Background:The allosteric interaction between agonists and guanylyl nucleotides reports on the interaction between G protein-coupled receptors and G proteins. Results: Such allostery differs in kind between reconstituted monomers and tetramers of the M 2 muscarinic receptor. Conclusion: Monomers and tetramers mediate allostery via different mechanisms. Significance: Only tetramers resemble muscarinic receptors in myocardial membranes in the nature of their sensitivity to guanylyl nucleotides.

We have reported previously that amiodarone interacts with muscarinic receptors via a novel allosteric site. This study presents mechanistic details on the nature of that interaction. Amiodarone enhanced the maximal level of agonist-stimulated release of arachidonic acid (AA) from Chinese hamster ovary cells that expressed M 3 muscarinic receptors; this enhancement was observed for acetylcholine and for the partial agonist pilocarpine. A similar effect of amiodarone was observed when pilocarpine was used to stimulate inositol phosphate (IP) metabolism, but not when acetylcholine was used. Subsequent studies showed that the IP response exhibited a much larger receptor reserve than the AA response, and reduction of that reserve by receptor alkylation unmasked amiodarone's enhancement of the maximal IP response to acetylcholine. Modulating the receptor reserve also revealed acetylcholine's greater affinity (K A ) for the conformation of the receptor that mediates the AA response. The amiodarone analog N-ethylamiodarone (NEA) did not alter maximal agonist response but merely reduced agonist potency (that is, it appeared to be an antagonist). However, the action of NEA could be clearly distinguished from the action of the orthosteric antagonist NMS. Demonstration of this point was facilitated by an elaboration of Hall's allosteric two-state model; this new model represents a system composed of two ligands that compete with each other at the orthosteric site and two ligands that compete with each other at the allosteric site. In conclusion, amiodarone competes with NEA at a novel, extracellular, allosteric site to enhance the maximal stimulation evoked by acetylcholine and pilocarpine in two different responses.

The M2 muscarinic receptor is the prototypic model of allostery in GPCRs, yet the molecular and the supramolecular determinants of such effects are unknown. Monomers and oligomers of the M2 muscarinic receptor therefore have been compared to identify those allosteric properties that are gained in oligomers. Allosteric interactions were monitored by means of a FRET-based sensor of conformation at the allosteric site and in pharmacological assays involving mutants engineered to preclude intramolecular effects. Electrostatic, steric, and conformational determinants of allostery at the atomic level were examined in molecular dynamics simulations. Allosteric effects in monomers were exclusively negative and derived primarily from intramolecular electrostatic repulsion between the allosteric and orthosteric ligands. Allosteric effects in oligomers could be positive or negative, depending upon the allosteric-orthosteric pair, and they arose from interactions within and between the constituent protomers. The complex behavior of oligomers is characteristic of muscarinic receptors in myocardial preparations.DOI: http://dx.doi.org/10.7554/eLife.11685.001

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.