title: Characterization of an amiloride binding region in the alpha-subunit of ENaC.
abstract: One of the defining characteristics of the epithelial sodium channel (ENaC) is its block by the diuretic amiloride. This study investigates the role of the extracellular loop of the alpha-subunit of ENaC in amiloride binding and stabilization. Mutations were generated in a region of the extracellular loop, residues 278-283. Deletion of this region, WYRFHY, resulted in a loss of amiloride binding to the channel. Channels formed from wild-type alpha-subunits or alpha-subunits containing point mutations in this region were examined and compared at the single-channel level. The open probabilities (Po) of wild-type channels were distributed into two populations: one with a high Po and one with a low Po. The mean open times of all the mutant channels were shorter than the mean open time of the wild-type (high-Po) channel. Besides mutations Y279A and H282D, which had amiloride binding affinities similar to that of wild-type alpha-ENaC, all other mutations in this region caused changes in the amiloride binding affinity of the channels compared with the wild-type channel. These data provide new insight into the relative position of the extracellular loop with respect to the pore of ENaC and its role in amiloride binding and channel gating.
authors.affiliation: Center for Cell and Molecular Signaling, Department of Physiology, Emory University School of Medicine, 615 Michael Street NE, Atlanta, GA 30322, USA.
authors.forename: Ollie
authors.initials: O
authors.lastname: Kelly
authors.forename: Chaomei
authors.initials: C
authors.lastname: Lin
authors.forename: Mohan
authors.initials: M
authors.lastname: Ramkumar
authors.forename: Nina C
authors.initials: NC
authors.lastname: Saxena
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Kleyman
authors.forename: Douglas C
authors.initials: DC
authors.lastname: Eaton
mesh_terms: D000584:Amiloride / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D016466:CHO Cells; D006224:Cricetinae; D004232:Diuretics / Q000378:metabolism* / Q000494:pharmacology; D053503:Epithelial Sodium Channels; D015640:Ion Channel Gating / Q000502:physiology*; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008954:Models, Biological; D016296:Mutagenesis; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D051381:Rats; D012964:Sodium / Q000378:metabolism; D015222:Sodium Channels / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D004232:Diuretics; D053503:Epithelial Sodium Channels; D015222:Sodium Channels; D000584:Amiloride; D012964:Sodium