Maturation of the Epithelial Na+ Channel Involves Proteolytic Processing of the α- and γ-Subunits*

  1. Thomas R. Kleyman
  1. Department of Medicine, Renal-Electrolyte Division, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213
  1. To whom correspondence should be addressed. Tel.: 412-647-3121; Fax: 412-648-9166; E-mail: kleyman{at}


The epithelial Na+ channel (ENaC) is a tetramer of two α-, one β-, and one γ-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 α-(95 kDa), β-(96 kDa), and γ-subunits (93 kDa) each produced a single band on SDS gels by immunoblotting. However, co-expression of αβγENaC subunits revealed a second band for each subunit (65 kDa for α, 110 kDa for β, and 75 kDa for γ) 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 α- and γ-subunits is also consistent with proteolytic cleavage. By using α- and γ-subunits with epitope tags at both the amino and carboxyl termini, proteolytic processing of the α- and γ-subunits was confirmed by isolation of an additional epitope-tagged fragment from the amino terminus (30 kDa for α and 18 kDa for γ) 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.

  • Received July 1, 2003.
  • Revision received July 17, 2003.
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This Article

  1. The Journal of Biological Chemistry 278, 37073-37082.
  1. All Versions of this Article:
    1. M307003200v1
    2. 278/39/37073 (most recent)

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