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A re‐examination of adult mouse nicotinic acetylcholine receptor channel activation kinetics
Author(s) -
Salamone Frank N.,
Zhou Ming,
Auerbach Anthony
Publication year - 1999
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1111/j.1469-7793.1999.0315r.x
Subject(s) - acetylcholine receptor , nicotinic acetylcholine receptor , complementary dna , mutant , protein subunit , chemistry , microbiology and biotechnology , acetylcholine , reaction rate constant , kinetics , alanine , biophysics , biology , receptor , biochemistry , gene , amino acid , endocrinology , physics , quantum mechanics
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.