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

Abstract

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. 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. 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. 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). 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. 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.

The γ-aminobutyric acid type A (GABA A ) receptor-associated protein (GABARAP) promotes GABA A receptor clustering and modulates the channel kinetics

“…channel closing upon agonist deoccupancy. Deactivation rates measured are consistent with literature values for GABA A (36,43,44) and glutamate or nicotinic receptors (21,45,46). These deactivation rates are slow (50-100 ms) compared with the time required to remove GABA from the recording chamber (Ͻ10 ms), so the differences measured are highly significant.…”

The dissociation of acetylcholine from open nicotinic receptor channels

“…To better appreciate the goodness of this fit, Fig. 3c The opening and closing rate constants of wild-type diliganded AChRs in the presence of ACh (␤2 ACh and ␣2 ACh) were taken as 50,000 s Ϫ1 and 2,000 s Ϫ1 , respectively, according to previously published values (10). The corresponding values for the mutants were calculated from the rate-constant estimates in the presence of choline (16) by applying the relation k mutant ACh͞kwild-type ACh ϭ kmutant choline͞kwild-type choline, where k is either the opening or closing rate constant (13).…”

“…and dissociates from closed diliganded receptors at Ϸ50,000 s Ϫ1 (10,14,15). Single-channel kinetic analysis, however, had not been exploited thus far to elucidate the openchannel agonist-binding kinetics.…”

Serum choline activates mutant acetylcholine receptors that cause slow channel congenital myasthenic syndromes

“…At high agonist concentrations, openings occur in clusters that reflect the activity of a single AChR (30). The cluster-based analysis method is described in detail elsewhere (21). Briefly, clusters were defined as a series of openings separated by closed intervals longer than some critical duration.…”

“…In adult wild-type mouse AChR, the two agonist-binding sites have similar equilibrium dissociation constants (21), and the following kinetic scheme was used to interpret the singlechannel currents:…”