Diversity of channels generated by different combinations of epithelial sodium channel subunits

The epithelial sodium channel is a multimeric protein formed by three homologous subunits: alpha, beta, and gamma; each subunit contains only two transmembrane domains. The level of expression of each of the subunits is markedly different in various Na+ absorbing epithelia raising the possibility that channels with different subunit composition can function in vivo. We have examined the functional properties of channels formed by the association of alpha with beta and of alpha with gamma in the Xenopus oocyte expression system using two-microelectrode voltage clamp and patch-clamp techniques. We found that alpha beta channels differ from alpha gamma channels in the following functional properties: (a) alpha beta channels expressed larger Na+ than Li+ currents (INa+/ILi+ 1.2) whereas alpha gamma channels expressed smaller Na+ than Li+ currents (INa+/ILi+ 0.55); (b) the Michaelis Menten constants (Km of activation of current by increasing concentrations of external Na+ and Li+ of alpha beta channels were larger (Km > 180 mM) than those of alpha gamma channels (Km of 35 and 50 mM, respectively); (c) single channel conductances of alpha beta channels (5.1 pS for Na+ and 4.2 pS for Li+) were smaller than those of alpha gamma channels (6.5 pS for Na+ and 10.8 pS for Li+); (d) the half-inhibition constant (Ki) of amiloride was 20-fold larger for alpha beta channels than for alpha gamma channels whereas the Ki of guanidinium was equal for both alpha beta and alpha gamma. To identify the domains in the channel subunits involved in amiloride binding, we constructed several chimeras that contained the amino terminus of the gamma subunit and the carboxy terminus of the beta subunit. A stretch of 15 amino acids, immediately before the second transmembrane domain of the beta subunit, was identified as the domain conferring lower amiloride affinity to the alpha beta channels. We provide evidence for the existence of two distinct binding sites for the amiloride molecule: one for the guanidium moiety and another for the pyrazine ring. At least two subunits alpha with beta or gamma contribute to these binding sites. Finally, we show that the most likely stoichiometry of alpha beta and alpha gamma channels is 1 alpha: 1 beta and 1 alpha: 1 gamma, respectively.