title: Maturation of the epithelial Na+ channel involves proteolytic processing of the alpha- and gamma-subunits.
abstract: The epithelial Na+ channel (ENaC) is a tetramer of two alpha-, one beta-, and one gamma-subunit, but little is known about its assembly and processing. Because co-expression of mouse ENaC subunits with three different carboxyl-terminal epitope tags produced an amiloride-sensitive sodium current in oocytes, these tagged subunits were expressed in both Chinese hamster ovary or Madin-Darby canine kidney type 1 epithelial cells for further study. When expressed alone alpha-(95 kDa), beta-(96 kDa), and gamma-subunits (93 kDa) each produced a single band on SDS gels by immunoblotting. However, co-expression of alphabetagammaENaC subunits revealed a second band for each subunit (65 kDa for alpha, 110 kDa for beta, and 75 kDa for gamma) that exhibited N-glycans that had been processed to complex type based on sensitivity to treatment with neuraminidase, resistance to cleavage by endoglycosidase H, and GalNAc-independent labeling with [3H]Gal in glycosylation-defective Chinese hamster ovary cells (ldlD). The smaller size of the processed alpha- and gamma-subunits is also consistent with proteolytic cleavage. By using alpha- and gamma-subunits with epitope tags at both the amino and carboxyl termini, proteolytic processing of the alpha- and gamma-subunits was confirmed by isolation of an additional epitope-tagged fragment from the amino terminus (30 kDa for alpha and 18 kDa for gamma) consistent with cleavage within the extracellular loop. The fragments remain stably associated with the channel as shown by immunoblotting of co-immunoprecipitates, suggesting that proteolytic cleavage represents maturation rather than degradation of the channel.
authors.affiliation: Department of Medicine, Renal-Electrolyte Division, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213, USA.
authors.forename: Rebecca P
authors.initials: RP
authors.lastname: Hughey
authors.forename: Gunhild M
authors.initials: GM
authors.lastname: Mueller
authors.forename: James B
authors.initials: JB
authors.lastname: Bruns
authors.forename: Carol L
authors.initials: CL
authors.lastname: Kinlough
authors.forename: Paul A
authors.initials: PA
authors.lastname: Poland
authors.forename: Keri L
authors.initials: KL
authors.lastname: Harkleroad
authors.forename: Marcelo D
authors.initials: MD
authors.lastname: Carattino
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Kleyman
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004285:Dogs; D053503:Epithelial Sodium Channels; D005260:Female; D008970:Molecular Weight; D011134:Polysaccharides / Q000378:metabolism; D021122:Protein Subunits; D015222:Sodium Channels / Q000737:chemistry / Q000378:metabolism*; D014981:Xenopus
chemical_list: D053503:Epithelial Sodium Channels; D011134:Polysaccharides; D021122:Protein Subunits; D015222:Sodium Channels