Distinct Structural Elements in the First Membrane-spanning Segment of the Epithelial Sodium Channel

• Published in: The Journal of biological chemistry 2006-10, Vol.281 (41), p.30455-30462
• Main Authors: Kashlan, Ossama B., Maarouf, Ahmad B., Kussius, Cassandra, Denshaw, Robert M., Blumenthal, Kenneth M., Kleyman, Thomas R.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Cell Membrane - metabolism; Epithelial Sodium Channels - chemistry; Epithelial Sodium Channels - genetics; Epithelial Sodium Channels - metabolism; Humans; Mice; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Patch-Clamp Techniques; Protein Conformation; Protein Structure, Secondary; Protein Structure, Tertiary; Tryptophan - chemistry
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M604615200

Epithelial Na+ channels (ENaCs) comprise three subunits that have been proposed to be arranged in either an α2βγ 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 αM1. 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 αM1 in the pore structure of ENaC, we performed tryptophan-scanning mutagenesis throughout αM1 (residues 110–130). We found that mutations within the amino-terminal part of αM1 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 αM1 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 αM1 is α-helical and packs against structural element(s) that contribute to the ENaC pore. Furthermore, these data suggest that the carboxyl-terminal half of αM1 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 αM1 is divided into two distinct regions.