title: Membrane topology of the epithelial sodium channel in intact cells.
abstract: The highly selective amiloride-sensitive epithelial sodium channel is formed of three homologous subunits termed alpha-, beta-, and gamma-rENaC. Each subunit has two putative transmembrane domains (M1 and M2), yielding a protein with a large (approximately 50 kDa) hydrophilic loop (between M1 and M2) and short hydrophilic NH2- and COOH-termini (9 and 10 kDa). All three subunits are glycosylated in a cell-free translation assay, demonstrating that they share in vitro a common pattern of membrane insertion. The membrane topology of the alpha-rENaC subunit in intact cells was studied in Xenopus laevis oocytes. We demonstrate that 1) all six potential N-linked glycosylation sites (N190, N259, N320, N339, N424, and N538) of the large hydrophilic loop are used in intact cells; 2) the glycosylation of alpha-rENaC does not play a significant role in the functional expression of the channel; and 3) the two hydrophobic domains M1 (A109-F131) and M2 (S588-L612) serve in intact cells as start- and stop-transfer signals, respectively. We conclude that alpha-rENaC spans the membrane twice with the short NH2- and COOH-terminal ends on the cytoplasmic side and a large hydrophilic loop in the extracellular space.
authors: Institut de Pharmacologie et de Toxicologie, Université de Lausanne, Switzerland.
authors: C M
authors: CM
authors: Canessa
authors: A M
authors: AM
authors: Merillat
authors: B C
authors: BC
authors: Rossier
mesh_terms: D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D002462:Cell Membrane / Q000737:chemistry*; D003599:Cytoskeleton / Q000502:physiology; D004285:Dogs; D006031:Glycosylation; D016297:Mutagenesis, Site-Directed; D011817:Rabbits; D015222:Sodium Channels / Q000737:chemistry* / Q000502:physiology; D013329:Structure-Activity Relationship; D014982:Xenopus laevis
chemical_list: D015222:Sodium Channels; D000584:Amiloride