Structural basis for the function and regulation of the epithelial sodium channel

Epithelial sodium channels (ENaC) mediate sodium transport across epithelia. Functional channels are assembled from three homologous &agr;, β and γ subunits with ∼30% similarity in amino acid sequence. Mutations in different subunits of this channel are responsible for diseases including Liddle's syndrome and type I pseudohypoaldosteronism. ENaC is synthesized on the ER membrane, aquires complex N-linked glycosylation in the Golgi and is trafficked to the plasma membrane where it is activated upon cleavage by numerous membrane-anchored and/or soluble serine proteases secreted into the extracellular milieu. Although it has been established that exogenous expression of all three subunits in oocytes is required for robust channel activity, the number and stoichiometry of subunits comprising one functional channel remains unclear. Different biophysical and electrophysiological studies have concluded that ENaC assembles as a trimer or a tetramer with possible larger molecular weight oligomers arising from higher order assembly of trimers or tetramers. Due to the lack of structural information on ENaC, the molecular aspects of channel activation and regulation of function remain less well understood. In the current study, using a battery of computational and experimental techniques, we address specific questions concerning the structural aspects of regulation of channel activation and function by constructing a structural model of the channel. Significant advances through this study include determination of oligomerization state of ENaC using native gel electrophoresis and identification of allosteric communication within the channel and modulating channel activity by rational mutagenesis of the identified allosteric sites. In this study, we conclude that ENaC assembles as both trimers and tetramers in the same cell. The amount of tetramers correlates well with increase in function and more importantly, the gamma subunit plays a crucial role in the formation of tetramers in oocytes. We believe that the results presented here would be immensely helpful in the future for understanding the cellular aspects of channel regulation and function at the molecular level.