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The voltage-activated H+ selective conductance of rat alveolar epithelial cells was studied using whole-cell and excised-patch voltage-clamp techniques. The effects of substituting deuterium oxide, D2O, for water, H2O, on both the conductance and the pH dependence of gating were explored. D+ was able to permeate proton channels, but with a conductance only about 50% that of H+. The conductance in D2O was reduced more than could be accounted for by bulk solvent isotope effects (i.e., the lower mobility of D+ than H+), suggesting that D+ interacts specifically with the channel during permeation. Evidently the H+ or D+ current is not diffusion limited, and the H+ channel does not behave like a water-filled pore. This result indirectly strengthens the hypothesis that H+ (or D+) and not OH– is the ionic species carrying current. The voltage dependence of H+ channel gating characteristically is sensitive to pHo and pHi and was regulated by pDo and pDi in an analogous manner, shifting 40 mV/U change in the pD gradient. The time constant of H+ current activation was about three times slower (
act was larger) in D2O than in H2O. The size of the isotope effect is consistent with deuterium isotope effects for proton abstraction reactions, suggesting that H+ channel activation requires deprotonation of the channel. In contrast, deactivation (tail) was slowed only by a factor 
1.5 in D2O. The results are interpreted within the context of a model for the regulation of H+ channel gating by mutually exclusive protonation at internal and external sites (Cherny, V.V., V.S. Markin, and T.E. DeCoursey. 1995. J. Gen. Physiol. 105:861–896). Most of the kinetic effects of D2O can be explained if the pKa of the external regulatory site is 
0.5 pH U higher in D2O.
Key Words: ion channels • proton transport • pH • pneumocytes • membrane transport
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