A re-examination of adult mouse nicotinic acetylcholine receptor channel activation kinetics

1. During routine sequencing of our mouse muscle alpha subunit acetylcholin e receptor channel (AChR) cDNA clones, we detected a discrepancy with the G enBank database entry (accession X03986). At nucleotides 1305-7 (residue 43 3, in the M4 domain) the database lists GTC which encodes a valine, while o ur putative 'wild-type' cDNA had the nucleotides GCC, which encodes an alan ine. No other sequence differences were found. 2. PCR amplification of genomic DNA confirmed that the BALB/C mouse alpha s ubunit gene has a T nucleotide at position 1306, and, therefore, that the p rotein has a V at position 433 in the M4 segment. 3. In order to determine the functional consequences of this difference, ei ther wild-type (V433) or mutant (A433) alpha subunits were co-expressed in HEK cells with mouse beta, epsilon and delta subunits. Single-channel curre nts were recorded in cell-attached patches, and rate and equilibrium consta nts were estimated from open and closed durations obtained from a range of ACh concentrations. No significant differences were found between the activ ation rate constants or equilibrium constants of the V433 and A433 variants . 4. Kinetic modelling of alpha V433 AChR suggests that the two transmitter b inding sites have similar dissociation equilibrium constants for acetylchol ine (similar to 160 mu M, in 142 mM extracellular KCl). 5. Diliganded AChRs occupy a closed state that has a lifetime of similar to 1 ms. The rate constants for entering and leaving this state do not vary w ith the ACh concentration. 6. The kinetics of a mutant AChR that causes a slow channel congenital myae sthenic syndrome, alpha G153S, was re-examined. The properties of this muta nt were similar with a V or an A at position alpha 433.