Identification of acetylcholine receptor channel-lining residues in the M1 segment of the alpha-subunit.
The muscle-type acetylcholine (ACh) receptor has the composition alpha 2 beta gamma delta. The subunits are arranged quasisymmetrically around a central, ion-conducting, water-filled channel. Each subunit has four membrane-spanning segments, M1-M4, and the channel through the membrane is formed among these segments. Substituting cysteine for each of the residues in and flanking the alpha M2 segment, we previously found that, at 10 of the 21 mutated positions, the cysteine was accessible to a small, positively charged, sulfhydryl-specific reagent, methanethiosulfonate ethylammonium (MTSEA), and inferred that the residues at these positions are exposed in the channel lumen. We have now applied the substituted-cysteine-accessibility method to alpha M1. We analyzed 15 consecutive residues, starting at alpha Pro211 at the extracellular end of M1. Wild-type alpha contains Cys222, which is inaccessible to MTSEA. We mutated each of the other 14 residues to cysteine and expressed the mutant alpha subunits, together with wild-type beta, gamma, and delta subunits, in Xenopus oocytes. Thirteen of the fourteen mutants gave robust ACh-induced currents. MTSEA irreversibly altered the ACh-induced response of seven cysteine-substitution mutants: alpha Y213C was susceptible to MSTEA added in the presence or the absence of ACh, alpha P211C, alpha I215C, alpha V216C, alpha N217C, and alpha I220C were susceptible in the absence of ACh, and alpha V218C was susceptible in the presence of ACh. These results imply that M1 is exposed in the channel, and its exposure changes during gating or desensitization.(ABSTRACT TRUNCATED AT 250 WORDS)
Akabas
M H
MH
Center for Molecular Recognition, College of Physicians and Surgeons, Columbia University, New York, New York 10032, USA.
Karlin
A
A
D000595:Amino Acid Sequence; D000818:Animals; D003545:Cysteine / Q000235:genetics; D015640:Ion Channel Gating; D007473:Ion Channels / Q000737:chemistry / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011950:Receptors, Cholinergic / Q000737:chemistry / Q000235:genetics*
D007473:Ion Channels; D011950:Receptors, Cholinergic; D003545:Cysteine