A novel fast-channel myasthenia caused by mutation in ß subunit of AChR reveals subunit-specific contribution of the intracellular M1-M2 linker to channel gating.

Genetic variants causing the fast-channel congenital myasthenic syndrome (CMS) have been identified in the α, δ, and ε but not the ß subunit of acetylcholine receptor (AChR). A 16-year-old girl with severe myasthenia had low-amplitude and fast-decaying miniature endplate potentials. Mutation analysis revealed two heteroallelic variants in CHRNB1 encoding the AChR ß subunit: a novel c.812C>T (p.P248L) variant in M1-M2 linker (p.P271L in HGVS nomenclature), and a ~430 bp deletion causing loss of exon 8 leading to frame-shift and a premature stop codon (p.G251Dfs*21). P248 is conserved in all ß subunits of different species, but not in other AChR subunits. Measurements of radio-labeled α-bungarotoxin binding show that ßP248L reduces AChR expression to 60% of wild-type. Patch clamp recordings of ACh-elicited single channel currents demonstrate that ßP248L shortens channel opening bursts from 3.3 ms to 1.2 ms, and kinetic analyses predict that the decay of the synaptic response is accelerated 2.4-fold due to reduced probability of channel reopening. Substituting ßP248 with threonine, alanine or glycine reduces the burst duration to 2.3, 1.7, and 1.5 ms, respectively. In non-ß subunits, substituting leucine for residues corresponding to ßP248 prolongs the burst duration to 4.5 ms in the α subunit, shortens it to 2.2 ms in the δ subunit, and has no effect in the ε subunit. Conversely, substituting proline for residues corresponding to ßP248 prolongs the burst duration to 8.7 ms in the α subunit, to 4.6 ms in the δ subunit, but has no effect in the ε subunit. Thus, this fast channel CMS is caused by the dual defects of ßP248L in reducing expression of the mutant receptor and accelerating the decay of the synaptic response. The results also reveal subunit-specific contributions of the M1-M2 linker to the durations of channel opening bursts.

Mutations causing congenital myasthenia reveal principal coupling pathway in the acetylcholine receptor ε-subunit.

We identify 2 homozygous mutations in the ε-subunit of the muscle acetylcholine receptor (AChR) in 3 patients with severe congenital myasthenia: εR218W in the pre-M1 region in 2 patients and εE184K in the ß8-ß9 linker in 1 patient. Arg218 is conserved in all eukaryotic members of the Cys-loop receptor superfamily, while Glu184 is conserved in the α-, δ-, and ε-subunits of AChRs from all species. εR218W reduces channel gating efficiency 338-fold and AChR expression on the cell surface 5-fold, whereas εE184K reduces channel gating efficiency 11-fold but does not alter AChR cell surface expression. Determinations of the effective channel gating rate constants, combined with mutant cycle analyses, demonstrate strong energetic coupling between εR218 and εE184, and between εR218 and εE45 from the ß1-ß2 linker, as also observed for equivalent residues in the principal coupling pathway of the α-subunit. Thus, efficient and rapid gating of the AChR channel is achieved not only by coupling between conserved residues within the principal coupling pathway of the α-subunit, but also between corresponding residues in the ε-subunit.