Interactions of Alcuronium, TMB-8, and Other Allosteric Ligands with Muscarinic Acetylcholine Receptors: Studies with Chimeric Receptors

Abstract

A series of ligands that allosterically modulate the binding of classical ligands to muscarinic receptors was evaluated at wild-type and chimeric receptors. All of the ligands studied had highest affinity toward the M(2) subtype and lowest affinity toward the M(5) subtype. The chimeric receptors were mostly M(5) sequence; the amount of M(2) sequence ranged from about 6 to just under 30%. Alcuronium and TMB-8 had much higher affinity for the chimeric receptor that included the M(2) second outer loop of the receptor plus flanking regions of TM4 and TM5 than for any of the other chimeric receptors (the affinities of which remained similar to that of the M(5) subtype). However, this chimera retained the negative cooperativity between alcuronium and the classical antagonist N-methylscopolamine that is characteristic of M(5) (these ligands are positively cooperative at M(2)). Verapamil, tetrahydroaminoacridine, and d-tubocurarine were also sensitive to that chimeric substitution, although verapamil and tetrahydroaminoacridine had even higher affinity for a chimera with M(2) sequence in TM7. None of these ligands shared gallamine's sensitivity to a region of the third outer loop, but studies in which obidoxime reversed the allosteric effects of gallamine and other ligands suggested that they nevertheless compete for a common site. In summary, although the present data are consistent with previous studies that have suggested that allosteric ligands bind to the outermost regions of muscarinic receptors, it appears that different allosteric ligands may derive subtype selectivity from different regions of the receptor.

Activation of Endogenous Nitric Oxide Synthase Coupled with Methacholine-Induced Exocytosis in Rat Parotid Acinar Cells

“…This inhibitor prevented the stimulation of amylase secretion by MCh ( Table 1), suggesting that activation of PLC by MCh contributes to Ca 2ϩ mobilization from intracellular stores and then to amylase secretion. Incubation of tissue cells with 15 M TMB-8, a muscarinic receptor antagonist (Ellis and Seidenberg, 2000), also inhibited amylase secretion induced by 1 M MCh (Table 1). The increase in [Ca 2ϩ ] i induced by the release of Ca 2ϩ through IP 3 -gated channels may in turn result in Ca 2ϩ release through ryanodine receptors.…”

Allosteric Modulation of the M3 Muscarinic Receptor by Amiodarone and N-Ethylamiodarone: Application of the Four-Ligand Allosteric Two-State Model

“…All of the muscarinic receptors are susceptible to allosteric modulation and, for a subset of allosteric ligands, a "common site" has been established. The ligands shown to act at this common site include gallamine, obidoxime, alcuronium, strychnine, and hexamethylene-bis-[dimethyl-(3-phthalimidopropyl)ammonium]dibromide (W84) (Ellis and Seidenberg, 2000;Trä nkle et al, 2003). Moreover, the high degree of sequence homology in the orthosteric acetylcholine binding site has impeded the development of useful subtype-selective orthosteric ligands (Jones et al, 1992).…”

Coupling of G Proteins to Reconstituted Monomers and Tetramers of the M2 Muscarinic Receptor

“…Nonspecific binding was measured in the presence of unlabeled atropine (3 M), which was premixed with the radioligand prior to the addition of the receptor. Other details have been described previously (31).…”