Faculty Collaboration Database

Fluorescence resonance energy transfer analysis of subunit stoichiometry of the epithelial Na+ channel. J Biol Chem 2004 Jun 25;279(26):27729-34

Date

04/21/2004

Pubmed ID

15096495

DOI

10.1074/jbc.M404169200

Scopus ID

2-s2.0-3042688447 (requires institutional sign-in at Scopus site)   64 Citations

Abstract

Activity of the epithelial Na(+) channel (ENaC) is rate-limiting for Na(+) (re)absorption across electrically tight epithelia. ENaC is a heteromeric channel comprised of three subunits, alpha, beta, and gamma, with each subunit contributing to the functional channel pore. The subunit stoichiometry of ENaC remains uncertain with electrophysiology and biochemical experiments supporting both a tetramer with a 2alpha:1beta:1gamma stoichiometry and a higher ordered channel with a 3alpha:3beta:3gamma stoichiometry. Here we used an independent biophysical approach based upon fluorescence resonance energy transfer (FRET) between differentially fluorophore-tagged ENaC subunits to determine the subunit composition of mouse ENaC functionally reconstituted in Chinese hamster ovary and COS-7 cells. We found that when all three subunits were co-expressed, ENaC contained at least two of each type of subunit. Findings showing that ENaC subunits interact with similar subunits in immunoprecipitation studies are consistent with these FRET results. Upon native polyacrylamide gel electrophoresis, moreover, oligomerized ENaC runs predominantly as a single species with a molecular mass of >600 kDa. Because single ENaC subunits have a molecular mass of approximately 90 kDa, these results also agree with the FRET results. The current results as a whole, thus, are most consistent with a higher ordered channel possibly with a 3alpha:3beta:3gamma stoichiometry.

Author List

Staruschenko A, Medina JL, Patel P, Shapiro MS, Booth RE, Stockand JD

MESH terms used to index this publication - Major topics in bold

Animals
CHO Cells
COS Cells
Cricetinae
Cricetulus
Epithelium
Fluorescence Resonance Energy Transfer
Gene Expression
Mice
Microscopy, Fluorescence
Models, Molecular
Molecular Weight
Protein Subunits
Recombinant Proteins
Sodium Channels