Brain Advance Access published online on December 22, 2004
Brain, doi:10.1093/brain/awh364
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1 Department of Neurology and Neuromuscular Research Laboratory, Mayo Clinic, Rochester, MN, USA
* To whom correspondence should be addressed. Summary We trace the cause of congenital myasthenic syndromes in two patients to mutations in the
Received August 12, 2004
Revised November 4, 2004
Accepted November 5, 2004
Article
Subunit-specific contribution to agonist binding and channel gating revealed by inherited mutation in muscle acetylcholine receptor M3-M4 linker
2 Department of Physiology and Biophysics and Receptor Biology Laboratory, Mayo Clinic, Rochester, MN, USA
Andrew G. Engel, E-mail: age{at}mayo.edu
Abstract 
subunit of the muscle acetylcholine receptor (AChR). Both patients harbour deletion of an asparagine residue in the subunit (N436del) 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 subunit shows that N346del determines the phenotype. Endplate studies show markedly reduced expression of the N346del-AChR and compensatory accumulation of fetal 
-AChR. Expression studies in HEK cells reveal decreased expression of N436del-AChR and abnormally brief channel openings. Thus, neuromuscular transmission is compromised by AChR deficiency, fast channel kinetics of the N346del-AChR and incomplete phenotypic rescue by -AChR. Single-channel kinetic analysis shows that the N436del 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, N436del shifts a negatively charged aspartic acid residue adjacent to M4; the effects of N436del 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 N346del are subunit-specific, we deleted residues that align with N436 in 
, 
and 
subunits. Each deletion mutant reduces AChR expression, but whereas the and mutants curtail channel open duration, the mutant strikingly prolongs open duration. Kinetic analysis reveals that the 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.
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