(+)-selective channel, like that of the F0 component of the H(+)-ATPase, is nearly constant over a wide range of pH encompassing the physiological range. To examine the possible contributions of buffer diffusion and buffer-channel proton transfer reactions to this phenomenon, the effects of buffer concentration on voltage-activated H+ currents were explored in voltage-clamped rat alveolar epithelial cells. Changes in the external buffer concentration ([B]o), evaluated using the whole-cell configuration, had only small effects on H+ currents (IH). Lowering [B]o from 100 to 1 mM did not alter the voltage-activation curve or reversal potential (Vrev) but reduced IH, typically by 10-30%. Changes in internal buffer concentration ([B]i), examined in inside-out patches, usually altered IH more distinctly and subtly changed the kinetics. Overall, the effects of changing buffer concentration were small and subtle. The maximum attenuation of the single-channel H+ current at 1 mM buffer was estimated to be approximately 20% at either mouth of the H+ channel. Therefore, the rate-determining step in H+ permeation is neither deprotonation of buffer at the inner mouth of the channel nor protonation of buffer at the external surface. Evidently the rate of H+ permeation through the channel is itself small enough that diffusion of buffer in bulk solution does not directly limit the conductance significantly.