Cooperativity and Oligomeric Status of Cardiac Muscarinic Cholinergic Receptors † (original) (raw)
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Effects of chain-length and unsaturation on affinity and selectivity at muscarinic receptors
British Journal of Pharmacology, 1990
Lengthening the chain in diphenylacetylcholine decreases affinity for muscarinic cholinoceptors in guinea-pig ileum. Diphenylacetoxypropyldimethylamine and its quaternary trimethylammonium salt are roughly equiactive: the dimethylamine and the piperidine have some selectivity for ileum compared with atria, but are not as active nor as selective as 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) methobromide (MeBr). With the weaker diphenylacetoxybutyl compounds the base is more active than the quaternary salt. 2 The diphenylacetoxybutyl-, cis-butenyl and trans-butenyl compounds have similar affinities. The quaternary salts are less active than the tertiary bases, but they are less selective than the butynyl analogues studied in earlier work. 3 1,1-Diphenyl-1-hydroxy-2,4-hexadiynyl dimethylamine and its trimethylammonium salt are inactive in concentrations below 100 uM, as are the (+-camphor-sulphonyl ester of 4-hydroxy-N-methyl piperidine and its methiodide. The (±)-phenylcyclopentylacetyl ester of 4-hydroxy-N-methylpiperidine methobromide is more active than its cyclohexyl analogue and than 4-DAMP MeBr but it is less selective than 4-DAMP MeBr. 4 The high selectivity of p-fluoro-hexahydrosila-diphenidol is confirmed but this compound has relatively low affinity (for ileum log K = 7.8). 5 The results indicate steric constraints to binding at muscarinic receptors which could be used to check molecular modelling of the receptor based on its known amino acid sequence. The group binding the charged nitrogen is probably at the mouth of a cavity which can accommodate two large rings (as in 4-DAMP MeBr) but with a depth less than about 7 A so that the rod-like hexadiynes cannot fit. Differences between types of receptor may only involve small changes in geometry secondary to differences in amino acids not directly involved in binding and the production of selectivity depends upon finding substituents which interfere with binding more at one type of receptor than at another.
Assessment of mechanistic proposals for the binding of agonists to cardiac muscarinic receptors
Biochemistry, 1986
Methylscopolamine has been used to characterize muscarinic receptors in crude homogenates prepared from hearts of Syrian golden hamsters. The Hill coefficient is one for specific binding of the radioligand itself and for its inhibition by muscarinic antagonists; markedly lower values are obtained for its inhibition by muscarinic agonists. The binding patterns of agonists have been analyzed in terms of a mixture of sites differing in affinity for the drug and reveal the following. (1) All agonists discern at least two classes of receptor in atrial and ventricular homogenates. (2) The number of classes and the relative size of each differ for different agonists in the same region and for the same agonist in different regions. ' Abbreviations: GMP-PNP, guanylyl imidodiphosphate; HEPES, N-(2-hydroxyethyl)piperazine-N'2-ethanesulfonic acid. C A R D I A C M U S C A R I N I C R E C E P T O R S V O L . 2 5 , N O . 2 2 , 1 9 8 6 6997 P carbachol LV(5), n = 2 RV(5), n = 2 K , , K2 F2 K I , K2, F2 arecoline AT(2), n = 2 LV(3), n = 2 K , , K2 F2 carbachol AT(5), n = 3 LV(5), n = 2 K
Evidence for a Tandem Two-site Model of Ligand Binding to Muscarinic Acetylcholine Receptors
Journal of Biological Chemistry, 2000
After short preincubations with N-[ 3 H]methylscopolamine ([ 3 H]NMS) or R(؊)-[ 3 H]quinuclidinyl benzilate ([ 3 H]QNB), radioligand dissociation from muscarinic M 1 receptors in Chinese hamster ovary cell membranes was fast, monoexponential, and independent of the concentration of unlabeled NMS or QNB added to reveal dissociation. After long preincubations, the dissociation was slow, not monoexponential, and inversely related to the concentration of the unlabeled ligand. Apparently, the unlabeled ligand becomes able to associate with the receptor simultaneously with the already bound radioligand if the preincubation lasts for a long period, and to hinder radioligand dissociation. When the membranes were preincubated with [ 3 H]NMS and then exposed to benzilylcholine mustard (covalently binding specific ligand), [ 3 H]NMS dissociation was blocked in wild-type receptors, but not in mutated (D99N) M 1 receptors. Covalently binding [ 3 H]propylbenzilylcholine mustard detected substantially more binding sites than [ 3 H]NMS. The observations support a model in which the receptor binding domain has two tandemly arranged subsites for classical ligands, a peripheral one and a central one. Ligands bind to the peripheral subsite first (binding with lower affinity) and translocate to the central subsite (binding with higher affinity). The peripheral subsite of M 1 receptors may include Asp-99. Experimental data on [ 3 H]NMS and [ 3 H]QNB association and dissociation perfectly agree with the predictions of the tandem two-site model.
Binding of N-methylscopolamine to the extracellular domain of muscarinic acetylcholine receptors
Scientific Reports
Interaction of orthosteric ligands with extracellular domain was described at several aminergic G protein-coupled receptors, including muscarinic acetylcholine receptors. The orthosteric antagonists quinuclidinyl benzilate (QNB) and N-methylscopolamine (NMS) bind to the binding pocket of the muscarinic acetylcholine receptor formed by transmembrane α-helices. We show that high concentrations of either QNB or NMS slow down dissociation of their radiolabeled species from all five subtypes of muscarinic acetylcholine receptors, suggesting allosteric binding. The affinity of NMS at the allosteric site is in the micromolar range for all receptor subtypes. Using molecular modelling of the M 2 receptor we found that E172 and E175 in the second extracellular loop and N419 in the third extracellular loop are involved in allosteric binding of NMS. Mutation of these amino acids to alanine decreased affinity of NMS for the allosteric binding site confirming results of molecular modelling. The allosteric binding site of NMS overlaps with the binding site of some allosteric, ectopic and bitopic ligands. Understanding of interactions of NMS at the allosteric binding site is essential for correct analysis of binding and action of these ligands.
Molecular Pharmacology, 1998
Muscarinic acetylcholine receptors bind allosteric modulators at a site apart from the orthosteric site used by conventional ligands. We tested in cardiac tissue whether modulator binding to ligand-occupied muscarinic M 2 receptors is a preferential event that can be detected using a radioactive allosteric agent. The newly synthesized dimethyl-W84 (N,NЈ-bis[3-(1,3-dihydro-1,3-dioxo-4-methyl-2H-isoindol-2-yl)propyl]-N,N,NЈ,NЈ-tetramethyl-1,6-hexanediaminium diiodide) has a particular high potency at M 2 receptors occupied by the conventional antagonist N-methylscopolamine (NMS); dissociation of [ 3 H]NMS is halfmaximally retarded at an EC 50,diss value of 3 nM. Using obidoxime as an "allosteric antagonist," evidence was found that dimethyl-W84 interacts with the postulated common allosteric site. Binding of [ 3 H]dimethyl-W84 (0.3 nM; specific activity, 168 Ci/mmol) was measured in porcine heart homogenates (4 mM Na 2 HPO 4 , 1 mM KH 2 PO 4 , pH 7.4, 23°) in the presence of 1 M NMS. Homologous competition experiments revealed two components of saturable radioligand binding: one with a high affinity (K D ϭ 2 nM) and small capacity (Ϸ30% of total saturable binding) and the other with a 20,000-fold lower affinity. The B max value of the high affinity sites (68 fmol/mg protein) matched muscarinic receptor density as determined by [ 3 H]NMS (79 fmol/mg). Prototype allosteric agents, alcuronium, W84 (the parent compound of the radioligand), and gallamine, displaced high affinity [ 3 H]dimethyl-W84 binding concentration-dependently (pK i values ϭ 8.62, 7.83, and 6.72, respectively). The binding affinities of the modulators were in excellent correlation with their potencies to allosterically stabilize NMS/ receptor complexes (EC 50,diss ϭ 8.40, 7.72, and 6.74, respectively). We conclude that high affinity binding of [ 3 H]dimethyl-W84 reflects occupation of the common allosteric site of M 2 receptors. This work was supported by the Deutsche Forschungsgemeinschaft und the Fonds der Chemischen Industrie, Germany. H.M.B.C. acknowledges a grant given by the Katholischer Akademischer Austauschdienst.
BJA: British Journal of Anaesthesia, 1995
Atropine and glycopyrronium are frequently used for premedication to reduce oral and respiratory secretions and prevent bradycardia. Glycopyrronium is said to have similar antisialagogue effects, but is less likely to cause significant tachycardia than atropine. Different antimuscarinic receptor selectivity patterns could explain the differences. The aim of this investigation was to determine the possible selectivity of glycopyrronium for M 2 and M 3 muscarinic receptor subtypes. Muscarinic receptor subtypes in Wistar rat ventricle and submandibular gland homogenates were characterized with [ 3 H]-/V-methylscopolamine ([ 3 H]-NMS) by ligand binding studies. Inhibition of [ 3 H]-NMS binding by non-labelled compounds showed the following order: in rat ventricle: glycopyrronium > atropine ^> otenzepad > hexahydrosiladiphenidol (HHSiD) > pirenzepine;inratsubmandibulargland:glycopyrronium > atropine ^> HHSiD > pirenzepine > otenzepad. These were similar to the expected order of frequency of M 2 and M 3 subtypes, respectively. Glycopyrronium showed similarly high affinities for both M 2 (K, = 1.889 (SEM 0.049) nmol litre" 1) and M 3 (K, = 1.686 (0.184) nmol litre" 1) subtypes. Glycopyrronium bound to a homogeneous population of binding sites in both tissues and showed no selectivity for M 2 or M 3 muscarinic receptor subtypes.
Biochemistry, 2007
FLAG-and HA-tagged M 2 muscarinic receptors from coinfected Sf9 cells have been purified in digitonin-cholate and reconstituted into phospholipid vesicles. The purified receptor was predominantly monomeric: it showed no detectable coimmunoprecipitation; it migrated as a monomer during electrophoresis before or after cross-linking with bis(sulfosuccinimidyl)suberate; and it bound agonists and antagonists in a manner indicative of identical and mutually independent sites. Receptor cross-linked after reconstitution or after reconstitution and subsequent solubilization in digitonin-cholate migrated almost exclusively as a tetramer. The binding properties of the reconstituted receptor mimicked those reported previously for cardiac muscarinic receptors. The apparent capacity for N-[ 3 H]methylscopolamine (NMS) was only 60% of that for [ 3 H]quinuclidinylbenzilate (QNB), yet binding at saturating concentrations of [ 3 H]QNB was inhibited fully and in a noncompetitive manner at comparatively low concentrations of unlabeled NMS. Reconstitution of the receptor with a saturating quantity of functional G proteins led to the appearance of three classes of sites for the agonist oxotremorine-M in assays with [ 3 H]QNB; GMP-PNP caused an apparent interconversion from highest to lowest affinity and the concomitant emergence of a fourth class of intermediate affinity. All of the data can be described quantitatively in terms of cooperativity among four interacting sites, presumably within a tetramer; the effect of GMP-PNP can be accommodated as a shift in the distribution of tetramers between two states that differ in their cooperative properties. Monomers of the M 2 receptor therefore can be assembled into tetramers with binding properties that closely resemble those of the muscarinic receptor in myocardial preparations.