The energetic consequences of loop 9 gating motions in acetylcholine receptor‐channels

Non technical summary Muscle cells have receptors that are activated by the neurotransmitter acetylcholine. The probability that these channels conduct ions across cell membranes increases dramatically when transmitter molecules are present at two binding sites, which are far from the region that regulates ionic conductance. In order to understand the molecular basis of receptor ‘gating’ we seek to learn how neurotransmitters and other small molecules activate this protein. We used single‐channel electrophysiology of receptors expressed in tissue‐cultured cells to study the effects of mutations at the C‐terminus of loop 9, a region near intra‐protein interfaces that are known to be important with regard to gating and assembly. We found that the mutation had modest but measurable effects on channel gating (mainly those in the epsilon subunit). We also found that mutations of loop 9 in the alpha subunit increase the kinetic heterogeneity of gating, which suggests that they alter the stability of the extracellular transmembrane domain interface. Acetylcholine receptor‐channels (AChRs) mediate fast synaptic transmission between nerve and muscle. In order to better‐understand the mechanism by which this protein assembles and isomerizes between closed‐ and open‐channel conformations we measured changes in the diliganded gating equilibrium constant (E2) consequent to mutations of residues at the C‐terminus of loop 9 (L9) in the α and ɛ subunits of mouse neuromuscular AChRs. These amino acids are close to two interesting interfaces, between the extracellular and transmembrane domain within a subunit (E–T interface) and between primary and complementary subunits (P–C interface). Most α subunit mutations modestly decreased E2 (mainly by slowing the channel‐opening rate constant) and sometimes produced AChRs that had heterogeneous gating kinetic properties. Mutations in the ɛ subunit had a larger effect and could either increase or decrease E2, but did not induce kinetic heterogeneity. There are broad‐but‐weak energetic interactions between αL9 residues and others at the αE–T interface, as well as between the ɛL9 residue and others at the P–C interface (in particular, the M2–M3 linker). These interactions serve, in part, to maintain the structural integrity of the AChR assembly at the E–T interface. Overall, the energy changes of L9 residues are significant but smaller than in other regions of the protein.