Persistent Binding and Functional Antagonism by Xanomeline at the Muscarinic M₅Receptor

Abstract

Xanomeline is a functionally selective M 1 /M 4 muscarinic acetylcholine receptor agonist. We have previously identified a novel mode of interaction of this ligand with the muscarinic M 1 receptor that involves persistent binding and activation of the receptor after extensive washout. In the present study, we tested the hypothesis that xanomeline also binds in a washresistant manner to muscarinic receptor subtypes where it exhibits low or no efficacy, such as the M 5 receptor subtype. A secondary hypothesis is that persistent binding of xanomeline to the M 5 receptor results in wash-resistant antagonism to the effects of full agonists. These hypotheses were tested in Chinese hamster ovary cells stably expressing the M 5 receptor. In these cells, xanomeline is a weak partial agonist and is able to inhibit carbachol-induced phosphoinositide hydrolysis to the maximal response of xanomeline in a concentration-dependent manner. Pretreatment with xanomeline followed by extensive washing resulted in a significant wash-resistant reduction in receptor affinity with no significant change in maximal cell surface receptor density. This was associated with wash-resistant antagonism of carbachol-induced activation of phosphoinositide hydrolysis at the M 5 receptor, reflected as decreased carbachol potency without a change in the maximal response. Similar experiments using the partial agonist pilocarpine demonstrated a reduction of pilocarpine potency as well as maximal response. Our results clearly indicate that wash-resistant binding of xanomeline to the muscarinic M 5 receptor is accompanied by persistent antagonism of receptor function. They also suggest a relationship between the efficacy of xanomeline and the functional consequences of its wash-resistant binding at different muscarinic receptor subtypes.

Immediate and Delayed Consequences of Xanomeline Wash-Resistant Binding at the M3 Muscarinic Receptor

“…In our current study, xanomeline was shown to bind with high-potency at the M3 receptor, similar to that obtained previously at the M1 [18] and M5 receptor [11] using similar expression systems. We have also shown that xanomeline binds to the M3 receptor in a wash-resistant manner similar to other muscarinic receptor subtypes [10, 18].…”

“…In contrast, its wash-resistant activation of the M1 receptor appears to involve the orthosteric receptor domain. It appears that xanomeline also has unique binding properties at the M2 [10] and M5 [11] receptors. However, it is unclear whether the molecular mechanism of xanomeline binding occurs in a similar manner at all the receptor subtypes.…”

Towards a muscarinic hypothesis of schizophrenia

“…143 Recent data suggest that xanomeline is also an antagonist at the M 5 receptor. 144 As muscarinic neurons carrying M 5 receptors have synaptic contact with dopaminergic neurons in the brainstem, the functional antagonism of xanomeline at M 5 receptors may offer an additional modulatory pathway on dopaminergic cell-firing.…”

Long-Term Activation upon Brief Exposure to Xanomleline Is Unique to M1 and M4 Subtypes of Muscarinic Acetylcholine Receptors

“…These findings have led to interest in xanomeline as a potential therapy for schizophrenia [12]–[15]. Besides its M 1 /M 4 preference, xanomeline binds to all muscarinic receptor subtypes in a way that is resistant to intensive washing and causes persistent receptor activation or antagonism [16]–[22].…”