Using a Radioalloster to Test Predictions of the Cooperativity Model for Gallamine Binding to the Allosteric Site of Muscarinic Acetylcholine M₂Receptors

Abstract

The muscarinic M(2) receptor contains an orthosteric and an allosteric site. Binding of an allosteric agent may induce a shift alpha of the equilibrium dissociation constant K(D) of a radioligand for the orthosteric site. According to the cooperativity model, the K(A) of alloster binding is expected to be shifted to an identical extent depending on whether the orthosteric site is occupied by the orthoster or not. Here, the novel radioalloster [(3)H]dimethyl-W84 (N,N'-bis[3-(1,3-dihydro-1, 3-dioxo-4-methyl-2H-isoindol-2-yl)propyl]-N,N,N',N'-tetramethyl-1, 6-hexanediaminium diiodide) was applied to directly measure the K(A) shift induced for the prototype allosteric modulator gallamine by binding of N-methylscopolamine (NMS) to the orthosteric site of porcine heart M(2) receptors (4 mM Na(2)HPO(4), 1 mM KH(2)PO(4), pH 7.4; 23 degrees C; data are means +/- S.E.). First, in the common way, the concentration-dependent inhibition by gallamine of [(3)H]NMS equilibrium binding was measured and analyzed using the cooperativity model, which yielded for the affinity of gallamine binding at free receptors a pK(A)= 8.35 +/- 0.09 and a cooperativity factor alpha = 46 (n = 5). The dissociation constant for gallamine binding at NMS-occupied receptors was predicted as p(alpha. K(A)) = 6.69. Labeling of the allosteric site by [(3)H]dimethyl-W84 allowed the measure of competitive displacement curves for gallamine. The K(i) for gallamine at free receptors amounted to pK(i,-NMS) = 8.27 +/- 0.39 (n = 5), which is in line with the prediction of the cooperativtiy model. In the presence of 1 microM NMS, to occupy the orthosteric site, gallamine displaced [(3)H]dimethyl-W84 with pK(i, +NMS) = 6.60 +/- 0.19 (n = 3). Thus, the NMS-induced pK(i) shift amounted to 47, which matches the predicted value of alpha = 46. These results validate the cooperativity model.

Mechanism of Corticotropin-Releasing Factor Type I Receptor Regulation by Nonpeptide Antagonists

“…For other GPCRs, allosteric modulation is consistent with a theoretical model, the allosteric ternary complex model (Stockton et al, 1983;Ehlert, 1988;Lazareno and Birdsall, 1995;Trankle et al, 1999;Leppik and Birdsall, 2000). In this model, the behavior of the allosteric ligand (e.g., NBI 35965) is defined by its affinity for the receptor and by the cooperativity between binding of allosteric and orthosteric ligand (e.g., CRF).…”

“…In particular, the data are fully consistent with (Tables 2 and 3; Appendix). This reciprocal relationship has been demonstrated for gallamine and N-methylscopolamine at the M 2 muscarinic acetylcholine receptor (Trankle et al, 1999).…”

“…As described above, the model predicts that the cooperative effect of N binding on the affinity of L for R is the same as the effect of L binding on the affinity of N for R (Trankle et al, 1999). This prediction was tested by measuring the effect of unlabeled peptides on equilibrium binding of [ 3 H]NBI 35965 to the R state (Fig.…”

[125I]2-(2-Chloro-4-iodo-phenylamino)-5-methyl-pyrroline (LNP 911), a High-Affinity Radioligand Selective for I1Imidazoline Receptors

“…LNP 911 behaves as an allosteric enhancer according to its physiological properties; conversely, an agonist (moxonidine) was shown to change the binding parameters of LNP 911. Such an agonist effect on the allosteric ligand binding has already been demonstrated on the muscarinic receptor (Trankle et al, 1999). LNP 911 might combine competitive binding and allosteric enhancement properties that look like the characteristics of allosteric modulators for adenosine or metabotropic glutamate 1 receptors (Gao et al, 2001; Knoflach et al, 2001).…”

Being mindful of seven‐transmembrane receptor ‘guests’ when assessing agonist selectivity

“…G-protein), the same is true in the reverse direction, namely the guest will increase the affinity of the receptor for the modulator. An example of this effect has been shown experimentally with [ 3 H]-dimethyl-W84 and [ 3 H]-NMS binding on muscarinic M2 receptors (Trankle et al, 1999). The influence of different coupling molecules (i.e.…”