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12496 Biochemistry 1995, 34, 12496-12500 Identification of Acetylcholine Receptor Channel-Lining Residues in the M1
 

Summary: 12496 Biochemistry 1995, 34, 12496-12500
Identification of Acetylcholine Receptor Channel-Lining Residues in the M1
Segment of the a-Subunit?
Myles H. Akabas* and Arthur Karlin*,s
Centerfor Molecular Recognition, and Departments of Biochemistry and Molecular Biophysics,Physiology and
Cellular Biophysics, Medicine, and Neurology, College of Physicians and Surgeons, Columbia University,
630 West 168th Street, New York, New York 10032
Received July 18, 1995; Revised Manuscript Received August 14, 1995@
ABSTRACT: The muscle-type acetylcholine (ACh) receptor has the composition a&6. The subunits are
arranged quasisymmetrically around a central, ion-conducting, water-filled channel. Each subunit has
four membrane-spanning segments, Ml-M4, and the channel through the membrane is formed among
these segments. Substituting cysteine for each of the residues in and flanking the aM2 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 aM1. We analyzed 15 consecutive residues, starting at aPro211 at the
extracellular end of M1. Wild-type a contains Cys222, which is inaccessible to MTSEA. We mutated
each of the other 14 residues to cysteine and expressed the mutant a subunits, together with wild-type p,
y, and 6 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: aY213C

  

Source: Akabas, Myles - Department of Physiology and Biophysics, Albert Einstein College of Medicine, Yeshiva University

 

Collections: Biology and Medicine