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• W1999647921 abstract "G protein-coupled receptors represent the largest superfamily of cell membrane-spanning receptors. We used allosteric small molecules as a novel approach to better understand conformational changes underlying the inactive-to-active switch in native receptors. Allosteric molecules bind outside the orthosteric area for the endogenous receptor activator. The human muscarinic M2 acetylcholine receptor is prototypal for the study of allosteric interactions. We measured receptor-mediated G protein activation, applied a series of structurally diverse muscarinic allosteric agents, and analyzed their cooperative effects with orthosteric receptor agonists. A strong negative cooperativity of receptor binding was observed with acetylcholine and other full agonists, whereas a pronounced negative cooperativity of receptor activation was observed with the partial agonist pilocarpine. Applying a newly synthesized allosteric tool, point mutated receptors, radioligand binding, and a three-dimensional receptor model, we found that the deviating allosteric/orthosteric interactions are mediated through the core region of the allosteric site. A key epitope is M2Trp422 in position 7.35 that is located at the extracellular top of transmembrane helix 7 and that contacts, in the inactive receptor, the extracellular loop E2. Trp 7.35 is critically involved in the divergent allosteric/orthosteric cooperativities with acetylcholine and pilocarpine, respectively. In the absence of allosteric agents, Trp 7.35 is essential for receptor binding of the full agonist and for receptor activation by the partial agonist. This study provides first evidence for a role of an allosteric E2/transmembrane helix 7 contact region for muscarinic receptor activation by orthosteric agonists. G protein-coupled receptors represent the largest superfamily of cell membrane-spanning receptors. We used allosteric small molecules as a novel approach to better understand conformational changes underlying the inactive-to-active switch in native receptors. Allosteric molecules bind outside the orthosteric area for the endogenous receptor activator. The human muscarinic M2 acetylcholine receptor is prototypal for the study of allosteric interactions. We measured receptor-mediated G protein activation, applied a series of structurally diverse muscarinic allosteric agents, and analyzed their cooperative effects with orthosteric receptor agonists. A strong negative cooperativity of receptor binding was observed with acetylcholine and other full agonists, whereas a pronounced negative cooperativity of receptor activation was observed with the partial agonist pilocarpine. Applying a newly synthesized allosteric tool, point mutated receptors, radioligand binding, and a three-dimensional receptor model, we found that the deviating allosteric/orthosteric interactions are mediated through the core region of the allosteric site. A key epitope is M2Trp422 in position 7.35 that is located at the extracellular top of transmembrane helix 7 and that contacts, in the inactive receptor, the extracellular loop E2. Trp 7.35 is critically involved in the divergent allosteric/orthosteric cooperativities with acetylcholine and pilocarpine, respectively. In the absence of allosteric agents, Trp 7.35 is essential for receptor binding of the full agonist and for receptor activation by the partial agonist. This study provides first evidence for a role of an allosteric E2/transmembrane helix 7 contact region for muscarinic receptor activation by orthosteric agonists. G protein-coupled receptors (GPCRs) 4The abbreviations used are: GPCR, G protein-coupled receptor; [35S]GTPγS, guanosine 5′-(γ-thio)triphosphate; NMS, N-methylscopolamine; [3H]NMS, [3H]N-methylscopolamine methylbromide; TM, transmembrane helix; CHO, Chinese hamster ovary. 4The abbreviations used are: GPCR, G protein-coupled receptor; [35S]GTPγS, guanosine 5′-(γ-thio)triphosphate; NMS, N-methylscopolamine; [3H]NMS, [3H]N-methylscopolamine methylbromide; TM, transmembrane helix; CHO, Chinese hamster ovary. have outstanding importance as targets for drug action (1Gether U. Endocr. Rev. 2000; 21: 90-113Crossref PubMed Scopus (1002) Google Scholar, 2Pierce K.L. Premont R.T. Lefkowitz R.J. Nat. Rev. Mol. Cell. Biol. 2002; 3: 639-650Crossref PubMed Scopus (2064) Google Scholar). Conformational changes underlying the inactive-to-active receptor switch in GPCRs are in the focus of current research. In general, the receptor transmembrane helices (TMs) rearrange, allowing the intracellular loop region to unfold and to stimulate neighboring G proteins (3Lu Z.-L. Saldanha J.W. Hulme E.C. Trends Pharmacol. Sci. 2002; 23: 140-146Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 4Schwartz T.W. Frimurer T.M. Holst B. Rosenkilde M.M. Elling C.E. Annu. Rev. Pharmacol. Toxicol. 2006; 46: 481-519Crossref PubMed Scopus (342) Google Scholar). Conformational changes include extracellular receptor regions, and a critical role of the second extracellular loop (E2) for GPCR activation and ligand binding has emerged (5Shi L. Javitch J.A. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 440-445Crossref PubMed Scopus (192) Google Scholar, 6Klco J.M. Wiegand C.B. Narzinski K. Baranski T.J. Nat. Struct. Mol. Biol. 2005; 12: 320-326Crossref PubMed Scopus (136) Google Scholar, 7Banères J.-L. Mesnier D. Martin A. Joubert L. Dumuis A. Bockaert J. J. Biol. Chem. 2005; 280: 20253-20260Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 8Scarselli M. Li B. Kim S.-K. Wess J. J. Biol. Chem. 2007; 282: 7385-7396Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 9Avlani V.A. Gregory K.J. Morton C.J. Parker M.W. Sexton P.M. Christopoulos A. J. Biol. Chem. 2007; 282: 25677-25686Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Rational development of agonistic drugs for GPCR activation requires deeper insight into such conformational changes. Because GPCRs are hardly accessible for crystallization, indirect approaches are applied that often involve modification of the receptor protein such as receptor mutagenesis, introduction of metal ion sites or disulfide bridges, or covalent linkage of moieties for fluorescence resonance energy transfer.Allosteric small molecules allow the study of native receptors. An increasing number of GPCRs is known to contain allosteric sites (10Christopoulos A. Kenakin T. Pharmacol. Rev. 2002; 54: 323-374Crossref PubMed Scopus (794) Google Scholar, 11Wess J. Mol. Pharmacol. 2005; 68: 1506-1509Crossref PubMed Scopus (47) Google Scholar); cinacalcet is the first allosteric GPCR modulator that has recently entered the market (12Brauner-Osborne H. Wellendorph P. Jensen A.A. Curr. Drug Targets. 2007; 1: 169-184Crossref Scopus (212) Google Scholar). Allosteric sites are located outside the orthosteric area that is occupied by the endogenous transmitter. Our strategy is based on the hypothesis that activation-related three-dimensional changes involve the whole receptor protein and thus include allosteric sites. Therefore, allosteric ligands should be useful to probe activation-related spatial rearrangement of the receptor protein outside the orthosteric binding area.Muscarinic acetylcholine receptors, especially the M2 subtype, have served as an excellent model system to study cooperative interactions that result from simultaneous binding of allosteric and orthosteric agents (11Wess J. Mol. Pharmacol. 2005; 68: 1506-1509Crossref PubMed Scopus (47) Google Scholar). The so-called common allosteric site of muscarinic receptors resides extracellular to the orthosteric binding site for the endogenous activator acetylcholine. Until now, the consequences of muscarinic allosteric-orthosteric interactions have been studied with a focus on binding affinity. Affinity shifts are cooperative, i.e. the allosteric and the orthosteric ligand change each other's binding affinity toward the same direction and to the same extent. Depending on whether affinities are increased, decreased, or left unaltered, cooperativity is positive, negative, or neutral. The starting point of the present study was a previous observation that an allosteric-orthosteric interaction involved muscarinic receptor activation (13Zahn K. Eckstein N. Tränkle C. Sadeé W. Mohr K. J. Pharmacol. Exp. Ther. 2002; 301: 720-728Crossref PubMed Scopus (49) Google Scholar). We wondered whether allosteric interactions can be exploited as a new strategy to gain insight into activation related conformational changes. Applying allosteric small molecules as probes, we provide evidence that the full agonist acetylcholine and a partial agonist induce divergent conformational changes in the extracellular receptor region. We identified an amino acid, M2Trp422, that plays a key role for allosteric/orthosteric binding and activation cooperativity. This amino acid forms a junction between E2 and TM7 that appears to be essentially involved in GPCR activation.EXPERIMENTAL PROCEDURESMaterialsTest Compounds—Acetylcholine iodide, pilocarpine hydrochloride, carbachol chloride, oxotremorine sesquifumarate, oxotremorine-M iodide, atropine sulfate, N-methylscopolamine bromide, and gallamine triethiodide were obtained from Sigma-Aldrich, and [35S]GTPγS and [3H]NMS were from PerkinElmer Life Sciences. Alcuronium chloride was generously provided by Hoffmann-La Roche AG (Grenzach Wyhlen, Germany). Naphmethonium and W84 are commercially available through AXXORA Deutschland (Grünberg, Germany). The synthesis of propyl-semiW84 has been described elsewhere (14Mohr M. Heller E. Ataie A. Mohr K. Holzgrabe U. J. Med. Chem. 2004; 47: 3324-3327Crossref PubMed Scopus (16) Google Scholar). The structural formulas of the test compounds applied in this study are shown in supplemental Fig. S1.Chemical Synthesis of Propyl-[3-[1,3-dioxo-1H,3H-benzo[de]isoquinoline-2-yl]-2,2-dimethylpropyl]dimethyl ammonium bromide = Seminaph—780 mg (0.5 mmol) of N-[3-(N′,N′-dimethylamino)-2,2-dimethylpropyl]naphthalimide (15Muth M. Sennwitz M. Mohr K. Holzgrabe U. J. Med. Chem. 2005; 48: 2212-2217Crossref PubMed Scopus (12) Google Scholar) were dissolved in 5 ml of 1-bromopropane and stirred at 80 °C for 8 days. The obtained precipitate was filtrated, washed three times with diethyl ether, and dried in vacuo to give 770 mg (71%) seminaph. Mp: 196 °C; 1H NMR (400 MHz, Me2SO-d6): δ 0.90 (t, J 7.2 Hz, -CH2CH3), 1.24 (s, C(CH3)2), 1.75 (m, -CH2CH3), 3.17 (s, N+(CH3)2), 3.36 (br, N+CH2CH2), 3.47 (s, CH2N+), 4.13 (s, NPhthCH2), 7.89 (t, J 7.7 Hz, CHar, 2H), 8.49 (m, CHar, 4H); 13C-NMR: 10.7 (-CH2CH3), 15.9 (-CH2CH3), 25.7 (C(CH3)2), 49.1 (C(CH3)2), 52.2 (N+(CH3)2), 68.7 (N+CH2CH2-), 71.9 (-CH2N+), 122.4, 127.6, 131.4, 127.4, 131.1, 134.5 (CHar), 164.8 (C = O); IR: 783, 937, 1234, 1342, 1587, 1656, 1704 cm−1; Analysis (calcd, found for C22H29BrN2O2 (433.4),) C (61.09, 61.25), H (6.76, 6.58), N (6.48, 6.57).Cell Culture, Transient Transfection, and Membrane Preparation—Unless otherwise indicated we used CHO cells that were stably transfected with the human M2 receptor gene (13Zahn K. Eckstein N. Tränkle C. Sadeé W. Mohr K. J. Pharmacol. Exp. Ther. 2002; 301: 720-728Crossref PubMed Scopus (49) Google Scholar) or wild-type CHO cells that were transiently transfected with wild-type or mutant M2 receptor genes as described previously (16Prilla S. Schrobang J. Ellis J. Höltje H.D. Mohr K. Mol. Pharmacol. 2006; 70: 181-193Crossref PubMed Scopus (54) Google Scholar). One day before transfection, the cells were grown in 10-cm dishes by seeding 1.6 × 106 cells/dish. 48 h after transfection cells were harvested. Stably transfected CHO cells grown to confluence were treated for 24 h with 5 mm sodium butyrate added to the culture medium to increase receptor expression before harvesting the cells. The cells were homogenized in ice-cold buffer (10 mm HEPES, 10 mm MgCl2, 100 mm NaCl, pH 7.4). After centrifugation membranes were resuspended and stored in aliquots at -80 °C. Receptor densities as determined by [3H]NMS binding experiments amounted to 3–5 pmol/mg membrane protein in case of stably transfected cells and to 0.3–0.6 pmol/mg protein in case of transiently transfected cells.[35S]GTPγS Binding Experiments—[35S]GTPγS binding experiments were carried out as described before (13Zahn K. Eckstein N. Tränkle C. Sadeé W. Mohr K. J. Pharmacol. Exp. Ther. 2002; 301: 720-728Crossref PubMed Scopus (49) Google Scholar). Using 96-well plates, the membranes (50 μl; final concentration, 50 μg protein/ml) were added to 400 μl of incubation buffer (final concentrations, 10 mm HEPES, 10 mm MgCl2, and 100 mm NaCl, pH 7.4) containing 10 μm GDP (final concentration) and the test compounds at the indicated concentrations. After adding 50 μl of 0.07 nm [35S]GTPγS (final concentration), the mixture was incubated for 60 min at 30 °C. Membrane-bound radioactivity was separated by vacuum filtration. After drying, glass fiber filter mats were melted with scintillation wax, and radioactivity was counted.[3H]NMS Binding Experiments—Binding of [3H]NMS (0.2 nm) was measured in the absence and presence of increasing concentrations of test compounds in 96-well plates under the conditions of the [35S]GTPγS binding experiments (10 mm HEPES, 10 mm MgCl2, 100 mm NaCl, 10 μm GDP, pH 7.4, 30 °C), unless otherwise indicated. An incubation time of 6 h was applied to ensure equilibrium binding conditions in these experiments. Nonspecific [3H]NMS binding was determined in the presence of 3 μm atropine. As described before (16Prilla S. Schrobang J. Ellis J. Höltje H.D. Mohr K. Mol. Pharmacol. 2006; 70: 181-193Crossref PubMed Scopus (54) Google Scholar), NMS binding affinity for wild-type and mutant receptors was determined in homologous [3H]NMS/NMS competition experiments. Allosteric probe binding affinity was derived from [3H]NMS/allosteric agent interaction experiments. Apparent affinities of orthosteric agonists were deduced from [3H]NMS/agonist interaction experiments performed either in the absence or presence of a fixed concentration of allosteric probe. Filtration and measurement of membrane-bound radioactivity was carried out as described above. [3H]NMS dissociation experiments were performed as described previously (16Prilla S. Schrobang J. Ellis J. Höltje H.D. Mohr K. Mol. Pharmacol. 2006; 70: 181-193Crossref PubMed Scopus (54) Google Scholar).Three-dimensional Modeling and Molecular Dynamics Simulations and Homology Modeling—The coordinates of the x-ray structure 1U19 (Protein Data Bank) of bovine rhodopsin in its inactive state with a resolution of 2.2 Å (17Okada T. Sugihara M. Bondar A.N. Elstner M. Entel P. Buss V. J. Mol. Biol. 2004; 342: 571-583Crossref PubMed Scopus (929) Google Scholar) were used as template for the homology model of the human muscarinic M2 receptor. Further details were as described previously (16Prilla S. Schrobang J. Ellis J. Höltje H.D. Mohr K. Mol. Pharmacol. 2006; 70: 181-193Crossref PubMed Scopus (54) Google Scholar).Receptor-Ligand Complex—Docking of N-methylscopolamine (NMS) has been described previously (16Prilla S. Schrobang J. Ellis J. Höltje H.D. Mohr K. Mol. Pharmacol. 2006; 70: 181-193Crossref PubMed Scopus (54) Google Scholar). The allosteric modulator seminaph was docked using the software package GOLD (18Jones G. Willett P. Glen R.C. J. Mol. Biol. 2005; 245: 43-53Crossref Scopus (1372) Google Scholar). The binding site was defined as a sphere with a diameter of 10 nm around the residue M2Asp175, which is located in the center of the allosteric binding pocket. 50 different complex geometries in three main clusters were written out and scored with GoldScore. The best scored protein-ligand complex, which was in accordance with mutation experiments, was used for the following molecular dynamics simulation.Molecular Dynamics Simulations—For further refinement and structure validation, several molecular dynamics simulations were carried out using the software package GROMACS (19Lindahl E. Hess B. van der Spoel D. J. Mol. Model. 2001; 7: 306-317Crossref Google Scholar). The simulations were performed for the ligand-free and the seminaph-occupied receptor and the ternary complex consisting of the M2 receptor, NMS, and seminaph, respectively. Further details including the model quality check have been described previously (16Prilla S. Schrobang J. Ellis J. Höltje H.D. Mohr K. Mol. Pharmacol. 2006; 70: 181-193Crossref PubMed Scopus (54) Google Scholar).Data and Statistical Analysis—Indicated are the mean values ± standard error. Nonlinear regression analysis was done using the software PRISM 4.03 and INSTAT 3.0 (Graph Pad, San Diego, CA). Data from [35S]GTPγS binding studies were analyzed according to a ternary complex model, which explicitly includes effects of the allosteric ligand on receptor activation (20Hall D.A. Mol. Pharmacol. 2000; 58: 1412-1423Crossref PubMed Scopus (173) Google Scholar).Ractive/Rt=L.(1+α.k.[A]+β.M.[B](1+α.γ.δ.k.[A]))1+L+M.[B](1+β.L)+K.[A](1+α.L+γ.M.[B](1+α.β.δ.L))(Eq. 1) Ractive/Rt is the fraction of active receptors relative to the total population of receptors. K and M are the association constants of the orthosteric agonist A and the allosteric agent B, respectively. L denotes the receptor isomerization constant. α and β indicate the intrinsic efficacy of A and B, respectively. γ represents the binding cooperativity between A and B, and δ represents the activation cooperativity between A and B. The zero level of muscarinic receptor dependent [35S]GTPγS binding was defined as the binding in the presence of 1 μm of the inverse agonist atropine, Ractive/Rt = 0.0. The maximum effect of the full agonist acetylcholine on [35S]GTPγS observed under control conditions in the absence of another test compound was set as Ractive/Rt = 1.0. Global nonlinear curve fitting of fractional receptor activation Ractive/Rt as the dependent variable with A and B as the independent variables yielded estimates for L, K, M, α, β, γ, and δ.Allosteric Probe Effects—The effect of naphmethonium on [3H]NMS equilibrium binding was analyzed as described (21Ehlert F.J. Mol. Pharmacol. 1988; 35: 187-194Google Scholar, 22Lazareno S. Birdsall N.J.M. Mol. Pharmacol. 1995; 48: 362-378PubMed Google Scholar). [3H]NMS binding data derived from inhibition experiments with an agonist in the absence and presence of allosteric agent were fitted globally according to the extended allosteric ternary complex model using Equation 34 in Ref. 22Lazareno S. Birdsall N.J.M. Mol. Pharmacol. 1995; 48: 362-378PubMed Google Scholar. Specifically, fractional occupancy was fitted as the dependent variable with the concentrations of the agonist and the allosteric agent as two independent variables. After fixing the affinities of the radioligand and the allosteric agent to parameter values having been determined in separate experiments, curve fitting yielded estimates of agonist affinity and agonist cooperativity with naphmethonium.RESULTSAgonist-dependent Receptor Sensitivity to Allosteric Probes—We used M2 receptors expressed in membranes from CHO cells transfected with the human receptor gene. Receptor activation leads to G protein stimulation, which was measured using the GTP analogue [35S]GTPγS that binds to an activated G protein but is not hydrolyzed (23Lazareno S. Methods Mol. Biol. 1999; 106: 231-245PubMed Google Scholar). To induce receptor activation we applied the endogenous full agonist acetylcholine, the partial agonist pilocarpine, and additional agonists as specified below. As allosteric probes we chose the flexible “spaghetti-like” bis(ammonio)alkane-type compounds naphmethonium and W84, their shortened derivatives seminaph and propyl-semiW84, the rigid “disk-like” alcuronium, and the smaller gallamine (all structures shown in supplemental Fig. S1).Naphmethonium (Fig. 1A) and the other probes hardly affected the basal, starting level of G protein activity but discriminated sensitively between pilocarpine- and acetylcholine-bound receptors (Fig. 1B, left and right panels, respectively). As expected, the partial agonist pilocarpine induced a significantly lower maximum response than did the endogenous full agonist acetylcholine. Naphmethonium reduced the maximum response of pilocarpine but not of acetylcholine. The potency as indicated by the inflection point of the curve was allosterically increased with pilocarpine but strongly decreased with acetylcholine (arrows in Fig. 1B indicate the inflection point shift to the left and right, respectively).To parameterize the findings obtained with naphmethonium (Fig. 1C) and the other probes (supplemental Fig. S2), we applied Hall's allosteric two-state model (20Hall D.A. Mol. Pharmacol. 2000; 58: 1412-1423Crossref PubMed Scopus (173) Google Scholar). The model assumes that receptors switch spontaneously between inactive and active (G protein stimulating) states (Fig. 2, gray and green panels, respectively). A ligand, either orthosteric or allosteric, may bind to these states with different affinities, thus affecting the equilibrium between inactive and active states. The model does not exclude that ligand-bound receptors differ in conformation depending on the type of bound ligand (20Hall D.A. Mol. Pharmacol. 2000; 58: 1412-1423Crossref PubMed Scopus (173) Google Scholar). When orthosteric ligand and allosteric probe are present simultaneously, reciprocal cooperative interactions may occur between these ligands with respect to both binding affinity and receptor activation (log γ and log δ in Fig. 1C). Depending on whether these parameters are increased, decreased, or left unchanged, cooperativity is positive, negative, or neutral, respectively.FIGURE 2Allosteric two state model of receptor activation by Hall (20Hall D.A. Mol. Pharmacol. 2000; 58: 1412-1423Crossref PubMed Scopus (173) Google Scholar). Possible states of receptor occupancy according to the ternary complex model of allosteric/orthosteric interactions are displayed for inactive (gray front panel) and active receptors (green background panel). In each state of occupancy, the receptor can switch between the inactive conformation R and the active conformation R*. The parameters are explained in the legend for Fig. 1C.View Large Image Figure ViewerDownload Hi-res image Download (PPT)With pilocarpine, all probes except gallamine displayed negative cooperativity of receptor activation, whereas binding cooperativity varied depending on the probe (Table 1). With acetylcholine, however, there was no evidence for impaired receptor activation with any of the probes, whereas binding cooperativity was strongly negative with all probes. The divergent cooperativities with allosteric probes suggest a pronounced difference in three-dimensional shape of the allosteric site between the acetylcholine-bound and the pilocarpine-bound receptor.TABLE 1Test compound actions in terms of the allosteric two state model of receptor activation Underlying experimental data are displayed in supplemental Fig. S2. The parameter values were obtained as described in the legend of Fig. 1.Spontaneous receptor activity: receptor isomerization constant (log L)Agonist/receptor interactionAllosteric probe/receptor interactionProbe/agonist/receptor interactionAgonist affinity for R (log K)Agonist intrinsic efficacy (log α)Allosteric probe affinity for R (log M)aNo significant difference in any pair of log M values for each given allosteric probe (t test, p > 0.05)Allosteric probe intrinsic efficacy (log β)Cooperativity of binding to inactive receptors (log γ)Cooperativity of receptor activation (log δ)Experiments with the partial agonist pilocarpine W84–0.80 ± 0.045.76 ± 0.061.08 ± 0.046.54 ± 0.10–0.34 ± 0.08–0.89 ± 0.14–0.64 ± 0.11bSignificantly smaller than zero indicating negative activation cooperativity (t test, p < 0.05) Propyl-semiW84–0.80 ± 0.045.92 ± 0.061.06 ± 0.045.68 ± 0.060.01 ± 0.05–1.77 ± 0.13–0.79 ± 0.07bSignificantly smaller than zero indicating negative activation cooperativity (t test, p < 0.05) Seminaph–0.83 ± 0.026.12 ± 0.031.09 ± 0.025.66 ± 0.110.07 ± 0.030.11 ± 0.12–0.89 ± 0.03bSignificantly smaller than zero indicating negative activation cooperativity (t test, p < 0.05) Alcuronium–0.82 ± 0.025.97 ± 0.061.08 ± 0.036.37 ± 0.12–0.10 ± 0.030.14 ± 0.14–0.84 ± 0.04bSignificantly smaller than zero indicating negative activation cooperativity (t test, p < 0.05) Gallamine–0.84 ± 0.045.99 ± 0.051.09 ± 0.046.18 ± 0.080.03 ± 0.05–2.06 ± 0.14–0.15 ± 0.07Experiments with the full agonist acetylcholine W84–0.73 ± 0.076.16 ± 0.182.00 ± 0.166.40 ± 0.09–0.59 ± 0.13–3.01 ± 0.57–0.26 ± 0.48 Propyl-semiW84–0.80 ± 0.146.30 ± 0.221.93 ± 0.205.68 ± 0.15–0.008 ± 0.16–3.02 ± 0.67–0.28 ± 0.56 Seminaph–0.79 ± 0.186.27 ± 0.682.65 ± 0.646.05 ± 0.17–0.003 ± 0.201–2.26 ± 0.88–13.80 ± 9.59 Alcuronium–0.76 ± 0.146.45 ± 0.242.12 ± 0.236.34 ± 0.130.09 ± 0.15–3.26 ± 1.050.02 ± 0.96 Gallamine–0.79 ± 0.146.26 ± 0.332.29 ± 0.316.06 ± 0.14–0.06 ± 0.16–2.08 ± 0.41–0.68 ± 0.36a No significant difference in any pair of log M values for each given allosteric probe (t test, p > 0.05)b Significantly smaller than zero indicating negative activation cooperativity (t test, p < 0.05) Open table in a new tab To check that the divergent sensitivities between acetylcholine and pilocarpine to the allosteric probes do not merely depend on differences in the chemical nature of the agonists, we compared responses of a number of agonists to high concentrations of the probes naphmethonium and alcuronium. Agonist concentration-effect curves for M2 receptor-mediated [35S]GTPγS binding were analyzed on a descriptive level with the inflection point and the upper plateau of the curve, indicating agonist potency and intrinsic efficacy, respectively. Control values obtained in the absence of allosteric probe are compiled in Table 2, allosteric changes of these parameters are illustrated in Fig. 3. The allosteric agents reduced the potency of the full agonists acetylcholine, carbachol, oxotremorine, and oxotremorine M to a similar extent by ∼2–3 orders of magnitude, whereas the potency of the partial agonist pilocarpine was not reduced or even increased (Fig. 3A). Vice versa, the maximum effect of the full agonists was hardly changed by the allosteric probes, whereas a strong reduction of efficacy was observed with the partial agonist (Fig. 3B). Therefore, we conclude that the difference in sensitivity between acetylcholine and pilocarpine to allosteric modulation is related to their full and partial agonist character, respectively.TABLE 2Potency and efficacy of the orthosteric agonists under control conditions in [35S]GTPγS binding experiments Pairwise recording of concentration-effect curves of the agonist under study and of acetylcholine for comparison. ACh, acetylcholine; CCh, carbachol; Oxo, oxotremorine; Oxo-M, oxotremorine M; Pilo, pilocarpine. Potency is the –log value of the inflection points of the curves. Efficacy is the maximum agonist effect expressed as percentages of the maximum effect of acetylcholine set as 100%. The data indicate the means ± S.E. of 2–27 experiments performed as quadruplicate determinations.AchCChOxoOxo-MPiloPotency7.69 ± 0.037.11 ± 0.148.01 ± 0.158.13 ± 0.106.35 ± 0.09Efficacy (%)100102 ± 6108 ± 4106 ± 479 ± 3aSignificantly different from acetylcholine efficacy (t test, p < 0.05)a Significantly different from acetylcholine efficacy (t test, p < 0.05) Open table in a new tab FIGURE 3Allosteric effects on the action of full agonists and the partial agonist pilocarpine explored in [35S]GTPγS binding experiments as shown in Fig. 1. A, difference in agonist potency (log concentration at the agonist curve inflection point) in the presence and absence of allosteric probe. B, difference in maximum effect (upper curve plateau) in the presence and absence of allosteric probe expressed in percentages of the agonist effect in the absence of probe (set as 100%). Allosteric probe concentrations were 100 μm naphmethonium (Naph) and 300 μm alcuronium (Alc) throughout except 30 and 100 μm, respectively, in case of pilocarpine because of pronounced efficacy loss. The data indicate the means ± S.E. derived from two to 14 concentration-effect-curves determined in quadruplicate. ACh, acetylcholine; CCh, carbachol; Oxo, oxotremorine; Oxo-M, oxotremorine M; Pilo, pilocarpine.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Previous binding experiments suggested that the allosteric site of the M2 receptor changes considerably in shape when the orthosteric site is occupied by an agonist instead of an antagonist (24Grossmüller M. Antony J. Tränkle C. Holzgrabe U. Mohr K. Naunyn-Schmiedeberg’s Arch. Pharmacol. 2006; 372: 267-276Crossref PubMed Scopus (18) Google Scholar). In that study we compared structure-binding relationships for stepwise shortened allosteric agents in M2 receptors whose orthosteric site was occupied by either the radioantagonist [3H]NMS or the radioagonist [3H]oxotremorine M. Being an inverse agonist NMS stabilizes the receptor in an inactive conformation. We predicted that the binding cooperativities found in the present study with acetylcholine should differ from binding cooperativities with the antagonist NMS, and we wondered how these cooperativities would compare with those found for pilocarpine.As shown in Fig. 4, there is no correlation between the binding cooperativities of the probes with acetylcholine and NMS, respectively. In contrast, probe/NMS binding cooperativities strongly correlate with probe/pilocarpine binding cooperativities" @default.
• W1999647921 created "2016-06-24" @default.
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• W1999647921 date "2007-11-01" @default.
• W1999647921 modified "2023-10-16" @default.
• W1999647921 title "Allosteric Small Molecules Unveil a Role of an Extracellular E2/Transmembrane Helix 7 Junction for G Protein-coupled Receptor Activation" @default.
• W1999647921 cites W1603274157 @default.
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