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 nonconducting resting states, conducting open states, and nonconducting desensitized states. We previously probed the structure of the mouse-muscle ACh receptor channel in the resting state obtained in the absence of agonist and in the 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 lesser extent by M1 from each of the subunits. We determined the rates of reaction of a small, sulfhydryl-specific, charged reagent, 2-aminoethyl methanethiosulfonate with cysteines substituted for residues in (alpha M2 and the alpha M1-M2 loop in the desensitized state and compared these rates to rates previously obtained in the resting and open states. The reaction rates of the substituted cysteines are different in the three functional states of the receptor, indicating significant structural differences. By comparing the rates of reaction of extracellularly and intracellularly added 2-aminoethyl methanethiosulfonate, we previously located the closed gate in the resting state between (alpha G240 and (alpha T244, in the predicted M1-M2 loop at the intracellular end of M2. Now, we have located the closed gate in the stable desensitized state between alpha G240 and alpha L251. The gate in the desensitized state includes the resting state gate and an extension further into M2.