Dronedarone Modulates M₁ and M₃ Muscarinic Receptors with Subtype Selectivity, Functional Selectivity, and Probe Dependence

We have previously reported that amiodarone interacts with a novel allosteric site on muscarinic receptors. Amiodarone's most striking effect is to enhance the maximal response elicited by muscarinic agonists at the M₁, M₃, and M₅ receptors. Furthermore, the quaternary analog N-ethylamiodarone (NEA) is inhibitory at these receptors and appears to compete with amiodarone at that allosteric site. In the present studies, we show that dronedarone also modulates Gq-mediated responses at M₁ and M₃, although in a more discriminating manner. For example, dronedarone markedly enhances pilocarpine-stimulated release of arachidonic acid from CHO cells, via the M₃ receptor subtype, but does not affect the acetylcholine-stimulated response. Such probe-dependent effects are diagnostic of an allosteric interaction. In comparison to these effects at M₃, dronedarone is strongly inhibitory toward both pilocarpine and acetylcholine at the M₁ subtype. The effects of dronedarone are consistent with an interaction at the amiodarone site: dronedarone inhibits the enhancement of acetylcholine's response produced by amiodarone at the M₃ subtype; also, NEA reverses the enhancement of pilocarpine's response at M₃ produced by either dronedarone or amiodarone. In studies with the M₁-selective allosteric agonist 4-[3-(4-butylpiperidin-1-yl)-propyl]-7-fluoro-4H-benzo[1,4]oxazin-3-one (AC-260584), amiodarone enhanced the maximal response observed, whereas dronedarone was inhibitory. On the other hand, benzyl quinolone carboxylic acid, the well-known allosteric ligand that dramatically enhances the potency of acetylcholine at the M₁ subtype, had no effect on the response profile of AC-260584. In summary, dronedarone acts at M₁ and M₃ muscarinic receptors in a manner that complements amiodarone and provides an additional tool with which to investigate this novel allosteric site.