title: Distinct structural elements in the first membrane-spanning segment of the epithelial sodium channel.
abstract: Epithelial Na+ channels (ENaCs) comprise three subunits that have been proposed to be arranged in either an alpha2betagamma or a higher ordered configuration. Each subunit has two putative membrane-spanning segments (M1 and M2), intracellular amino and carboxyl termini, and a large extracellular loop. We have used the TOXCAT assay (a reporter assay for transmembrane segment homodimerization) to identify residues within the transmembrane segments of ENaC that may participate in important structural interactions within ENaC, with which we identified a candidate site within alphaM1. We performed site-directed mutagenesis at this site and found that, although the mutants reduced channel activity, ENaC protein expression at the plasma membrane was unaffected. To deduce the role of alphaM1 in the pore structure of ENaC, we performed tryptophan-scanning mutagenesis throughout alphaM1 (residues 110-130). We found that mutations within the amino-terminal part of alphaM1 had effects on activity and selectivity with a periodicity consistent with a helical structure but no effect on channel surface expression. We also observed that mutations within the carboxyl-terminal part of alphaM1 had effects on activity and selectivity but with no apparent periodicity. Additionally, these mutants reduced channel surface expression. Our data support a model in which the amino-terminal half of alphaM1 is alpha-helical and packs against structural element(s) that contribute to the ENaC pore. Furthermore, these data suggest that the carboxyl-terminal half of alphaM1 may be helical or assume a different conformation and may be involved in tertiary interactions essential to proper channel folding or assembly. Together, our data suggest that alphaM1 is divided into two distinct regions.
authors.affiliation: Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.
authors.forename: Ossama B
authors.initials: OB
authors.lastname: Kashlan
authors.forename: Ahmad B
authors.initials: AB
authors.lastname: Maarouf
authors.forename: Cassandra
authors.initials: C
authors.lastname: Kussius
authors.forename: Robert M
authors.initials: RM
authors.lastname: Denshaw
authors.forename: Kenneth M
authors.initials: KM
authors.lastname: Blumenthal
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Kleyman
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D053503:Epithelial Sodium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D014364:Tryptophan / Q000737:chemistry
chemical_list: D053503:Epithelial Sodium Channels; D014364:Tryptophan