Changes of Cooperativity between N-Methylscopolamine and Allosteric Modulators Alcuronium and Gallamine Induced by Mutations of External Loops of Muscarinic M3 Receptors

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Vol. 60, Issue 4, 761-767, October 2001

Changes of Cooperativity between N-Methylscopolamine and Allosteric Modulators Alcuronium and Gallamine Induced by Mutations of External Loops of Muscarinic M₃ Receptors

Alena Krej $c$ í and Stanislav Tu $c$ ek

Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic

To clarify the involvement of specific domains of muscarinic receptors in the action of allosteric modulators, muscarinicM₃ receptors (on which allosteric interactions are weak) weregenetically modified to become more similar to M₂ receptors (onwhich allosteric interactions are strong) and were expressed inCOS-7 cells. Affinity for allosteric modulator gallamine was enhanced25- to 50-fold by modifications of the third external loop (o3)and the negative effect of gallamine on the affinity for classicalantagonist N-[³H]methylscopolamine ([³H]NMS) was augmented. Affinity for alcuronium became 3-fold higherafter the o3 loop of M₃ receptors was made identical with theo3 loop of M₂ receptors, and alcuronium acquired positive influenceon the affinity for [³H]NMS. This is the first instance of inducing positive cooperativityon muscarinic receptors by genetic manipulation. Transferringwhole o2 loop from M₂ to M₃ receptors substantially enhanced affinitiesfor gallamine and alcuronium without augmenting their negativeaction on [³H]NMS binding. In contrast, effects of simply adding two negativecharges into the o2 loop of M₃ receptors were small. Removal ofArg from o1 loop abolished the negative effect of gallamine butnot of alcuronium on [³H]NMS binding at equilibrium. Data point to an important roleof o3 loop in the mechanism of the positive and negative cooperativitybetween [³H]NMS and alcuronium and gallamine, respectively, and in the bindingof both modulators to M₂ receptors and reveal independence betweenmutation-induced changes in the affinity for a modulator and inthe magnitude and direction of the allosteric effect of themodulator.

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