Identification of an amiloride binding domain within the α-subunit of the epithelial Na+ channel

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, Thomas R. Kleyman

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Abstract

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 amine acid residues that contact amiloride, and identified a putative amiloride binding domain WYRFHY (residues 278-283) within the extracellular domain of αrENaC. Mutations were generated to examine the role of this sequence in amiloride binding. Functional analyses of wild type (wt) and mutant αrENaCs were performed by cRNA expression in Xenopus oocytes and by reconstitution into planar lipid bilayers. Wild type αrENaC was inhibited by amiloride with a K(i) of 169 nM. Deletion of the entire WYRFHY tract (αrENaC Δ278-283) 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 αrENaC or αrENaC Δ278-283 were co-expressed with wt β- and γrENaC (K(i) values of 155 nM and 22.8 μM, respectively). Moreover, αrENaC H282D was insensitive to sub-micromolar concentrations of amiloride (K(i) = 6.52 μM), whereas αrENaC 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 WYRFY participate in amiloride binding. Our results, in conjunction with recent studies demonstrating that mutations within the membrane-spanning domains of αrENaC and mutations preceding the second membrane-spanning domains of α-, β-, and γrENaC alters amiloride's K(i), suggest that selected regions of the extracellular loop of αrENaC may be in close proximity to residues within the channel pore.

Original languageEnglish
Pages (from-to)21075-21083
Number of pages9
JournalJournal of Biological Chemistry
Volume272
Issue number34
DOIs
Publication statusPublished - Aug 22 1997
Externally publishedYes

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Epithelial Sodium Channels
Amiloride
Mutation
Membranes
Complementary RNA
Lipid bilayers
Lipid Bilayers
Xenopus
Oocytes
Amines
Anti-Idiotypic Antibodies

ASJC Scopus subject areas

  • Biochemistry

Cite this

Ismailov, I. I., Kieber-Emmons, T., Lin, C., Berdiev, B. K., Shlyonsky, V. G., Patton, H. K., ... Kleyman, T. R. (1997). Identification of an amiloride binding domain within the α-subunit of the epithelial Na+ channel. Journal of Biological Chemistry, 272(34), 21075-21083. https://doi.org/10.1074/jbc.272.34.21075

Identification of an amiloride binding domain within the α-subunit of the epithelial Na+ channel. / Ismailov, Iskander I.; Kieber-Emmons, Thomas; Lin, Chaomei; Berdiev, Bakhram K.; Shlyonsky, Vadim Gh; Patton, Holly K.; Fuller, Catherine M.; Worrell, Roger; Zuckerman, Jonathan B.; Sun, Weijing; Eaton, Douglas C.; Benos, Dale J.; Kleyman, Thomas R.

In: Journal of Biological Chemistry, Vol. 272, No. 34, 22.08.1997, p. 21075-21083.

Research output: Contribution to journalArticle

Ismailov, II, Kieber-Emmons, T, Lin, C, Berdiev, BK, Shlyonsky, VG, Patton, HK, Fuller, CM, Worrell, R, Zuckerman, JB, Sun, W, Eaton, DC, Benos, DJ & Kleyman, TR 1997, 'Identification of an amiloride binding domain within the α-subunit of the epithelial Na+ channel', Journal of Biological Chemistry, vol. 272, no. 34, pp. 21075-21083. https://doi.org/10.1074/jbc.272.34.21075
Ismailov II, Kieber-Emmons T, Lin C, Berdiev BK, Shlyonsky VG, Patton HK et al. Identification of an amiloride binding domain within the α-subunit of the epithelial Na+ channel. Journal of Biological Chemistry. 1997 Aug 22;272(34):21075-21083. https://doi.org/10.1074/jbc.272.34.21075
Ismailov, Iskander I. ; Kieber-Emmons, Thomas ; Lin, Chaomei ; Berdiev, Bakhram K. ; Shlyonsky, Vadim Gh ; Patton, Holly K. ; Fuller, Catherine M. ; Worrell, Roger ; Zuckerman, Jonathan B. ; Sun, Weijing ; Eaton, Douglas C. ; Benos, Dale J. ; Kleyman, Thomas R. / Identification of an amiloride binding domain within the α-subunit of the epithelial Na+ channel. In: Journal of Biological Chemistry. 1997 ; Vol. 272, No. 34. pp. 21075-21083.
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AU - Kieber-Emmons, Thomas

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AU - Berdiev, Bakhram K.

AU - Shlyonsky, Vadim Gh

AU - Patton, Holly K.

AU - Fuller, Catherine M.

AU - Worrell, Roger

AU - Zuckerman, Jonathan B.

AU - Sun, Weijing

AU - Eaton, Douglas C.

AU - Benos, Dale J.

AU - Kleyman, Thomas R.

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