Novel determinants of epithelial sodium channel gating within extracellular thumb domains.
Activity of the epithelial Na(+) channel (ENaC) is modulated by Na(+) self-inhibition, an allosteric down-regulation of channel open probability by extracellular Na(+). We searched for determinants of Na(+) self-inhibition by analyzing changes in this inhibitory response resulting from specific mutations within the extracellular domains of mouse ENaC subunits. Mutations at gammaMet(438) altered the Na(+) self-inhibition response in a substitution-specific manner. Fourteen substitutions (Ala, Arg, Asp, Cys, Gln, Glu, His, Ile, Phe, Pro, Ser, Thr, Tyr, and Val) significantly suppressed Na(+) self-inhibition, whereas three mutations (Asn, Gly, and Leu) moderately enhanced the inhibition. Met to Lys mutation did not alter Na(+) self-inhibition. Mutations at the homologous site in the alpha subunit (G481A, G481C, and G481M) dramatically increased the magnitude and speed of Na(+) self-inhibition. Mutations at the homologous betaAla(422) resulted in minimal or no change in Na(+) self-inhibition. Low, high, and intermediate open probabilities were observed in oocytes expressing alphaG481Mbetagamma, alphabetagammaM438V, and alphaG481M/betagammaM438V, respectively. This pair of residues map to thealpha5 helix in the extracellular thumb domain in the chicken acid sensing ion channel 1 structure. Both residues likely reside near the channel surface because both alphaG481Cbetagamma and alphabetagammaM438C channels were inhibited by an externally applied and membrane-impermeant sulfhydryl reagent. Our results demonstrate that alphaGly(481) and gammaMet(438) are functional determinants of Na(+) self-inhibition and of ENaC gating and suggest that the thumb domain contributes to the channel gating machinery.
Maarouf
Ahmad B
AB
Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh, PA 15261, USA.
Sheng
Nan
N
Chen
Jingxin
J
Winarski
Katie L
KL
Okumura
Sora
S
Carattino
Marcelo D
MD
Boyd
Cary R
CR
Kleyman
Thomas R
TR
Sheng
Shaohu
S
D019943:Amino Acid Substitution; D000818:Animals; D002478:Cells, Cultured; D053503:Epithelial Sodium Channels / Q000235:genetics / Q000378:metabolism*; D015640:Ion Channel Gating / Q000502:physiology*; D017136:Ion Transport / Q000502:physiology; D051379:Mice; D009154:Mutation; D009865:Oocytes; D017433:Protein Structure, Secondary / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D014981:Xenopus
D053503:Epithelial Sodium Channels; D021122:Protein Subunits; D012964:Sodium