Fluorescence resonance energy transfer analysis of subunit stoichiometry of the epithelial Na+ channel.
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.
Staruschenko
Alexander
A
Department of Physiology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229, USA.
Medina
Jorge L
JL
Patel
Pravina
P
Shapiro
Mark S
MS
Booth
Rachell E
RE
Stockand
James D
JD
D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003412:Cricetulus; D004848:Epithelium / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D015870:Gene Expression; D051379:Mice; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D008970:Molecular Weight; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015222:Sodium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D021122:Protein Subunits; D011994:Recombinant Proteins; D015222:Sodium Channels