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Volume 272, Number 34, Issue of August 22, 1997 pp. 21075-21083
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Identification of an Amiloride Binding Domain within the -Subunit of the Epithelial Na⁺ Channel

(Received for publication, June 28, 1996, and in revised form, June 4, 1997)

Iskander I. Ismailov , Thomas Kieber-Emmons ^§ , Chaomei Lin ^¶ , Bakhram K. Berdiev , Vadim Gh. Shlyonsky , Holly K. Patton , Catherine M. Fuller , Roger Worrell ^** , Jonathan B. Zuckerman ^¶ , Weijing Sun , Douglas C. Eaton ^** , Dale J. Benos and Thomas R. Kleyman ^¶§§

From the  Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35294, the Departments of ^¶ Medicine, ^§ Pathology, and ^§§ Physiology and  Institute for Neurological Sciences, University of Pennsylvania and Veterans Administration Medical Center, Philadelphia, Pennsylvania 19104, and the ^** Department of Physiology, Emory University, Atlanta, Georgia 30322

Limited information is available regarding domains within the epithelial Na⁺ channel (ENaC) which participate in amiloride binding. We previously utilized the anti-amiloride antibody (BA7.1) as a surrogate amiloride receptor to delineate amino acid residues that contact amiloride, and identified a putative amiloride binding domain WYRFHY (residues 278-283) within the extracellular domain of rENaC. Mutations were generated to examine the role of this sequence in amiloride binding. Functional analyses of wild type (wt) and mutant rENaCs were performed by cRNA expression in Xenopus oocytes and by reconstitution into planar lipid bilayers. Wild type rENaC was inhibited by amiloride with a K_i of 169 nM. Deletion of the entire WYRFHY tract (rENaC 278-283) resulted in a loss of sensitivity of the channel to submicromolar concentrations of amiloride (K_i = 26.5 µM). Similar results were obtained when either rENaC or rENaC 278-283 were co-expressed with wt - and rENaC (K_i values of 155 nM and 22.8 µM, respectively). Moreover, rENaC H282D was insensitive to submicromolar concentrations of amiloride (K_i = 6.52 µM), whereas rENaC H282R was inhibited by amiloride with a K_i of 29 nM. These mutations do not alter ENaC Na⁺:K⁺ selectivity nor single-channel conductance. These data suggest that residues within the tract WYRFHY participate in amiloride binding. Our results, in conjunction with recent studies demonstrating that mutations within the membrane-spanning domains of rENaC and mutations preceding the second membrane-spanning domains of -, -, and rENaC alters amiloride's K_i, suggest that selected regions of the extracellular loop of rENaC may be in close proximity to residues within the channel pore.

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