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Identification of Various Allosteric Interaction Sites on M₁ Muscarinic Receptor Using ¹²⁵I-Met35-Oxidized Muscarinic Toxin 7

Commissariat à l'Energie Atomique, Département d'Ingénierie et d'Etude des Protéines, Gif-sur-Yvette, France

Monoiodinated, Met35-oxidized muscarinic toxin 7 (MT7ox) was synthesized, and its affinity constants for free or N-methyl scopolamine (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, except for the MT1 toxin, all of the ligands studied [NMS, atropine, gallamine, brucine, tacrine, staurosporine, and (9S,10S,12R)-2,3,9,10,11-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid hexyl ester (KT5720)] interact allosterically with muscarinic toxin 7. 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 (pK_x 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 identified previously for "classic" or "atypical" allosteric agents and highlight the potential of this new allosteric tracer in studying allosterism at muscarinic receptors.

Address correspondence to: Dr. D. Servent, CEA, Département d'Ingénierie et d'Etude des Protéines, 91191 Gif-sur-Yvette, France. E-mail: denis.servent{at}cea.fr

This article has been cited by other articles:

				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]