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A highly conserved leucine is found in the middle of the porelining (M2) domain of the members of the ligand-gated ion channel family. Two very different roles have been proposed for this leucine. In one model, this residue swings into the lumen of the channel during desensitization to form the nonconducting desensitized state, whereas in the other model, the leucines from each subunit interact with each other to form a constriction in the channel that constitutes the actual gate of the channel. We examined the role of this leucine in the muscle-type acetylcholine receptor by replacing it with the polar amino acid threonine. Replacement of the leucine in any one subunit slows desensitization and shifts the dose-response relationship toward lower concentrations. Replacement of leucines in additional subunits leads to progressively larger shifts in the dose-response curves. The shift depends only on the number of leucines replaced, not on which particular subunits contain the mutation; in other words, the mutations act independently. At the single-channel level, the mutation greatly increases the channel mean open time. We conclude that the role of the conserved leucine is to set the mean open time of the channel through interactions with other regions of the receptor rather than to serve as the gate per se of the ion channel.

Alteration of the ligand-binding domain of the nicotinic acetylcholine receptor through site-directed mutagenesis offers a powerful approach to the elucidation of structure-function relations in the receptor. Several conserved tyrosine residues in the large extracellular amino terminus of the alpha subunit of the receptor have been implicated by both chemical labeling and mutagenesis studies as playing an important role in the interaction of acetylcholine with the receptor. We and others have previously shown that substitution of phenylalanine for tyrosine at position 198 of the alpha subunit (alpha Y198F) leads to a rightward shift in the dose-response curve for acetylcholine-elicited currents. We have further investigated this particular mutation by examining the interaction of the competitive antagonist d-tubocurarine (curare) with the receptor. In contrast to the effect on the interaction of agonists with the receptor, this mutation leads to a marked increase in the affinity of the receptor for curare. Furthermore, this enhancement in affinity is selective for curare and is not seen with other competitive antagonists (pancuronium, beta-erythroidine, and gallamine). Examination of the structures of these competitive antagonists leads to the proposal that this enhancement is due to the formation of an aromatic-aromatic interaction between the phenylalanine ring at position alpha 198 in the mutant and one of the aromatic rings of curare and that this can provide information about the spatial arrangement of this residue in the binding site.