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

1. During routine sequencing of our mouse muscle α subunit acetylcholine receptor channel (AChR) cDNA clones, we detected a discrepancy with the GenBank database entry (accession X03986). At nucleotides 1305-7 (residue 433, in the M4 domain) the database lists GTC which encodes a valine, while our putative 'wild-type' cDNA had the nucleotides GCC, which encodes an alanine. No other sequence differences were found. 2. PCR amplification of genomic DNA confirmed that the BALB/C mouse α subunit gene has a T nucleotide at position 1306, and, therefore, that the protein has a V at position 433 in the M4 segment. 3. In order to determine the functional consequences of this difference, either wild-type (V433) or mutant (A433) α subunits were co-expressed in HEK cells with mouse β, ε and δ subunits. Single-channel currents were recorded in cell-attached patches, and rate and equilibrium constants were estimated from open and closed durations obtained from a range of ACh concentrations. No significant differences were found between the activation rate constants or equilibrium constants of the V433 and A433 variants. 4. Kinetic modelling of αV433 AChR suggests that the two transmitter binding sites have similar dissociation equilibrium constants for acetylcholine (~ 160 μM, in 142 mM extracellular KCl). 5. Diliganded AChRs occupy a closed state that has a lifetime of ~ 1 ms. The rate constants for entering and leaving this state do not vary with the ACh concentration. 6. The kinetics of a mutant AChR that causes a slow channel congenital myaesthenic syndrome, αG153S, was re-examined. The properties of this mutant were similar with a V or an A at position α433.