Novel Allosteric Effects of Amiodarone at the Muscarinic M₅Receptor

Abstract

Allosteric sites on muscarinic receptors may present superior therapeutic targets for several central nervous system disorders, due to the potential of allosteric ligands to provide more selective modulation and to preserve the spatiotemporal patterning that is characteristic of synaptic transmission. We have found that the antiarrhythmic drug amiodarone interacts allosterically with M 1 and M 5 muscarinic receptors. At both M 1 and M 5 , amiodarone was only able to partially inhibit the binding of the orthosteric antagonist [3 H]N-methylscopolamine (NMS). In addition, amiodarone was able to alter the rate of dissociation of [ 3 H]NMS from M 1 and M 5 receptors. These findings suggest that NMS and amiodarone are able to bind to the receptor simultaneously. The pharmacology of the effect on NMS dissociation demonstrated that amiodarone was not interacting at the "common" site at which gallamine, obidoxime, and many other muscarinic allosteric ligands are known to bind. In functional studies, amiodarone enhanced the ability of acetylcholine (at EC 20 ) to activate the M 5 receptor; however, under the same conditions, amiodarone did not enhance M 1 activation. More detailed studies at M 5 found that the effect of amiodarone was to enhance the efficacy of acetylcholine, without increasing its potency. This report describes the first demonstration of allosteric enhancement of efficacy at the M 5 receptor, and the first demonstration of enhancement of efficacy but not potency at any muscarinic receptor. In summary, amiodarone has been shown to be a novel positive allosteric modulator of muscarinic receptors that is selective for the M 5 subtype, relative to M 1 .

Allosteric Modulation of the M3 Muscarinic Receptor by Amiodarone and N-Ethylamiodarone: Application of the Four-Ligand Allosteric Two-State Model

“…The ability of amiodarone to inhibit the binding of [ 3 H]NMS to M 1 , M 2 , and M 5 muscarinic receptors has been reported previously (Stahl and Ellis, 2010). The present work extends the study of amiodarone's effects on binding to include the M 3 subtype (Fig.…”

“…For binding experiments, amiodarone was dissolved in dimethyl sulfoxide, the concentration of which was maintained below 1% for all assays; all other reagents were dissolved in buffer or deionized water. Equilibrium binding studies were performed as described previously by Ellis and Seidenberg (1999) and Stahl and Ellis (2010). In brief, these studies were performed in PBS with 1 mM CaCl 2 and 1 mM MgCl 2 , pH 7.4, for 1 h at 25°C.…”

“…Measurement of [ 3 H]AA release was performed as described in Stahl and Ellis (2010). CHO cells were seeded on 48-well plates (Greiner Bio-One GmbH, Frickenhausen, Germany) at a density of 2.9 ϫ 10 4 cells/well in 0.25 ml of F-12 medium.…”

“…We have characterized the allosteric effects of amiodarone on muscarinic receptors and established that it interacts with a novel site (Stahl and Ellis, 2010). An enhancement of the M 5 response was observed for a number of full and partial muscarinic agonists, including ACh.…”

Role of M1, M3, and M5 muscarinic acetylcholine receptors in cholinergic dilation of small arteries studied with gene-targeted mice

“…For example, pharmacological activation of M 5 receptors may be useful to increase cerebral perfusion in certain pathophysiological conditions, such as Alzheimer's disease and cerebral ischemia (37,41). Interestingly, several agents have been described recently that can selectivity enhance signaling through M 1 or M 5 receptors (4,8,27,40). Such ligands may become relevant for the development of novel muscarinic drugs aimed at modulating local perfusion.…”