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

Abstract

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-

Wash-Resistantly Bound Xanomeline Inhibits Acetylcholine Release by Persistent Activation of Presynaptic M2and M4Muscarinic Receptors in Rat Brain

“…Brain cortical and striatal prisms were prepared from 2-to 3-month-old male Wistar rats using a McIlwain tissue chopper set at a width of 0.35 mm. Superfusion experiments were done essentially as described previously (Lazareno et al, 2004). In brief, brain cortical slices were loaded with [ 3 H]choline (specific radioactivity, 82 Ci/mmol; Amersham, Little Chalfont, Buckinghamshire, UK) in Krebs' buffer (138 mM NaCl, 3 mM KCl, 1.2 mM CaCl 2 , 1 mM MgCl 2 1, 1.2 mM NaH 2 PO 4 , 25 mM NaHCO 3 , and 10 mM glucose; for noradrenaline release experiments in addition 0.03 mM EDTA and 0.06 mM ascorbic acid) for 30 min.…”

International Union of Basic and Clinical Pharmacology. XC. Multisite Pharmacology: Recommendations for the Nomenclature of Receptor Allosterism and Allosteric Ligands

“…For instance, the structural features that govern the binding of allosteric ligands can be different from those that determine the interaction of orthosteric ligands with a receptor. Allosteric ligands also offer the potential for greater receptor subtype-selectivity because of higher sequence divergence in allosteric sites across receptor subtypes relative to the conserved orthosteric domain or because of selective modulation of orthosteric ligand actions at a given subtype to the exclusion of others (Christopoulos, 2002;Lazareno et al, 2004). Importantly, ligands that bind to allosteric sites will modify the conformation of a receptor such that its interactive properties toward orthosteric ligands or coupling proteins may change.…”

Asparagine, Valine, and Threonine in the Third Extracellular Loop of Muscarinic Receptor Have Essential Roles in the Positive Cooperativity of Strychnine-Like Allosteric Modulators

“…Binding of allosteric ligands can induce conformational changes of the receptor that result in changes in receptor affinity for orthosteric agonists and antagonists (Tuček et al, 1990Proška and Tuček, 1994;Dong et al, 1995;Jakubík et al, 1995Jakubík et al, , 1997Lazareno and Birdsall, 1995). These effects exhibit remarkable receptor subtype selectivity that is also reflected in the functional outcome of receptor stimulation by orthosteric agonists Doležal and Tuček, 1998;Birdsall et al, 1999;Lazareno et al, 2004). Allosteric interactions are usually more pronounced at M 2 and M 4 than at M 1 , M 3 , or M 5 subtypes of muscarinic receptors (Christopoulos et al, 1999;Christopoulos and Kenakin, 2002), and at least in the case of the allosteric modulator gallamine this difference between M 2 and M 3 subtypes is not due to preferential coupling with G protein (Trä nkle et al, 2001), which is largely determined by the third intracellular loop.…”