Is the AChR a Cuckoo-Clock? Local and Remote Interactions of the α M2-3 Linker in Gating

The αM2-3 linker of pentameric ligand-gated ion channels (18-residues in AChRs) is at the interface of the extracellular and transmembrane domains, which twist relative to each other early in the channel-opening process (phi∼0.7). Phi-analyses of mouse muscle AChRs indicate that residues in this linker and those at the transmitter binding sites share a common, even-earlier position in the gating transition state (phi∼0.9). To understand the mechanism for the similar behavior of these two separated regions (∼30 A), whose combined motions appear to trigger channel-opening, we used single-channel free-energy mutant cycle analyses (A-A pairs) to measure the extents to which α-subunit linker residues (265-268) interact with other amino acids. The significant interactions (>0.5 kcal/mol) were as follows (n pairs tested; energies in kcal/mol): i) αloop2 (7), αE45-αP265 ∼+3.5 (positive is unfavorable); ii) αcys-loop (2), αV132-αS268 ∼+1; iii) agonist sites (3), αS268 and αY93 or αY198 showed no coupling but the interaction with αY190 was ∼-1.2 (favorable); iv) intra-linker (4), no significant coupling; v) eloop9 (6), eG183-αS268 and eG183-αT267 ∼+1.4; vi) epre-M1 (1), eL221-αT267, no coupling. The large, unfavorable interaction between αP265 and its loop 2 neighbor αE45 (∼5 A separation in GLIC) suggests that these side chains share a common energetic environment that is perturbed by the gating conformational change (twist). The long-distance, favorable interaction between αS268A in the linker and αY190A at the agonist site is more surprising. We hope to elucidate a network of apposed residues whose shared energy changes might be associated with the twist. More importantly, we are exploring the possibility that there is a long-distance energy transfer between the agonist binding site and transmembrane domain via the backbone that is not by a cuckoo-clock-like mechanical linkage.