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Mutational Disruption of a Conserved Disulfide Bond in Muscarinic Acetylcholine Receptors Attenuates Positive Homotropic Cooperativity between Multiple Allosteric Sites and Has Subtype-Dependent Effects on the Affinities of Muscarinic Allosteric Ligands

Departments of Psychiatry and Pharmacology, Hershey Medical Center, Penn State University College of Medicine, Hershey, Pennsylvania

The 2nd outer loop (o2) of muscarinic acetylcholine receptors (mAChRs) contains a highly conserved cysteine residue that is believed to participate in a disulfide bond and is flanked on either side by epitopes that are critical to the binding of many muscarinic allosteric modulators. We determined the allosteric binding parameters of the modulators gallamine, W84, and tetrahydroaminoacridine (THA) at M₂ and M₃ mAChRs in which these cysteine residues had been mutated to alanines. THA is known to bind to mAChRs with a strong positive homotropic cooperativity (a Hill slope of approximately 2) that implies that it must interact with multiple allosteric sites. The disulfide cysteine mutations in M₂ receptors reduced the allosteric potencies of the tested modulators as if the critical adjacent residue (Tyr177) itself had been mutated. However, in M₃ receptors, the disulfide cysteine mutations had no effect on the potencies of gallamine or W84 and even increased the potency of THA. It was most interesting that the strong, positive, homotropic interactions of THA at both M₂ and M₃ receptors were markedly reduced by the cysteine mutations. In addition, gallamine also displayed positive homotropic cooperativity in its interactions with M₃ receptors (but not M₂ receptors), and this cooperativity was not evident in the cysteine mutants. Thus, it seems that these cysteine residues play a role in linking cooperating allosteric sites, although it is not currently possible to say whether these multiple sites lie within one receptor or on two linked receptors of a dimer or higher order oligomer.

Address correspondence to: John Ellis, Department of Psychiatry, H073, Hershey Medical Center, Penn State University College of Medicine, 500 University Drive, Hershey, PA 17033. E-mail: johnellis{at}psu.edu

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