Membrane topology of the amiloride-sensitive epithelial sodium channel

Peter M Snyder; Fiona J McDonald; John B Stokes; Michael J Welsh

doi:10.1016/S0021-9258(19)51094-8

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Membrane topology of the amiloride-sensitive epithelial sodium channel

Journal article

Open access

Peer reviewed

Membrane topology of the amiloride-sensitive epithelial sodium channel

Peter M Snyder, Fiona J McDonald, John B Stokes and Michael J Welsh

The Journal of biological chemistry, Vol.269(39), pp.24379-24383

09/30/1994

DOI: 10.1016/S0021-9258(19)51094-8

PMID: 7929098

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Abstract

The amiloride-sensitive epithelial sodium channel (ENaC) is involved in fluid and electrolyte absorption across a number of epithelia, and cloning of several ENaC subunits has begun to facilitate investigation of the structure, function, and regulation of this channel. Analysis of the amino acid sequence has revealed two potential membrane-spanning domains, but little else is known about the structure of ENaC. To investigate the membrane topology of one subunit, alpha rENaC, we used in vitro transcription, translation, and translocation into microsomal membranes. This generated a glycosylated protein of 93 kDa. Sequence analysis also revealed eight potential sites for N-glycosylation, six of which were found to be glycosylated (Asn190, Asn259, Asn320, Asn339, Asn424, and Asn538), indicating that they are extracellular. The C terminus was localized as intracellular based on antibody recognition and protease sensitivity of a tagged epitope at the C terminus. The N terminus was also found to be intracellular, based on its protease sensitivity. Similar results were obtained by expression in Xenopus oocytes. Together, these results support a model of alpha rENaC consisting of an intracellular N terminus and C terminus, a large N-glycosylated extracellular domain, and two membrane-spanning domains that each pass once through the plasma membrane. Because of their sequence similarity, it is likely that this structure is shared by other ENaC subunits and possibly the degenerins of Caenorhabditis elegans as well.

Amino Acid Sequence

Epithelium - drug effects

Intracellular Membranes - physiology

Xenopus laevis

Cells, Cultured

Molecular Sequence Data

Glycosylation

Sodium Channels - drug effects

Sodium Channels - physiology

Recombinant Proteins

Intracellular Membranes - chemistry

Oocytes

Animals

Membrane Potentials

Sodium Channels - chemistry

Amiloride - pharmacology

Female

Protein Conformation

Epithelium - physiology

Details

Title: Subtitle: Membrane topology of the amiloride-sensitive epithelial sodium channel
Creators: Peter M Snyder - Howard Hughes Medical Institute, Department of Internal Medicine, University of Iowa College of Medicine, Iowa City 52242
Fiona J McDonald
John B Stokes
Michael J Welsh
Resource Type: Journal article
Publication Details: The Journal of biological chemistry, Vol.269(39), pp.24379-24383
DOI: 10.1016/S0021-9258(19)51094-8
PMID: 7929098
NLM abbreviation: J Biol Chem
ISSN: 0021-9258
eISSN: 1083-351X
Publisher: United States
Grant note: HL07344 / NHLBI NIH HHS
Language: English
Date published: 09/30/1994
Academic Unit: Neurology; Molecular Physiology and Biophysics; Pulmonary, Critical Care, and Occupational Medicine; Cardiovascular Medicine; Fraternal Order of Eagles Diabetes Research Center; Neurosurgery; Medicine Administration; Internal Medicine
Record Identifier: 9984020712102771

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