Identification of various allosteric interaction sites on M1 muscarinic receptor using 125I-Met35-oxidized muscarinic toxin 7

Monoiodinated, Met35-oxidized muscarinic toxin 7 (MT7ox) was synthesized, and its affinity constants for free or N-methyl scopolamine (NMS)-occupied hM 1 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 [H-3] 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 I-125-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 I-125-MT7ox and [H-3] 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 hM(1) 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.