The cation-conducting channel of the nicotinic acetylcholine (ACh) receptor is lined by the first (M1) and second (M2) membrane-spanning segments of each of its five subunits. Six consecutive residues, αS239 to αT244, in the α subunit M1–M2 loop and at the intracellular end of M2 were mutated to cysteine. The accessibility of the substituted cysteines were probed with small, cationic, sulfhydryl-specific reagents added extracellularly and intracellularly. In the closed state of the channel, there is a barrier to these reagents added from either side between αG240 and αT244. ACh induces the removal of this barrier, which acts as an activation gate. The residues αG240, αE241, αK242, and αT244 line a narrow part of the channel, in which this gate is located.
