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J. Biol. Chem., Vol. 281, Issue 12, 8233-8241, March 24, 2006
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-Subunit Confers Novel Biophysical Features to 
-Human Epithelial Sodium Channel (ENaC) via a Physical Interaction*
112
From the
Departments of Anesthesiology and Physiology and Biophysics, University of Alabama at Birmingham, Alabama 35205 and the ¶Institut de Pharmacologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique UPR411, 06560 Sophia Antipolis, France
Native amiloride-sensitive Na+ channels exhibit a variety of biophysical properties, including variable sensitivities to amiloride, different ion selectivities, and diverse unitary conductances. The molecular basis of these differences has not been elucidated. We tested the hypothesis that co-expression of 
-epithelial sodium channel (ENaC) underlies, at least in part, the multiplicity of amiloride-sensitive Na+ conductances in epithelial cells. For example, the -subunit may form multimeric channels with 
-ENaC. Reverse transcription-PCR revealed that -ENaC is co-expressed with -subunits in cultured human lung (H441 and A549), pancreatic (CFPAC), and colonic epithelial cells (Caco-2). Indirect immunofluorescence microscopy revealed that -ENaC is co-expressed with -, -, and -ENaC in H441 cells at the protein level. Measurement of current-voltage that cation selectivity ratios for the revealed relationships Na+/Li+/K+/Cs+/Ca2+/Mg2+, the apparent dissociation constant (Ki) for amiloride, and unitary conductances for -ENaC differed from those of both - and -ENaC (n = 6). The contribution of the subunit to PLi/PNa ratio and unitary Na+ conductance under bi-ionic conditions depended on the injected cRNA concentration. In addition, the EC50 for proton activation, mean open and closed times, and the self-inhibition time of -ENaC differed from those of - and -ENaC. Co-immunoprecipitation of -ENaC with - and -subunits in H441 and transfected COS-7 cells suggests an interaction among these proteins. We, therefore, concluded that the interactions of -ENaC with other subunits could account for heterogeneity of native epithelial channels.
Received for publication, November 16, 2005 , and in revised form, January 10, 2006.
* The work was supported by Cystic Fibrosis Foundation Grants JI04G0, DK37206, DK56596, HL075540, and HL51173. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Both authors equally contributed to the study.
2 To whom correspondence should be addressed: Dept. of Physiology and Biophysics, University of Alabama at Birmingham, 1918 University Blvd., 704 MCLM, Birmingham, AL 35294-0005. Fax: 205-934-2377; E-mail: benos{at}physiology.uab.edu.
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