Journal Article

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

Xin-Ming Shen, Kinji Ohno, Steven M. Sine and Andrew G. Engel

in Brain

Published on behalf of The Guarantors of Brain

Volume 128, issue 2, pages 345-355
Published in print February 2005 | ISSN: 0006-8950
Published online December 2004 | e-ISSN: 1460-2156 | DOI: http://dx.doi.org/10.1093/brain/awh364
Subunit-specific contribution to agonist binding and channel gating revealed by inherited mutation in muscle acetylcholine receptor M3–M4 linker

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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 (εN436del) 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 εN436del-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.

Keywords: acetylcholine receptor; congenital myasthenic syndrome; M3–M4 loop; mutagenesis; single-channel patch-clamp recordings; ACh = acetylcholine; AChR = acetylcholine receptor; α-bgt = α-bungarotoxin; CMS = congenital myasthenic syndrome; EP = endplate; HEK = human embryonic kidney; M = transmembrane domain; MEPC = miniature endplate current

Journal Article.  7054 words.  Illustrated.

Subjects: Neurology ; Neuroscience

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