Identification of Various Allosteric Interaction Sites on M₁Muscarinic Receptor Using¹²⁵I-Met35-Oxidized Muscarinic Toxin 7

Abstract

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 [3 H]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 125 I-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:3Ј,2Ј,1Ј-kl]pyrrolo [3,4-i][1,6]benzodiazocine-10-carboxylic acid hexyl ester (KT5720)] interact allosterically with muscarinic toxin 7. Equilibrium binding experiments with 125 IMT7ox and [3 H]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.An increasing body of evidence now shows that many Gprotein-coupled receptors are susceptible to allosteric modulation (Christopoulos and Kenakin, 2002), and this phenomenon is particularly well documented for the five muscarinic acetylcholine receptor subtypes (Tucek and Proska, 1995;Birdsall et al., 1997;Ellis, 1997;Lazareno et al., 2004;Birdsall and Lazareno, 2005). These receptors are characterized by at least two distinct binding sites. Agonists and competitive antagonists bind to the orthosteric site, located inside the transmembrane domain, whereas allosteric agents induce a positive or a negative alteration of the orthosteric ligand affinity by interacting with an allosteric site, located more extracellularly (Stockton et al., 1983;Ellis et al., 1993;Ellis, 1997;Trankle and Mohr, 1997;Christopoulos et al., 1998;Christopoulos and Kenakin, 2002). Furthermore, recent publications report the presence of at least two distinct allosteric sites on muscarinic receptors selective for distinct classes of allosteric agents, suggesting that it is possible for three small ligands to bind simultaneously to a muscarinic receptor (Birdsall et al., 2001;Lazareno et al., 2002). For instance, staurosporine and other indolocarbazole compounds have been shown to interact at a second allosteric site on hM 1 receptor, associated with a positive cooperativity with ACh (Lazareno et al., 20...

Different Interactions between MT7 Toxin and the Human Muscarinic M1 Receptor in Its Free and N-Methylscopolamine-Occupied States

“…3 H]NMS, respectively (Fruchart-Gaillard et al, 2006). The similar results obtained using the two approaches confirm the reciprocity of the NMS-MT7 cooperative interaction and validate the allosteric ternary complex model used to study the MT7-hM 1 interaction.…”

“…Current evidence points to the high affinity, large subtype selectivity, and allosteric nature of the interaction of the MT7 toxin with the hM 1 receptor (Max et al, 1993;Carsi and Potter, 2000;Olianas et al, 2000;Mourier et al, 2003;Fruchart-Gaillard et al, 2006). To better understand how MT7 binds to the free and NMS-occupied hM 1 receptor and to progress toward the structural modeling of these interactions, we have investigated how different single amino acid modifications introduced into the MT7 sequence altered its potency, determined using both equilibrium and dissociation kinetics experiments as well as functional assays.…”

“…Expression of recombinant MT7 in the baculovirus vector-Sf9 insect cell system (Nasman et al, 2000) and in Pichia pastoris (Krajewski et al, 2001) has resulted in relatively low yields. However, using a one-step solid-phase chemical synthesis, we have been able to achieve good yields of muscarinic toxins (Mourier et al, 2003;Fruchart-Gaillard et al, 2006) and have shown that synthetic MT1 and MT7 toxins possess physicochemical and pharmacological profiles identical to those of purified natural toxins. Furthermore, we have been able to demonstrate that the two toxins seem to recognize the hM 1 receptor differently (Mourier et al, 2003).…”

Exploration of the Orthosteric/Allosteric Interface in Human M1 Muscarinic Receptors by Bitopic Fluorescent Ligands

“…Brucine was a very partial displacer, featuring almost neutral binding cooperativity with [ 3 H]NMS. Analyses according to the allosteric ternary complex model yielded affinity (K x ) and cooperativity factor (a) estimates (Table 2) in agreement with literature reports for gallamine (Matsui et al, 1995;Lazareno et al, 2000;Fruchart-Gaillard et al, 2006) and brucine Fruchart-Gaillard et al, 2006). Bo(10)Pz and Bo(15)Pz displayed an apparent competitive mode of interaction (Fig.…”

“…As expected, drug affinity constants at the free receptor do not significantly vary with tracer type or underlying binding mechanism. They are in overall agreement with affinity values reported in the literature ([ 3 H]NMS binding, M1-enriched membrane preparations) for atropine and pirenzepine (Caulfield and Birdsall, 1998), gallamine (Matsui et al, 1995;Lazareno et al, 2000;Fruchart-Gaillard et al, 2006), and brucine Fruchart-Gaillard et al, 2006 (Birdsall and Lazareno, 2005;May et al, 2007). Slope factors for all inhibition curves did not statistically differ from 1.…”