Subunit-specific contribution to agonist binding and channel gating revealed by inherited mutation in muscle acetylcholine receptor M3-M4 linker

We trace the cause of congenital myasthenic syndromes in two patients to mutations in the ε subunit of the muscle acetylcholine receptor (AChR). Both patients harbour deletion of an asparagine residue in the ε subunit (sN436del) at the C-terminus of the cytoplasmic loop linking the third (M3) and fourth (M4) transmembrane domains. The presence of a null mutation in the second allele of the ε subunit shows that εN346del determines the phenotype. Endplate studies show markedly reduced expression of the εN346del-AChR and compensatory accumulation of fetal γ-AChR. Expression studies in HEK cells reveal decreased expression of γN436del-AChR and abnormally brief channel openings. Thus, neuromuscular transmission is compromised by AChR deficiency, fast channel kinetics of the εN346del-AChR and incomplete phenotypic rescue by γ-AChR. Single-channel kinetic analysis shows that the εN436del shortens channel openings by reducing stability of the diliganded receptor: rates of channel closing and of ACh dissociation are increased and the rate of channel opening is decreased. In addition to shortening the M3-M4 loop, εN436del shifts a negatively charged aspartic acid residue adjacent to M4; the effects of εN436del are shown to result from shortening of the M3-M4 loop and not from juxtaposition of a negative charge to M4. To determine whether the consequences of εN346del are subunit-specific, we deleted residues that align with εN436 in β, δ and α subunits. Each deletion mutant reduces AChR expression, but whereas the β and δ mutants curtail channel open duration, the α mutant strikingly prolongs open duration. Kinetic analysis reveals that the α mutant increases the stability of the diliganded receptor: rates of channel closing and of ACh dissociation are decreased and the rate of channel opening is increased. The overall studies reveal subunit asymmetry in the contributions of the M3-M4 loops in optimizing AChR activation through allosteric links to the channel and the agonist binding site.