Deuterium isotope effects on permeation and gating of proton channels in rat alveolar epithelium

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 DC 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 pH(0) and pH(i) and was regulated by pD(0) and pD(i) in an analogous manner, shifting 40 mV/U change in the pD gradient, The time constant of HC current activation was about three times slower (tau(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 Hi channel activation requires deprotonation of the channel, In contrast, deactivation (tau tail) was slowed only by a factor less than or equal to 1.5 in D2O The results are interpreted within the context of a model for the regulation of Hi 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 pK(a) of the external regulatory site is similar to 0.5 pH U higher in D(2)0.