Positive cooperativity of acetylcholine and other agonists with allosteric ligands on muscarinic acetylcholine receptors

It is well known that allosteric modulators of muscarinic acetylcholine receptors can both diminish and increase the affinity of receptors for their antagonists. We investigated whether the allosteric modulators can also increase the affinity of receptors for their agonists. Twelve agonists and five allosteric medulators were tested in experiments on membranes of CHO cells that had been stably transfected with genes for the M-1-M-4 receptor subtypes. Allosterically induced changes in the affinities for agonists were computed from changes in the ability of a fixed concentration of each agonist to compete with [H-3]N-methylscopolamine for the binding to the receptors in the absence and the presence of varying concentrations of allosteric modulators. The effects of allosteric modulators varied greatly depending on the agonists and the subtypes of receptors. The affinity for acetylcholine was augmented by (-)-eburnamonine on the M-2 and M-4 receptors and by brucine on the M-1 and M-3 receptors. Brucine also enhanced the affinities for carbachol, bethanechol, furmethide, methylfurmethide, pilocarpine, 3-(3-pentylthio-1 ,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1 -methylpyridine (pentylthio-TZTP), oxotremorine-M, and McN-A-343 on the M-1, M-3, and M-4 receptors, for pentylthio-TZTP on the M-2 receptors, and for arecoline on the M-3 receptors. (-)-Eburnamonine enhanced the affinities for carbachol, bethanechol, furmethide, methylfurmethide, pentylthio-TZTP, pilocarpine, oxotremorine and oxotremorine-M on the M-2 receptors and for pilocarpine on the M-4 receptors. Vincamine, strychnine, and alcuronium displayed fewer positive allosteric interactions with the agonists, but each allosteric modulator displayed positive cooperativity with at least one agonist on at least one muscarinic receptor subtype. The highest degrees of positive cooperativity were observed between (-)-eburnamonine and pilocarpine and (-)-eburnamonine and oxotremorine-M on the M-2 receptors (25- and 7-fold increases in affinity, respectively) and between brucine and pentylthio-TZTP on the M-2 and brucine and carbachol on the M-1 receptors (8-fold increases in affinity). The discovery that it is possible to increase the affinity of muscarinic receptors for their agonists by allosteric modulators offers a new way to subtype-specific pharmacological enhancement of transmission at cholinergic (muscarinic) synapses.