Protons regulate electrogenic sodium absorption in a variety of epithelia, including the cortical collecting duct, frog skin, and urinary bladder. Recently, three subunits (, , ) coding for the epithelial sodium channel (ENaC) were cloned. However, it is not known whether pH regulates Na⁺ channels directly by interacting with one of the three ENaC subunits or indirectly by interacting with a regulatory protein. As a first step to identifying the molecular mechanisms of proton-mediated regulation of apical membrane Na⁺ permeability in epithelia, we examined the effect of pH on the biophysical properties of ENaC. To this end, we expressed various combinations of -, -, and -subunits of ENaC in Xenopus oocytes and studied ENaC currents by the two-electrode voltage-clamp and patch-clamp techniques. In addition, the effect of pH on the -ENaC subunit was examined in planar lipid bilayers. We report that ,,-ENaC currents were regulated by changes in intracellular pH (pH_i) but not by changes in extracellular pH (pH_o). Acidification reduced and alkalization increased channel activity by a voltage-independent mechanism. Moreover, a reduction of pH_i reduced single-channel open probability, reduced single-channel open time, and increased single-channel closed time without altering single-channel conductance. Acidification of the cytoplasmic solution also inhibited ,-ENaC, ,-ENaC, and -ENaC currents. We conclude that pH_i but not pH_o regulates ENaC and that the -ENaC subunit is regulated directly by pH_i.

cortical collecting duct; amiloride; patch clamp; hydrogen ion; Xenopus oocyte

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