Allosteric Interactions of Staurosporine and Other Indolocarbazoles withN-[methyl-³H]Scopolamine and Acetylcholine at Muscarinic Receptor Subtypes: Identification of a Second Allosteric Site

Abstract

We have studied the interactions of five indolocarbazoles with N-[methyl-(3)H]scopolamine (NMS) and unlabeled acetylcholine at M(1)-M(4) muscarinic receptors, using equilibrium and nonequilibrium radioligand binding studies. The results are consistent with an allosteric model in which the primary and allosteric ligands bind simultaneously to the receptor and modify each other's affinities. The compounds were generally most active at M(1) receptors. [(3)H]NMS binding was enhanced by staurosporine, KT5720, and KT5823 at M(1) and M(2) receptors, and by K-252a at M(1) receptors. Gö 7874 reduced [(3)H]NMS affinity by up to threefold for all subtypes. A range of cooperative effects with acetylcholine was seen, and, at the M(1) receptor, KT5720 had a log affinity of 6.4 and enhanced acetylcholine affinity by 40%. The compounds inhibited the dissociation of [(3)H]NMS to different extents across the receptor subtypes, with the largest effects at M(1) receptors. In equilibrium binding studies the inhibitory potency of gallamine at M(1) receptors was not affected by KT5720, indicating that these agents bind to two distinct allosteric sites and have neutral cooperativity with each other. In contrast, gallamine and staurosporine had a negatively cooperative or competitive interaction at M(1) receptors. Similarly, the potency and relative effectiveness of KT5720 for inhibiting [(3)H]NMS dissociation from M(1) receptors were not affected by gallamine or brucine, but were affected in a complex manner by staurosporine. These results demonstrate that there are at least two distinct allosteric sites on the M(1) receptor, both of which can support positive cooperativity with acetylcholine.

Identification of multiple allosteric sites on the M1 muscarinic acetylcholine receptor

“…In contrast, KT5720 did not affect the potency of gallamine to inhibit N-methylscopolamine binding or dissociation from M 1 receptors, suggesting that gallamine, KT5720 and N-methylscopolamine could simultaneously bind to the receptor. Furthermore, when K75720 is bound to the receptor free receptor (as is our study), there is neutral cooperativity between this allosteric agent and gallamine [8,9]. In our study, we found that both staurosporine and KT5720 were able to bind to both intra-and extracellular faces of the receptor, suggesting that they can compete with gallamine.…”

“…These facts, together with the studies performed with gallamine, suggest that the second allosteric site proposed by Birdsall and co-workers [9] is located in the intracellular face of the receptor, very close to the intracellular loop 3.…”

“…In this study, we performed docking simulations of staurosporine and four indolocarbazoles (Chart 1) on the rhodopsin-based homology model of the M 1 receptor. It has been experimentally shown that these compounds have a complex allosteric effect on M 1 -M 4 muscarinic receptors, involving more than one allosteric site [9]. In this contribution, we show the existence of three allosteric sites involved not only in the positive cooperativity with the orthosteric site, but also in the activation of a possible acetylcholine-independent G-protein coupling to the receptor.…”

“…Experimentally, staurosporine inhibited the potency of gallamine to bind to M 1 receptors and modulated the ability of KT5720 to inhibit N-methylscopolamine dissociation, but it was not possible to determine whether staurosporine occluded the binding of both gallamine and KT5720 or whether staurosporine bound to the same site as K5720 and had negative cooperativity with gallamine [8,9]. In contrast, KT5720 did not affect the potency of gallamine to inhibit N-methylscopolamine binding or dissociation from M 1 receptors, suggesting that gallamine, KT5720 and N-methylscopolamine could simultaneously bind to the receptor.…”

Pathophysiology of respiratory failure following acute dichlorvos poisoning in a rodent model

“…Gallamine is a known antagonist of muscarinic agonists and acts non-competitively via allosteric binding of M1 and M2 muscarinic receptors in the brain and lung [28,29]. Both M1 and M2 receptors are present in the pre-Botzinger complex [30] Gaspari and Paydarfar Page 7…”