Asymmetric organization of the pore region of the epithelial sodium channel.
Epithelial sodium channels (ENaCs) are composed of three homologous subunits that have regions preceding the second transmembrane domain (also referred as pre-M2) that form part of the channel pore. To identify residues within this region of the beta-subunit that line the pore, we systematically mutated residues Gln(523)-Ile(536) to cysteine. Wild type and mutant mouse ENaCs were expressed in Xenopus oocytes, and a two-electrode voltage clamp was used to examine the properties of mutant channels. Cysteine substitutions of 9 of 13 residues significantly altered Li(+) to Na(+) current ratios, whereas only cysteine replacement of beta Gly(529) resulted in K(+)-permeable channels. Besides beta G525C, large increases in the inhibitory constant of amiloride were observed with mutations at beta Gly(529) and beta Ser(531) within the previously identified 3-residue tract that restricts K(+) permeation. Cysteine substitution preceding (beta Phe(524) and beta Gly(525)), within (beta Gly(530)) or following (beta Leu(533)) this 3-residue tract, resulted in enhanced current inhibition by external MTSEA. External MTSET partially blocked channels with cysteine substitutions at beta Gln(523), beta Phe(524), and beta Trp(527). MTSET did not inhibit alpha beta G525C gamma, although previous studies showed that channels with cysteine substitutions at the corresponding sites within the alpha- and gamma-subunits were blocked by MTSET. Our results, placed in context with previous observations, suggest that pore regions from the three ENaC subunits have an asymmetric organization.
Li
Jinqing
J
Department of Physiology, University of Pennsylvania, Philadelphia 19104 , USA.
Sheng
Shaohu
S
Perry
Clint J
CJ
Kleyman
Thomas R
TR
D000584:Amiloride / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002412:Cations; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D053503:Epithelial Sodium Channels; D005020:Ethyl Methanesulfonate / Q000031:analogs & derivatives* / Q000494:pharmacology; D007202:Indicators and Reagents / Q000494:pharmacology; D007700:Kinetics; D008094:Lithium / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D011188:Potassium / Q000378:metabolism; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000378:metabolism; D012964:Sodium / Q000378:metabolism / Q000494:pharmacology; D015222:Sodium Channels / Q000737:chemistry* / Q000378:metabolism; D013997:Time Factors; D014981:Xenopus
D002412:Cations; D018076:DNA, Complementary; D053503:Epithelial Sodium Channels; D007202:Indicators and Reagents; D015222:Sodium Channels; C093171:methanethiosulfonate ethylammonium; D012694:Serine; D000584:Amiloride; D008094:Lithium; D005020:Ethyl Methanesulfonate; D012964:Sodium; D003545:Cysteine; D011188:Potassium