Congenital myasthenic syndrome caused by prolonged acetylcholine receptor channel openings due to a mutation in the M2 domain of the epsilon subunit.

Abstract

In a congenital myasthenic syndrome with a severe endplate myopathy, patch-clamp studies revealed markedly prolonged acetylcholine receptor (AChR) channel openings. Molecular genetic analysis of AChR subunit genes demonstrated a heterozygous adenosine-to-cytosine transversion at nucleotide 790 in exon 8 of the E-subunit gene, predicting substitution of proline for threonine at codon 264 and no other mutations in the entire coding sequences of genes encoding the a, f3, 8, and £ subunits. Genetically engineered mutant AChR expressed in a human embryonic kidney fibroblast cell line also exhibited markedly prolonged openings in the presence of agonist and even opened in its absence. The Thr-264 -> Pro mutation in the E subunit involves a highly conserved residue in the M2 domain lining the channel pore and is likely to disrupt the putative M2 a-helix. Our findings indicate that a single mutation at a critical site can greatly alter AChR channel kinetics, leading to a congenital myasthenic syndrome. This observation raises the possibility that mutations involving subunits of other ligand-gated channels may also exist and be the basis of various other neurologic or psychiatric disorders.Congenital myasthenic syndromes (CMS) are inherited disorders in which the safety margin of neuromuscular transmission is compromised by one or more specific mechanisms. In contrast to myasthenia gravis, antibodies against the acetylcholine (ACh) receptor (AChR) are absent. Until now, the CMS have been characterized by clinical, morphological, and electrophysiological criteria. Those CMS identified to date include endplate (EP) acetylcholinesterase deficiency, presynaptic abnormalities that effect the release or size of transmitter quanta, or AChR deficiency with or without an associated kinetic abnormality of AChR (1). In the case of a kinetic abnormality detected at the single-channel level, a mutation involving an AChR subunit is likely. Identification of such mutations is of interest because they can provide insights into structure-function relationships of AChR and allow for genetic counseling and prevention. We report here discovery of a spontaneous mutation in human AChR. We show that the mutation leads to markedly prolonged openings of the AChR channel and is the cause of a CMS. MATERIALS AND METHODSClinical Data. A woman, now 20 years old, had myasthenic symptoms since the neonatal period, a decremental electromyographic response on stimulation of motor nerves, negative tests for anti-AChR antibodies, and no history of similarly affected relatives. Previous studies of an intercostal muscle specimen from this patient at age 17 (2) revealed an EP myopathy with many degenerating junctional folds, abundant EP acetylcholinesterase, a reduced number (39%) of AChRs per EP, normal quantal release by nerve impulse, and reduced amplitude and a markedly prolonged and biexponential decay of miniature EP currents. Spectral analysis of AChR-induced current noise was best fitted by the sum of two lorentzians, consistent with two chan...

Increased neuromuscular activity causes axonal defects and muscular degeneration

“…Furthermore, several SCCMS mutations in the M2 segment have been shown to result in the normal expression and assembly of AChRs, but generate prolonged channel openings by stabilizing the open state and decreasing the rate of channel closure (Croxen et al, 1997;Engel et al, 1996;Milone et al, 1997;Ohno et al, 1995). These observations support our interpretation that the nic-1 twister dbn12 L258P substitution functions as a gain-of-function kinetic mutation.…”

“…Alternatively, postsynaptic defects might occur even before the first growth cones arrive, as the mutant AChR channel may open for extended periods in the absence of ACh ligand. In fact, mutations in the M2 segment of the human α, β and ε subunits have been shown to cause unusually high rates of spontaneous channel openings (Engel et al, 1996;Milone et al, 1997;Ohno et al, 1995). Although we have not determined whether the αL258P mutation renders the AChR channel leaky, the severe myofibril and AChR cluster defects observed in 17-hpf homozygous mutants, despite the presence of only few synaptic contacts, are consistent with the possibility that nic-1 twister dbn12 channel opens extensively in a ligand-independent manner.…”

Agonist activation of α7 nicotinic acetylcholine receptors via an allosteric transmembrane site

“…For example, increased levels of spontaneous opening of nAChRs have been reported in several mutated nAChRs (29,40,41) and as a result of the omission of subunits in heterologously expressed muscle nAChRs (42). In the same way, it is plausible that stabilization of the open conformation of a nAChR might occur as a result of ligands binding to multiple distinct sites.…”

Co-expression of the neuronal α 7 and L247T α 7 mutant subunits yields hybrid nicotinic receptors with properties of both wild-type α 7 and α 7 mutant homomeric receptors

“…In the last few years, a considerable number of mutations of single amino acids, some of which may be the structural determinants of neurological disorders, have been found to affect nAcChoR function greatly (15)(16)(17). In particular, a large body of evidence demonstrates that mutations of highly conserved leucines in the leucine ring appreciably alter the function of both neuronal ␣ 7 and muscle ␣␤␥␦ nAcChoRs (4)(5)(6)(7)(8).…”