Characterization of an amiloride binding region in the α-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 ␣-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 ␣-subunits or ␣-subunits containing point mutations in this region were examined and compared at the single-channel level. The open probabilities (P o) of wild-type channels were distributed into two populations: one with a high P o and one with a low P o. The mean open times of all the mutant channels were shorter than the mean open time of the wild-type (high-P o) channel. Besides mutations Y279A and H282D, which had amiloride binding affinities similar to that of wild-type ␣-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. 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. The expression of ␣-subunits alone, rather than the expression of all three subunits together, depends on the environmental conditions to which renal epithelial cells (A6) (7) or alveolar type II cells (14) are exposed. However, regardless of which channel type is expressed, one of the defining characteristics of all these channels is their block by the diuretic amiloride, a substituted pyrazinoylguanidine. The tertiary structure of all the subunits is similar: each subunit is predicted to span the membrane twice, to have a large extracellular loop, and to have short intracellular NH 2 and COOH termini (2, 6). Because amiloride blocks the channel from the extracellular surface of the channel protein, it is possible that one or more of the extracellular loops could play a role in amiloride's interaction with and block of the channel by stabilizing amiloride in the channel pore.About 70% of each ENaC subunit is extracellular. Structure-function studies of the extracellular loop have focused primarily on the region immediately preceding and including the second transmembrane (M2) domain of ␣-ENaC. These data have implicated these regions in binding to amiloride and also play a role in...

Small Molecule Activator of the Human Epithelial Sodium Channel

“…Amiloride priming experiments, performed to synchronize hENaC in an open conformation with an occluded pore, revealed that S3969 activation of hENaC was rapid (t1 ⁄ 2 ϳ 5 s) following amiloride removal. The majority of hENaC channels in the oocyte plasma membrane are closed or in a quiescent, nonconducting state (18,19,58), consistent with reports that ENaC exhibits long closed times on the order of seconds (27,59,60 , and % ENaC activation by S3969 (D) in oocytes expressing wild-type ␣␤␥ (WT), ␣␤G37S␥ (␤G37S), ␣R508X␤␥ (␣R508X), or ␤␥ hENaC channels, or no hENaC channels (uninjected) are shown. S3969 strongly activated wild-type ␣␤␥ and ␣␤G37S␥ hENaC, weakly activated ␣R508X␤␥ hENaC, and failed to activate ␤␥ hENaC or uninjected oocytes.…”

“…Amiloride is thought to occlude the pore of ␣ENaC subunits in an open conformation; thus, amiloride functions as an openstate blocker (27) (Fig. 2B, treatment b).…”

Extracellular Histidine Residues Crucial for Na+ Self-inhibition of Epithelial Na+ Channels

“…A six residue tract (WYRFHY) within the ECL of the ␣-subunit was identified as a putative amiloride binding site based on its homology with the antigen binding domain of an anti-amiloride antibody (19,24). Specific mutations within this tract altered the sensitivity of ␣-subunit channels to amiloride, and altered gating characteristics of both ␣ channels as well as ␣␤␥ channels (16,19,20). We and other investigators (16,25) recently reported that Ni 2ϩ is an external inhibitor of ENaC expressed in Xenopus oocytes.…”

“…His residues have been found to have key functional roles in enzymes, receptors and ion channels (37)(38)(39)(40). The changes in Na ϩ self-inhibition due to mutations at both His sites raise a question whether they may interact with each other at an ␣-␥ subunit interface and whether this proposed interaction is important for ENaC activity and channel regulation by extracellular factors including metals, amiloride, pH, temperature, and fluid flow (16,19,20,25,(41)(42)(43). In the context of previous studies and our current results, we suggest that a site in the proximity of these His residues has a role in modulating ENaC function.…”

Functional Role of Extracellular Loop Cysteine Residues of the Epithelial Na+ Channel in Na+ Self-inhibition

“…Theoretically, these Cys residues could form intrasubunit or intersubunit disulfide bonds that normally facilitate proper folding in the endoplasmic reticulum and maintain structural integrity (20). Firsov et al (14) investigated the roles of the ECL Cys residues by a systematic mutation of all 16 Cys residues in the rat ␣ECL, as well as selected mutations of Cys residues in the ␤ECL and ␥ECL. The authors concluded that the Cys-1 and Cys-6 residues of CRD-I in all three subunits and the Cys-11 and Cys-12 of CRD-II in ␣ and ␤ (but not in ␥) ECLs are required for efficient expression of ENaC at the plasma membrane.…”