title: Functional Roles of Clusters of Hydrophobic and Polar Residues in the Epithelial Na+ Channel Knuckle Domain.
abstract: The extracellular regions of epithelial Na(+) channel subunits are highly ordered structures composed of domains formed by α helices and β strands. Deletion of the peripheral knuckle domain of the α subunit in the αβγ trimer results in channel activation, reflecting an increase in channel open probability due to a loss of the inhibitory effect of external Na(+) (Na(+) self-inhibition). In contrast, deletion of either the β or γ subunit knuckle domain within the αβγ trimer dramatically reduces epithelial Na(+) channel function and surface expression, and impairs subunit maturation. We systematically mutated individual α subunit knuckle domain residues and assessed functional properties of these mutants. Cysteine substitutions at 14 of 28 residues significantly suppressed Na(+) self-inhibition. The side chains of a cluster of these residues are non-polar and are predicted to be directed toward the palm domain, whereas a group of polar residues are predicted to orient their side chains toward the space between the knuckle and finger domains. Among the mutants causing the greatest suppression of Na(+) self-inhibition were αP521C, αI529C, and αS534C. The introduction of Cys residues at homologous sites within either the β or γ subunit knuckle domain resulted in little or no change in Na(+) self-inhibition. Our results suggest that multiple residues in the α subunit knuckle domain contribute to the mechanism of Na(+) self-inhibition by interacting with palm and finger domain residues via two separate and chemically distinct motifs.
authors: From the Renal-Electrolyte Division, Department of Medicine.
authors: Jingxin
authors: J
authors: Chen
authors: From the Renal-Electrolyte Division, Department of Medicine.
authors: Evan C
authors: EC
authors: Ray
authors: the Department of Biological Sciences.
authors: Megan E
authors: ME
authors: Yates
authors: the Department of Biological Sciences.
authors: Teresa M
authors: TM
authors: Buck
authors: the Department of Biological Sciences.
authors: Jeffrey L
authors: JL
authors: Brodsky
authors: From the Renal-Electrolyte Division, Department of Medicine.
authors: Carol L
authors: CL
authors: Kinlough
authors: From the Renal-Electrolyte Division, Department of Medicine.
authors: Katie L
authors: KL
authors: Winarski
authors: From the Renal-Electrolyte Division, Department of Medicine, the Department of Cell Biology, the Department of Microbiology and Molecular Genetics, and.
authors: Rebecca P
authors: RP
authors: Hughey
authors: From the Renal-Electrolyte Division, Department of Medicine, the Department of Cell Biology, the Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261 kleyman@pitt.edu.
authors: Thomas R
authors: TR
authors: Kleyman
authors: From the Renal-Electrolyte Division, Department of Medicine.
authors: Shaohu
authors: S
authors: Sheng
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D053503:Epithelial Sodium Channels / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D017434:Protein Structure, Tertiary; D051381:Rats; D012964:Sodium / Q000494:pharmacology
chemical_list: D053503:Epithelial Sodium Channels; D012964:Sodium
keywords: acid-sensing ion channel (ASIC); allosteric regulation; epithelial sodium channel (ENaC); ion channel; sodium channel