Interactions of Orthosteric and Allosteric Ligands with  [<sup>3</sup>H]Dimethyl-W84 at the Common Allosteric Site of Muscarinic  M<sub>2</sub> Receptors

Thiochrome (2,7-dimethyl-5H-thiachromine-8-ethanol), an oxidation product and metabolite of thiamine, has little effect on the equilibrium binding of L-[ 3 3 H]ACh from potassium-stimulated slices of rat striatum, which contain autoinhibitory presynaptic M 4 receptors, but not from hippocampal slices, which contain presynaptic M 2 receptors. We conclude that thiochrome is a selective M 4 muscarinic receptor enhancer of ACh affinity and has neutral cooperativity with ACh at M 1 to M 3 receptors; it therefore demonstrates a powerful new form of selectivity, "absolute subtype selectivity", which is derived from cooperativity rather than from affinity.Most receptor-active ligands bind to the same site as the endogenous ligand, the so-called orthosteric site. Agonists mimic the actions of the endogenous ligand, whereas antagonists physically prevent the endogenous ligand from binding but lack its actions. The property of a ligand that determines its effect when bound to a receptor is called its efficacy. Different degrees of efficacy lead to so-called full agonists, partial agonists with a smaller maximal effect than full agonists; neutral antagonists, which occupy the active site without exerting any effect; and inverse agonists, which reduce the activity of constitutively active receptors (Kenakin, 2002). The selectivity of an orthosteric ligand for one receptor or receptor subtype is determined by its affinity for the receptor and its efficacy at that receptor. The difference in affinity between the target receptor and other receptors must be large for an orthosteric ligand to have useful selectivity. This can be difficult to achieve for receptors that show close homology at the orthosteric binding region such as the five subtypes of muscarinic receptor (M 1 -M 5 ) (Hulme et al., 1990). Selectivity derived from efficacy can also be hard to achieve, because the effect of a partial agonist depends on properties of the tissue, such as receptor density and downstream amplification mechanisms, which vary between cells and tissues, so a ligand with no apparent functional effect on tissues in vitro may nevertheless activate tissues in vivo, leading to unacceptable side effects (Terry et al., 2002).An alternative approach for developing selective ligands is to look for allosteric ligands that bind to a site on the receptor which is different from the site to which the endogenous ligand binds. This allows both types of ligand to bind simultaneously. If the affinity (or efficacy) of the endogenous ligand is different when it is bound to the allosteric liganded receptor compared with when it is bound to the free receptor, then the allosteric ligand exhibits positive or negative coop-

Section: Discussionmentioning

confidence: 99%

Thiochrome Enhances Acetylcholine Affinity at Muscarinic M₄Receptors: Receptor Subtype Selectivity via Cooperativity Rather than Affinity

Lazareno

Doležal²,

Popham³

et al. 2004

“…All of the muscarinic receptors are susceptible to allosteric modulation and, for a subset of allosteric ligands, a "common site" has been established. The ligands shown to act at this common site include gallamine, obidoxime, alcuronium, strychnine, and hexamethylene-bis-[dimethyl-(3-phthalimidopropyl)ammonium]dibromide (W84) (Ellis and Seidenberg, 2000;Trä nkle et al, 2003). Moreover, the high degree of sequence homology in the orthosteric acetylcholine binding site has impeded the development of useful subtype-selective orthosteric ligands (Jones et al, 1992).…”

Section: Introductionmentioning

confidence: 99%

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

Stahl¹,

Elmslie²,

Ellis³

2011

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.

“…As occasionally speculated (Avissar el al., 1983;Wreggett and Wells, 1995;Lazareno et al, 1998;Trankle et al, 2003;Urizar at al., 2005), it is readily obvious that the available experimental evidence points out the impossibility of ex-F.C. holds a Ramón y Cajal research contract with the Ministerio de Ciencia y Tecnología.…”

mentioning

confidence: 99%

The Two-State Dimer Receptor Model: A General Model for Receptor Dimers

Franco

Casadó²,

Mallol³

et al. 2006

Nonlinear Scatchard plots are often found for agonist binding to G-protein-coupled receptors. Because there is clear evidence of receptor dimerization, these nonlinear Scatchard plots can reflect cooperativity on agonist binding to the two binding sites in the dimer. According to this, the "two-state dimer receptor model" has been recently derived. In this article, the performance of the model has been analyzed in fitting data of agonist binding to A 1 adenosine receptors, which are an example of receptor displaying concave downward Scatchard plots. Analysis of agonist/antagonist competition data for dopamine D 1 receptors using the two-state dimer receptor model has also been performed. Although fitting to the two-state dimer receptor model was similar to the fitting to the "two-independent-site receptor model", the former is simpler, and a discrimination test selects the two-state dimer receptor model as the best. This model was also very robust in fitting data of estrogen binding to the estrogen receptor, for which Scatchard plots are concave upward. On the one hand, the model would predict the already demonstrated existence of estrogen receptor dimers. On the other hand, the model would predict that concave upward Scatchard plots reflect positive cooperativity, which can be neither predicted nor explained by assuming the existence of two different affinity states. In summary, the two-state dimer receptor model is good for fitting data of binding to dimeric receptors displaying either linear, concave upward, or concave downward Scatchard plots