Mutations in the M4 domain of the Torpedo californica nicotinic acetylcholine receptor alter channel opening and closing.

We studied the functional effects of single amino acid substitutions in the postulated M4 transmembrane domains of Torpedo californica nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes at the single-channel level. At low ACh concentrations and cold temperatures, the replacement of wild-type alpha418Cys residues with the large, hydrophobic amino acids tryptophan or phenylalanine increased mean open times 26-fold and 3-fold, respectively. The mutation of a homologous cysteine in the beta subunit (beta447Trp) had similar but smaller effects on mean open time.

Slow-channel transgenic mice: a model of postsynaptic organellar degeneration at the neuromuscular junction.

The slow-channel congenital myasthenic syndrome (SCCMS) is a dominantly inherited disorder of neuromuscular transmission characterized by delayed closure of the skeletal muscle acetylcholine receptor (AChR) ion channel and degeneration of the neuromuscular junction. The identification of a series of AChR subunit mutations in the SCCMS supports the hypothesis that the altered kinetics of the endplate currents in this disease are attributable to inherited abnormalities of the AChR. To investigate the role of these mutant AChR subunits in the development of the synaptic degeneration seen in the SCCMS, we have studied the properties of the AChR mutation, epsilonL269F, found in a family with SCCMS, using both in vitro and in vivo expression systems.

Mouse-Torpedo chimeric alpha-subunit used to probe channel-gating determinants on the nicotinic acetylcholine receptor primary sequence.

1. To determine if structural domains are important for nicotinic acetylcholine receptor (nAChr) channel function, six mouse-Torpedo chimeric alpha-subunits were constructed (Fig. 2) and coexpressed with Torpedo californica beta-, gamma-, and delta-subunits in Xenopus laevis oocytes.

Tryptophan substitutions at the lipid-exposed transmembrane segment M4 of Torpedo californica acetylcholine receptor govern channel gating.

Our previous amino acid substitutions at the postulated lipid-exposed transmembrane segment M4 of the Torpedo californica acetylcholine receptor (AChR) focused on the alpha C418 position. A tryptophan substitution on the alpha C418 produced a 3-fold increase in normalized macroscopic response to acetylcholine in voltage-clamped Xenopus laevis oocytes (Lee et al., 1994).

Effects of antibody binding on structural transitions of the nicotinic acetylcholine receptor.

Patch-clamping and photoaffinity-labeling techniques were used to study the effects of binding of monoclonal antibodies (mAbs) on the function of Torpedo californica nicotinic acetylcholine receptor (nAChR). The rat anti-Torpedo nAChR mAbs examined here are known to inhibit ligand binding to either the high-affinity (mAb 247) or both the high- and low-affinity binding sites (mAb 370 and mAb 387) [Mihovilovic, M. & Richman, D.