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1 Department of Medicine, University of Pittsburgh, Pittsburgh, PA, USA
2 Department of Medicine, University of Pennsylvania, Philadelphia, PA, USA
3 Department of Medicine, University of Pittsburgh, Pittsburgh, PA, USA; Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, PA, USA
* To whom correspondence should be addressed. E-mail: kleyman{at}pitt.edu.
Epithelial sodium channels (ENaCs) are composed of three structurally related subunits that form a tetrameric channel. The Xenopus oocyte expression system was used to identify regions within the ENaC 
-subunit that confer a dominant negative phenotype on functional expression of 
ENaC in order to define domains that have a role in subunit-subunit interactions. Co-expression of full length mouse ENaC with either (i) the -subunit first membrane spanning domain and short downstream hydrophobic domain (M1H1), (ii) M1H1 and its downstream hydrophilic extracellular loop (M1H1-ECL), (iii) the membrane spanning domain of a control type 2 transmembrane protein (GT) fused to ECL (GT-ECL), (iv) the extracellular domain of a control type 1 transmembrane protein (Tac) fused to the -subunit second membrane spanning domain and short upstream hydrophobic domain (Tac-H2M2), or (v) the -subunit cytoplasmic C-terminus (Ct) significantly reduced amiloride-sensitive Na+ currents in Xenopus oocytes. Functional expression of Na+ channels was not inhibited when full length ENaC was co-expressed with either (i) the ECL lacking a signal-anchor sequence, (ii) M1H1 and its cytoplasmic C-terminus expressed as a fusion protein, (iii) full length GT or (iv) full length Tac. Furthermore, the expression of ROMK channels was not inhibited when full length ROMK was co-expressed with either M1H1-ECL or the -subunit cytoplasmic C-terminus. Full-length FLAG-tagged -, - or ENaC co-immunoprecipitated with myc-tagged M1H1-ECL, whereas wild type GT did not. These data suggest that multiple sites within the -subunit participate in subunit-subunit interactions that are required for proper assembly of the hetero-oligomeric ENaC complex.
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