A Re-examination of Adult Mouse Nicotinic Acetylcholine Receptor Channel Activation Kinetics

1. During routine sequencing of our mouse muscle alpha 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 alpha 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) alpha subunits were co-expressed in HEK cells with mouse beta, epsilon and delta 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 alphaV433 AChR suggests that the two transmitter binding sites have similar dissociation equilibrium constants for acetylcholine ( approximately 160 microM in 142 mM extracellular KCl). 5. Diliganded AChRs occupy a closed state that has a lifetime of approximately 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, alphaG153S, was re-examined. The properties of this mutant were similar with a V or an A at position alpha433.