Examining the Role of the M1-M2 Loop in Cys-Loop Receptor Desensitization and Gating

Abstract

The intracellular M1-M2 loop of Cys-loop receptors connects the pore-lining M2 α-helix with the adjacent M1 α-helix, and it is located near the narrowest constriction of the open pore. In ganglionic acetylcholine receptors (AChRs), oxidation of the −4’ cysteine in this linker greatly affects the kinetics of recovery from desensitization; in glycine receptors (GlyRs), a naturally occurring mutation significantly alters the biophysical properties of these receptors, leading to hyperekplexia in humans. However, despite these findings, the M1-M2 loop's role in gating and desensitization of Cys-loop receptors has yet to receive thorough investigation. To this end, we engineered a series of mutations to the M1-M2 loops of the human α3β4 AChR, adult muscle AChR, and α1 GlyR. Mutations to the −4’ cysteine of α3β4 AChRs had only small effects on deactivation and responses to low-frequency repetitive stimulation in the whole-cell configuration. In the GlyR, alanine and threonine scans of the M1-M2 loop did not consistently change receptor kinetics, as measured in fast-perfused outside-out patches; however, the large patch-to-patch variability in GlyR behavior complicated our comparison across mutants. In the muscle AChR, alanine, glycine, proline, and valine scans were performed on the M1-M2 loop of the α1 subunit, and their effects were measured in fast-perfused outside-out patches. Remarkably, just as mutations to M2 consistently lead to a prolongation of the rate of deactivation, so too mutations to the M1-M2 loop invariably led to an increase of the rate of entry into desensitization; thus, just as residues in M2 very likely change microenvironments during channel gating, so too do residues in the M1-M2 loop during desensitization. This result suggests that the desensitization gate of the AChR is located near the narrowest constriction of the open channel.