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First published on January 26, 2006; DOI: 10.1124/mol.105.020883

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Received for publication November 18, 2005.
Revised January 24, 2006.
Accepted for publication January 24, 2006.

Identification of various allosteric interaction sites on M1 muscarinic receptor using [125I]MT7ox toxin

Carole Fruchart-Gaillard 1, Gilles Mourier 1, Catherine Marquer 1, Andre Menez 1, Denis Servent 1*

1 CEA

* Address correspondence to: E-mail: denis.servent{at}cea.fr

Abstract

A monoiodinated MT7ox toxin was synthesized and its affinity constants for free or NMS-occupied hM1 receptor were measured directly by equilibrium and kinetic binding experiments. Identical values were obtained with the two types of assay methods, 14 pM and 0.9 nM in free or NMS-liganded receptor states, respectively, highlighting a strong negative cooperativity between this allosteric toxin and NMS. Identical results were obtained with indirect binding experiments with [3H]NMS using the ternary complex model, clearly demonstrating the reciprocal nature of this cooperativity. Furthermore, the effects of various orthosteric and allosteric agents on the dissociation kinetic of [125I]MT7ox were measured and show that, with the exception of the MT1 toxin, all the ligands studied (NMS, atropine, gallamine, brucine, tacrine, staurosporine and KT5720) interact allosterically with MT7 toxin. Equilibrium binding experiments with [125I]MT7ox and [3H]NMS were conducted to reveal the effects of these ligands on the free receptor and affinity constants (pKx values) were calculated using the allosteric ternary complex model. Our results suggest that MT7 toxin interacts with hM1 receptor at a specific allosteric site which may partially overlap those previously identified for "classical" or "atypical" allosteric agents and highlight the potential of this new allosteric tracer in studying allosterism at muscarinic receptors.


Key words: Muscarinic cholinergic, Receptor binding studies


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C. Fruchart-Gaillard, G. Mourier, C. Marquer, E. Stura, N. J. M. Birdsall, and D. Servent
Different Interactions between MT7 Toxin and the Human Muscarinic M1 Receptor in Its Free and N-Methylscopolamine-Occupied States
Mol. Pharmacol., December 1, 2008; 74(6): 1554 - 1563.
[Abstract] [Full Text] [PDF]


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