Modal gating of muscle nicotinic acetylcholine receptors
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Many ion channels exhibit multiple patterns of kinetic activity in single-channel currents. This behavior is rare in WT mouse muscle nicotinic acetylcholine receptors (AChRs), where A 2 C↔A 2 O gating events are well-described by single exponentials. Also, single-channel open probability (PO) is essentially homogeneous at a given agonist concentration in the WT receptors. Here I report that perturbations of almost all the residues in loop C (α188-α199, at the agonist binding site) generate heterogeneity in PO (‘modes’). Such unsettled activity was apparent with an alanine substitution at all positions in loop C (except αY190 and αY198) and with different side chain substitutions at αP197 for both adult- and fetal-type AChRs. I used single channel electrophysiology along with site-directed mutagenesis to study modal gating in AChRs consequent to mutations/deletions in loop C. The multiple patterns of kinetic activity arose from the difference in agonist affinity rather than in intrinsic AChR gating. Out of the four different agonists used to study the modal behavior, acetylcholine (ACh) showed a higher degree of kinetic heterogeneity compared to others. The time constant for switching between modes was long (~mins), suggesting that they arise from alternative, stable protein conformations. By studying AChRs having only 1 functional binding site, I attempted to find the source of the affinity difference, which was traced mainly to the αδ agonist site. Affinity at the neurotransmitter binding site is mainly determined by a core of five aromatic residues (αY93, αW149, αY190, αY198 and δW57). Phenylalanine substitutions at all aromatic residues except αY93 resulted in elimination of modes. Modes were also eliminated by alanine mutation at δW57 on the complementary side but not at other aromatics. Also, by substituting four γ subunit residues into the δ subunit on the complementary β sheet, I found that modes were reduced. Based on our results, we propose that WT loop C has an important role in determining resting affinity, in part by making stable interactions with the complementary surface of the αδ binding pocket. We suggest a possible structural basis for the fluctuations caused by loop C perturbations and propose that at the αδ agonist binding site, both loop C and the complementary subunit surface can adopt alternative conformations and interact with each other with respect to the aromatic core, to cause the variations in affinity.