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. 1995 Jul 1;486(Pt 1):189–206. doi: 10.1113/jphysiol.1995.sp020802

Activation of recombinant mouse acetylcholine receptors by acetylcholine, carbamylcholine and tetramethylammonium.

Y Zhang 1, J Chen 1, A Auerbach 1
PMCID: PMC1156508  PMID: 7562635

Abstract

1. The kinetic properties of cloned mouse embryonic nicotinic acetylcholine receptors (AChRs) expressed in HEK 293 cells or Xenopus oocytes were examined using high concentrations of acetylcholine (ACh), carbamylcholine (CCh), or tetramethylammonium (TMA). The rate constants of agonist binding and channel gating were estimated by fitting kinetic models to idealized open and closed intervals over a range of agonist concentrations. 2. Once doubly liganded, TMA-activated receptors open at approximately 3000 s-1. The equilibrium binding constants for TMA are 525 and 12,800 microM. Doubly liganded CCh-activated receptors open at approximately 11,500 s-1; the equilibrium binding constants for this agonist are 14 and 570 microM. If we assume that doubly liganded, ACh-activated receptors open at 60,000 s-1, then the equilibrium binding constants for ACh are 20 and > 650 microM, similar to those for CCh. For all three agonists the higher affinity site both binds and releases agonists more slowly than does the lower affinity site. 3. ACh and CCh bind to the two sites equally rapidly, at approximately 2 x 10(7) and 4 x 10(7) M-1 s-1 at the first and second binding sites, respectively. Compared with ACh, the TMA association rate is approximately 100 times slower at the first binding site, and approximately 30 times slower at the second binding site. These results indicate that at both binding sites the association rate of TMA is not limited by diffusional or steric factors. 4. All three agonists dissociate from the receptor binding sites at similar rates. The dissociation rate for all agonists was approximately 40 times slower at the first binding site than at the second. These results suggest that the interaction of the quarternary amine moiety with the receptor determines the rate of release of the agonist, and that the nature of this interaction is quite different at the two binding sites. 5. Although the channel opening rates for the three agonists varied approximately 20-fold, the channel closing rates were not strongly agonist dependent, and varied less than 3-fold. We speculate that the ester moiety of the agonist promotes both rapid binding and fast opening of the ligand receptors, and that interactions of the quarternary amine moiety of the agonist with the receptor determine the channel closing rate constant.

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Selected References

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