Intracellular H+ regulates the alpha-subunit of ENaC, the epithelial Na+ channel

Protons regulate electrogenic sodium absorption in a variety of epithelia, including the cortical collecting duct, frog skin, and urinary bladder. Rec ently three subunits (alpha, beta, gamma) coding for the epithelial sodium channel (ENaC) were cloned. However, it is not known whether pH regulates N a+ channels directly by interacting with one of the three ENaC subunits or indirectly by interacting with a regulatory protein. As a first step to ide ntifying the molecular mechanisms of proton-mediated regulation of apical m embrane Na+ permeability in epithelia, we examined the effect of pH on the biophysical properties of ENaC. To this end, we expressed various combinati ons of alpha-, beta-, and gamma-subunits of ENaC in Xenopus oocytes and stu died ENaC currents by the two-electrode voltage-clamp and patch-clamp techn iques. In addition, the effect of pH on the alpha-ENaC subunit was examined in planar lipid bilayers. We report that alpha,beta,gamma-ENaC currents we re regulated by changes in intracellular pH (pH(i)) but not by changes in e xtracellular pH (pH(o)). Acidification reduced and alkalization increased c hannel activity by a voltage-independent mechanism. Moreover, a reduction o f pH(i) reduced single-channel open probability, reduced single-channel ope n time, and increased single-channel closed time without altering single-ch annel conductance. Acidification of the cytoplasmic solution also inhibited alpha,beta-ENaC, alpha,gamma-ENaC, and alpha-ENaC currents. We conclude th at pH(i) but not pH(o) regulates ENaC and that the alpha-ENaC subunit is re gulated directly by pH(i).