Acetylcholine receptor channel structure in the resting, open, and desensitized states probed with the substituted-cysteine-accessibility method

The nicotinic acetylcholine (ACh) receptors cycle among classes of noncondu cting resting states, conducting open states, and nonconducting desensitize d states. We previously probed the structure of the mouse-muscle ACh recept or channel in the resting state obtained in the absence of agonist and in t he open states obtained after brief exposure to ACR. We now have probed the structure in the stable desensitized state obtained after many minutes of exposure to ACh. Muscle-type receptor has the subunit composition (alpha (2 )beta gamma delta. Each subunit has four membrane-spanning segments, M1-M4. The channel lumen in the membrane domain is lined largely by M2 and to a l esser extent by M1 from each of the subunits. We determined the rates of re action of a small, sulfhydryl-specific, charged reagent, 2-aminoethyl metha nethiosulfonate with cysteines substituted for residues in (alpha M2 and th e alpha M1-M2 loop in the desensitized state and compared these rates to ra tes previously obtained in the resting and open states. The reaction rates of the substituted cysteines are different in the three functional states o f the receptor, indicating significant structural differences. By comparing the rates of reaction of extracellularly and intracellularly added 2-amino ethyl methanethiosulfonate, we previously located the closed gate in the re sting state between (alpha G240 and (alpha T244, in the predicted M1-M2 loo p at the intracellular end of M2. Now, we have located the closed gate in t he stable desensitized state between alpha G240 and alpha L251. The gate in the desensitized state includes the resting state gate and an extension fu rther into M2.