MEMBRANE TOPOLOGY OF THE AMILORIDE-SENSITIVE EPITHELIAL SODIUM-CHANNEL

The amiloride-sensitive epithelial sodium channel (ENaC) is involved i n fluid and electrolyte absorption across a number of epithelia, and c loning of several ENaC subunits has begun to facilitate investigation of the structure, function, and regulation of this channel. Analysis o f the amino acid sequence has revealed two potential membrane-spanning domains, but little else is known about the structure of ENaC. To inv estigate the membrane topology of one subunit, alpha rENaC, we used in vitro transcription, translation, and translocation into microsomal m embranes. This generated a glycosylated protein of 93 kDa. Sequence an alysis also revealed eight potential sites for N-glycosylation, six of which were found to be glycosylated (Asn(190), Asn(259), Asn(320), As n(339) Asn(424), and Asn(538)), indicating that they are extracellular . The C terminus was localized as intracellular based on antibody reco gnition and protease sensitivity of a tagged epitope at the C terminus . The N terminus was also found to be intracellular, based on its prot ease sensitivity. Similar results were obtained by expression in Xenop us oocytes. Together, these results support a model of alpha rENaC con sisting of an intracellular N terminus and C terminus, a large N-glyco sylated extracellular domain, and two membrane-spanning domains that e ach pass once through the plasma membrane. Because of their sequence s imilarity, it is likely that this structure is shared by other ENaC su bunits and possibly the degenerins of Caenorhabditis elegans as well.