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 M₂ subtype and lowest affinity toward the M₅ subtype. The chimeric receptors were mostly M₅ sequence; the amount of M₂ sequence ranged from about 6 to just under 30%. Alcuronium and TMB-8 had much higher affinity for the chimeric receptor that included the M₂ 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 M₅subtype). However, this chimera retained the negative cooperativity between alcuronium and the classical antagonistN-methylscopolamine that is characteristic of M₅ (these ligands are positively cooperative at M₂). Verapamil, tetrahydroaminoacridine, andd-tubocurarine were also sensitive to that chimeric substitution, although verapamil and tetrahydroaminoacridine had even higher affinity for a chimera with M₂ 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.