A Deep Non-Conducting State in the Nicotinic Acetylcholine Receptor

Abstract

We studied the effect of systematically introducing aromatic residues in the pore domain of the muscle nicotinic acetylcholine receptor (AChR). Specifically, we mutated positions 9′, 13′, 16′, and 20′ of the second transmembrane segments (M2), four positions that face the channel's lumen in the open conformation. Mutations were engineered one position at a time in individual subunits or as combinations of mutations in multiple subunits. We used single-channel and ensemble-type current recordings to monitor the single-channel conductance and the kinetics of gating-desensitization, and used equilibrium 125I alpha-bungarotoxin binding to estimate cell-surface expression. Kinetic analysis revealed that most of these mutations prolong the mean duration of diliganded bursts of openings (and, as expected, slow the deactivation time course of macroscopic currents) whether present in one or all five subunits, consistent with a ‘gain-of-function’ effect that is typical of mutations in M2. The effect on the kinetics of entry into desensitization varied among mutants, but was generally mild. The most striking effect of these mutations was observed at positions 16′ and 20′, and consists of a marked decrease in the magnitude of the peak currents in response to single ACh-concentration jumps (from zero to 100 uM) to an extent that cannot be accounted for by the number of mutated AChRs on the cell surface, their single-channel conductance, or their open probability at 100-uM ACh. We conclude that these mutants can enter some sort of refractory state even in the absence of bound ACh, in such a way that only a small fraction of the receptors in the membrane remain activatable. Analysis of the effects of single and multiple mutations at these positions indicate that this desensitized-like state results directly from the interaction among same-ring aromatic side chains (very likely through pi-pi interactions).