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

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.

Aromatic Residues ∈Trp-55 and δTrp-57 and the Activation of Acetylcholine Receptor Channels

“…4B) are evidence that the association of ACh to the transmitter binding site is not diffusionlimited, in which case perturbation of K d should mainly arise from changes in the dissociation rate constant rather than the association rate constant (⌽ binding ϳ 0). Similarly, high ⌽ binding values were found previously for a mutational series of AChR residue ␣Asp-152 (37), and for different agonists of WT AChRs (where agonist K d values are mainly determined by the k ϩ rate constant) (38). Further support for the idea that agonist binding requires a conformational change is that the association rate constant is not diffusion-limited.…”

“…Further support for the idea that agonist binding requires a conformational change is that the association rate constant is not diffusion-limited. The smaller agonist, tetramethylammonium ion, associates ϳ50 times more slowly than the larger ACh molecule (38). The experimental observations that agonists and the above-mentioned residues are bound-like in energy at the transition state for binding indicates that there is a conformational change associated with ligand binding, and at this transition state for this reaction (when the channel is still nonconducting) the ligand is in intimate contact with the protein.…”

The nicotinic receptor ligand binding domain

“…The early findings showed that the fully occupied receptor switches between closed and open states very rapidly, occurring in tens of microseconds (Colquhoun and Sakmann, 1981), but there was uncertainty in assigning exponential components in the dwell time distributions to particular states in a kinetic scheme Steinbach, 1986, 1987). Eventually, by fitting descriptions such as Scheme 1 to dwell times obtained over a wide range of ACh concentrations, a complete set of rate constants governing receptor activation was achieved (Sine et al, 1990;Zhang et al, 1995). The measured rate constants indicated that ACh binds at a rate some 10-fold slower than the rate of diffusion, the rate of ACh dissociation from the Torpedo receptor differed markedly at the two binding sites (Sine et al, 1990), and opening of the receptor channel occurred within tens of microseconds (Maconochie et al, 1995).…”

A fluorophore attached to nicotinic acetylcholine receptor βM2 detects productive binding of agonist to the αδ site

“…Kinetic analyses of single-channel and macroscopic function suggests that in the resting state, the affinity of ACh for the two sites differs by a factor of 30 (8). Experiments with partially assembled ␣␥ and ␣␦ dimers or with fully assembled, pentameric ␥-less or ␦-less receptors show that the ␣␦ site has the higher affinity for ACh or carbamylcholine (9)(10)(11).…”