Fluorescence resonance energy transfer analysis of subunit stoichiometry of the epithelial Na+ channel

Alexander Staruschenko, Jorge L. Medina, Pravina Patel, Mark S Shapiro, Rachell E. Booth, James D Stockand

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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, α, β, and γ, 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 2α:1β:1γ stoichiometry and a higher ordered channel with a 3α:3β:3γ 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 ∼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 3α:3β:3γ stoichiometry.

Original languageEnglish (US)
Pages (from-to)27729-27734
Number of pages6
JournalJournal of Biological Chemistry
Volume279
Issue number26
DOIs
StatePublished - Jun 25 2004

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Epithelial Sodium Channels
Fluorescence Resonance Energy Transfer
Stoichiometry
Molecular mass
Electrophysiology
Native Polyacrylamide Gel Electrophoresis
Fluorophores
COS Cells
Cricetulus
Electrophoresis
Immunoprecipitation
Ovary
Epithelium

ASJC Scopus subject areas

  • Biochemistry

Cite this

Fluorescence resonance energy transfer analysis of subunit stoichiometry of the epithelial Na+ channel. / Staruschenko, Alexander; Medina, Jorge L.; Patel, Pravina; Shapiro, Mark S; Booth, Rachell E.; Stockand, James D.

In: Journal of Biological Chemistry, Vol. 279, No. 26, 25.06.2004, p. 27729-27734.

Research output: Contribution to journalArticle

Staruschenko, Alexander ; Medina, Jorge L. ; Patel, Pravina ; Shapiro, Mark S ; Booth, Rachell E. ; Stockand, James D. / Fluorescence resonance energy transfer analysis of subunit stoichiometry of the epithelial Na+ channel. In: Journal of Biological Chemistry. 2004 ; Vol. 279, No. 26. pp. 27729-27734.
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