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

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 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 M). 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 M, respectively). Moreover, ␣rENaC H282D was insensitive to submicromolar concentrations of amiloride (K i ‫؍‬ 6.52 M), 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.The diuretic amiloride is a prototypic inhibitor of epithelial Na ϩ channels (ENaCs) 1 (1), although amiloride and its various derivatives inhibit many Na ϩ -selective transport proteins. Several laboratories have recently identified domains within the epithelial Na ϩ channel and the Na ϩ /H ϩ exchanger that appear to participate in amiloride binding. Residues within the second membrane-spanning domain of ␣rENaC may interact with amiloride, as mutations of a serine residue at position 589 result in a large decrease of the apparent K i for amiloride and the amiloride analog benzamil, as well as alter cation selectivity (2). Selected mutations of residues within a hydrophobic region, termed H2 (3), immediately preceding the second membrane-spanning domains of the ␣-, ␤-, and ␥-subunits of rENaC (i.e. Trp-␣582, Ser-␣583, Gly-␤525, Gly-␥537) and the ␣-subunit of bovine ENaC (Lys-504, Lys-515) affect the K i for amiloride, and several of these mutations affect single-channel conductance (4, 5). Snyder and co-workers have identified splice variants of ␣rENaC in which the C-terminal 199 or 216 amino acid residues, including the second membrane-spanning domain, are truncated (6). These splice variants are not functional when expressed in Xenopus oocytes, but retain amiloride and phenamil binding activity, s...

A Pore Segment in DEG/ENaC Na+ Channels

“…5C). However, it has been reported that mutation of residues at a site in the extracellular domain also disrupted block by amiloride, raising the possibility that amiloride might block the channel indirectly through an allosteric mechanism (35). If this is the case, then the site between Gly 533 and Gly 536 might not be a binding site for amiloride, but rather the site of a conformational change that occludes the pore in response to the binding of amiloride to a distant site.…”

Characterization of an amiloride binding region in the α-subunit of ENaC

“…However, previous studies have identified a region in the extracellular loop of rat ␣-ENaC between amino acids 278 and 283, with the sequence WYRFHY, that interacts with amiloride (12). When this stretch of amino acids, located just COOH terminal to the first transmembrane-spanning domain within a cysteinerich domain, is deleted or mutated, amiloride binding of these channels expressed in a bilayer system is dramatically reduced.…”

“…open probability; extracellular loop; channel pore; sodium channel; single-channel recording EPITHELIAL SODIUM CHANNELS (ENaC) play a critical role in the control of blood pressure and regulation of total body sodium balance. Although the original work in which the channel components were cloned suggested that functional channels consist of three subunits, ␣, ␤, and ␥ (5), under appropriate circumstances, ␣-subunits alone can form sodium-permeable channels (12,13,17). It has been proposed that some of the diversity in conductance, gating, and selectivity of functional ENaC may be due to different subunit combinations.…”

Mutations in the extracellular loop of α-rENaC alter sensitivity to amiloride and reactive species

“…Recent work examining the properties of channels composed solely of ␣-subunits identified a domain within the extracellular loop of ␣-rENaC (residues 278 -283) where mutations or deletions affect amiloride block as well as the open probability of the channels (14,17). Furthermore, selected mutations within this region of ␣-ENaC or within the corresponding region of ␥-ENaC altered the channel's response to external Ni 2ϩ (27) and to changes in extracellular Na ϩ (26).…”

“…In channels composed solely of ␣-subunits, mutations in this domain affect both channel gating and channel block by amiloride (14,17). This region within the ␣-subunit includes residues 278 -283 (WYRFHY).…”