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Mutational Analysis and Molecular Modeling of the Allosteric Binding Site of a Novel, Selective, Noncompetitive Antagonist of the Metabotropic Glutamate 1 Receptor

2003; Elsevier BV; Volume: 278; Issue: 10 Linguagem: Inglês

10.1074/jbc.m211759200

1083-351X

Pari Malherbe, Nicole A. Kratochwil, Frédéric Knoflach, Marie‐Thérèse Zenner, James N.C. Kew, Claudia Kratzeisen, Hans Peter Maerki, Geo Adam, Vincent Mutel,

Photoreceptor and optogenetics research

A model of the rmGlu1 seven-transmembrane domain complexed with a negative allosteric modulator, 1-ethyl-2-methyl-6-oxo-4-(1,2,4,5-tetrahydro-benzo[d]azepin-3-yl)- 1,6-dihydro-pyrimidine-5-carbonitrile (EM-TBPC) was constructed. Although the mGlu receptors belong to the family 3 G-protein-coupled receptors with a low primary sequence similarity to rhodopsin-like receptors, the high resolution crystal structure of rhodopsin was successfully applied as a template in this model and used to select residues for site-directed mutagenesis. Three mutations, F8016.51A, Y8056.55A, and T8157.39M caused complete loss of the [3H]EM-TBPC binding and blocked the EM-TBPC-mediated inhibition of glutamate-evoked G-protein-coupled inwardly rectifying K+ channel current and [Ca2+]i response. The mutation W7986.48F increased the binding affinity of antagonist by 10-fold and also resulted in a marked decrease in the IC50value (4 versus 128 nm) compared with wild type. The V7575.47L mutation led to a dramatic reduction in binding affinity by 13-fold and a large increase in the IC50 value (1160 versus 128 nm). Two mutations, N74745.51A and N75045.54A, increased the efficacy of the EM-TBPC block of the glutamate-evoked [Ca2+]i response. We observed a striking conservation in the position of critical residues. The residues Val-7575.47, Trp-7986.48, Phe-8016.51, Tyr-8056.55, and Thr-8157.39 are critical determinants of the EM-TBPC-binding pocket of the mGlu1 receptor, validating the rhodopsin crystal structure as a template for the family 3 G-protein-coupled receptors. In our model, the aromatic ring of EM-TBPC might interact with the cluster of aromatic residues formed from Trp-7986.48, Phe-8016.51, and Tyr-8056.55, thereby blocking the movement of the TM6 helix, which is crucial for receptor activation. A model of the rmGlu1 seven-transmembrane domain complexed with a negative allosteric modulator, 1-ethyl-2-methyl-6-oxo-4-(1,2,4,5-tetrahydro-benzo[d]azepin-3-yl)- 1,6-dihydro-pyrimidine-5-carbonitrile (EM-TBPC) was constructed. Although the mGlu receptors belong to the family 3 G-protein-coupled receptors with a low primary sequence similarity to rhodopsin-like receptors, the high resolution crystal structure of rhodopsin was successfully applied as a template in this model and used to select residues for site-directed mutagenesis. Three mutations, F8016.51A, Y8056.55A, and T8157.39M caused complete loss of the [3H]EM-TBPC binding and blocked the EM-TBPC-mediated inhibition of glutamate-evoked G-protein-coupled inwardly rectifying K+ channel current and [Ca2+]i response. The mutation W7986.48F increased the binding affinity of antagonist by 10-fold and also resulted in a marked decrease in the IC50value (4 versus 128 nm) compared with wild type. The V7575.47L mutation led to a dramatic reduction in binding affinity by 13-fold and a large increase in the IC50 value (1160 versus 128 nm). Two mutations, N74745.51A and N75045.54A, increased the efficacy of the EM-TBPC block of the glutamate-evoked [Ca2+]i response. We observed a striking conservation in the position of critical residues. The residues Val-7575.47, Trp-7986.48, Phe-8016.51, Tyr-8056.55, and Thr-8157.39 are critical determinants of the EM-TBPC-binding pocket of the mGlu1 receptor, validating the rhodopsin crystal structure as a template for the family 3 G-protein-coupled receptors. In our model, the aromatic ring of EM-TBPC might interact with the cluster of aromatic residues formed from Trp-7986.48, Phe-8016.51, and Tyr-8056.55, thereby blocking the movement of the TM6 helix, which is crucial for receptor activation. metabotropic glutamate human mGlu transmembrane seven-transmembrane seven-transmembrane domain 1-ethyl-2-methyl-6-oxo-4-(1,2,4,5-tetrahydro-benzo[d]azepin-3-yl)-1,6-dihydro-pyrimidine-5-carbonitrile G-protein-coupled receptor G-protein-coupled inwardly rectifying K+ channel wild type γ-aminobutyric acid, type B extracellular loop 7-(hydroxyimino)cyclopropan[b]chromen-1a-carboxylic acid ethyl ester 2-methyl-6-(phenylethynyl)pyridine [(3aS,6aS)-6a-naphtalen-2-ylmethyl-5-methyliden-hexahydro-cyclopental[c]furan-1-on] (S)-2-(4-fluorophenyl)-1-(toluene-4-sulfonyl)-pyrrolidine diphenylacetyl-carbamic acid ethyl ester (9H-xanthene-9-carbonyl)-carbamic acid butyl ester 2,6-di-tert-butyl-4-(3-hydroxy-2,2-dimethylpropyl)-phenol N-[2-[(2-chloro-6-fluorobenzyl)thio]ethyl]2-thiophenecarboxamide Chinese hamster ovary 5-hydroxytryptamine type 4 The mGlu1 receptor family currently comprises eight receptors that are divided into three classes on the basis of their sequence similarities, signal transduction, and agonist rank order of potency. Group I (mGlu1 and -5) receptors are coupled to the stimulation of phosphoinositide hydrolysis; group II (mGlu2 and -3) and group III receptors (mGlu4, -6, -7, and -8) are negatively coupled to cAMP production (1Conn P.J. Pin J.P. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 205-237Google Scholar, 2Pin J.P. De Colle C. Bessis A.S. Acher F. Eur. J. Pharmacol. 1999; 375: 277-294Google Scholar, 3De Blasi A. Conn P.J. Pin J.P. Nicoletti F. Trends Pharmacol. Sci. 2001; 22: 114-120Google Scholar). Many studies have demonstrated the involvement of mGlu receptors in the modulation of synaptic transmission, ion channel activity, and synaptic plasticity (4Holscher C. Gigg J. O'Mara S.M. Neurosci. Biobehav. Rev. 1999; 23: 399-410Google Scholar, 5Nakanishi S. Neuron. 1994; 13: 1031-1037Google Scholar), and dysfunction of these receptors has been implicated in psychiatric and neurological diseases (6Bordi F. Ugolini A. Prog. Neurobiol. 1999; 59: 55-79Google Scholar). The mGlu receptors belong to the family 3 of G-protein-coupled receptors (GPCRs). Other members of this family include the GABAB, Ca2+-sensing, vomeronasal, pheromone, and putative taste receptors (7Bockaert J. Pin J.P. EMBO J. 1999; 18: 1723-1729Google Scholar). The family 3 GPCRs shares a low sequence similarity with the other families. In contrast to family 1, the family 3 receptors are characterized by two distinctly separated topological domains: an exceptionally long extracellular amino-terminal domain (500–600 amino acids), which forms the agonist-binding pocket (8O'Hara P.J. Sheppard P.O. Thogersen H. Venezia D. Haldeman B.A. McGrane V. Houamed K.M. Thomsen C. Gilbert T.L. Mulvihill E.R. Neuron. 1993; 11: 41-52Google Scholar, 9Galvez T. Prezeau L. Milioti G. Franek M. Joly C. Froestl W. Bettler B. Bertrand H.O. Blahos J. Pin J.P. J. Biol. Chem. 2000; 275: 41166-41174Google Scholar, 10Kunishima N. Shimada Y. Tsuji Y. Sato T. Yamamoto M. Kumasaka T. Nakanishi S. Jingami H. Morikawa K. Nature. 2000; 407: 971-977Google Scholar), and the 7TM helical segments involved in receptor activation and G-protein coupling (11Parmentier M.L. Prezeau L. Bockaert J. Pin J.P. Trends Pharmacol. Sci. 2002; 23: 268-274Google Scholar). Compounds acting at group I mGlu receptors can be grouped into two categories. Category one comprises competitive agonists and antagonists. These compounds are phenylglycine derivatives or rigidified analogs of glutamate (12Schoepp D.D. Jane D.E. Monn J.A. Neuropharmacology. 1999; 38: 1431-1476Google Scholar), which logically bind to the glutamate-binding domain. Competitive group I ligands have achieved only limited subtype selectivity and potency, perhaps due to the high sequence homology of the mGlu receptor family agonist-binding site supported by the three-dimensional structure of mGlu1 amino-terminal domain (10Kunishima N. Shimada Y. Tsuji Y. Sato T. Yamamoto M. Kumasaka T. Nakanishi S. Jingami H. Morikawa K. Nature. 2000; 407: 971-977Google Scholar). However, recent development of more sensitive technologies for functional screening of GPCRs has resulted in the discovery of a second category of compounds. These novel compounds, which interact within the 7TMD of group I mGlu receptor, act as positive or negative allosteric modulators (13Gasparini F. Kuhn R. Pin J.P. Curr. Opin. Pharmacol. 2002; 2: 43-49Google Scholar). CPCCOEt was the first non-amino acid derivative, subtype-selective antagonist of the mGlu1 receptor (IC50 = 6.5 μm at hmGlu1b) to be described (14Annoura H. Fukunaga A. Uesugi M. Tatsuoka T. Horikawa Y. Bioorg. Med. Chem. Lett. 1996; 6: 763-766Google Scholar). Litschig et al. (15Litschig S. Gasparini F. Rueegg D. Stoehr N. Flor P.J. Vranesic I. Prezeau L. Pin J.P. Thomsen C. Kuhn R. Mol. Pharmacol. 1999; 55: 453-461Google Scholar) elucidated the site of action of CPCCOEt, which binds within the 7TMD of mGlu1, in close contact with the residues Thr-815 and Ala-818 of TM7. Similarly, methyl-6-(phenylethynyl)pyridine (MPEP), which was the first noncompetitive, highly potent, mGlu5-selective antagonist (IC50 = 36 nm at hmGlu5a) to be described (16Gasparini F. Lingenhohl K. Stoehr N. Flor P.J. Heinrich M. Vranesic I. Biollaz M. Allgeier H. Heckendorn R. Urwyler S. Varney M.A. Johnson E.C. Hess S.D. Rao S.P. Sacaan A.I. Santori E.M. Velicelebi G. Kuhn R. Neuropharmacology. 1999; 38: 1493-1503Google Scholar), was suggested to make close contact with the amino acid residues Ala-810 in TM7 and Pro-655 and Ser-658 in TM3 of the mGlu5 receptor. Moreover, it has been demonstrated that the CPCCOEt and MPEP interact with residues that appear to form overlapping binding pockets in homologous regions of the 7TMD of the mGlu1 and -5 receptors, respectively (17Pagano A. Ruegg D. Litschig S. Stoehr N. Stierlin C. Heinrich M. Floersheim P. Prezeau L. Carroll F. Pin J.P. Cambria A. Vranesic I. Flor P.J. Gasparini F. Kuhn R. J. Biol. Chem. 2000; 275: 33750-33758Google Scholar). Recently, BAY36-7620, another highly potent mGlu1-selective antagonist (IC50 = 160 nm at rmGlu1a) that interacts within the 7TMD, has been reported (18Carroll F.Y. Stolle A. Beart P.M. Voerste A. Brabet I. Mauler F. Joly C. Antonicek H. Bockaert J. Muller T. Pin J.P. Prezeau L. Mol. Pharmacol. 2001; 59: 965-973Google Scholar). Knoflach et al. (19Knoflach F. Mutel V. Jolidon S. Kew J.N.C. Malherbe P. Vieira E. Wichmann J. Kemp J.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13402-13407Google Scholar) have described a novel class of ligands RO 67-7476, RO 01-6128, and RO 67-4853 acting as positive allosteric modulators of the mGlu1 receptor. Interestingly, their binding pocket appears to be also located within the 7TMD of mGlu1. Furthermore, the mutational analysis revealed (19Knoflach F. Mutel V. Jolidon S. Kew J.N.C. Malherbe P. Vieira E. Wichmann J. Kemp J.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13402-13407Google Scholar) that RO 67-7476 binding site in the TM3 region of mGlu1 appears to overlap with that of the MPEP binding site in the homologous region of the mGlu5 receptor. Urwyler et al. (20Urwyler S. Mosbacher J. Lingenhoehl K. Heid J. Hofstetter K. Froestl W. Bettler B. Kaupmann K. Mol. Pharmacol. 2001; 60: 963-971Google Scholar) concomitantly reported on the identification of CGP7930 and its aldehyde analog CGP13501, as positive modulators of GABAB receptor function. In the present study, we have probed the allosteric antagonist-binding site of mGlu1 using molecular modeling, site-directed mutagenesis, [3H]1-ethyl-2-methyl-6-oxo-4-(1,2,4,5-tetrahydro-benzo[d]azepin-3-yl)-1,6-dihydro-pyrimidine-5-carbonitrile (EM-TBPC) binding, Ca2+ imaging, and G-protein-coupled inwardly rectifying K+ channel (GIRK) current activation. [3H]EM-TBPC is a highly potent, subtype-selective, noncompetitive antagonist of rmGlu1 receptor (21.Adam, G., Binggeli, A., Maerki, H. P., Mutel, V., Wilhelm, M., and Wostl, W. (2001) European Patent Application, pp. 1–85, Priority EP 99–1074549 A2.Google Scholar, 22Knoflach F. Mutel V. Kew J.N.C. Waselle L. Vieira E. Jolidon S. Wichmann J. Malherbe P. Kemp J.A. Soc. Neurosci. Abstr. 2001; 705: 13Google Scholar). Amino acid residues in the TM3, -5, -6, and -7 and extracellular loop 2 (EC2) regions, initially identified from an alignment of the 7TMD of rmGlu1 with bovine rhodopsin, were demonstrated by mutational analysis to be important determinants of the noncompetitive antagonist binding pocket of the mGlu1 receptor. A homology model constructed based on the x-ray crystal of bovine rhodopsin (23Palczewski K. Kumasaka T. Hori T. Behnke C.A. Motoshima H. Fox B.A. Le Trong I. Teller D.C. Okada T. Stenkamp R.E. Yamamoto M. Miyano M. Science. 2000; 289: 739-745Google Scholar) visualizes these findings and suggests a possible binding mode of EM-TBPC. EM-TBPC andN-[2-[(2-chloro-6-fluorobenzyl)thio]ethyl]2-thiophenecarboxamide (CFBTE-TPC) were synthesized at Hoffmann-La Roche Ltd. (21.Adam, G., Binggeli, A., Maerki, H. P., Mutel, V., Wilhelm, M., and Wostl, W. (2001) European Patent Application, pp. 1–85, Priority EP 99–1074549 A2.Google Scholar). [3H]EM-TBPC (specific activity 33.4 Ci/mmol) was synthesized by Dr. P. Huguenin at the Roche chemical and isotope laboratories (21.Adam, G., Binggeli, A., Maerki, H. P., Mutel, V., Wilhelm, M., and Wostl, W. (2001) European Patent Application, pp. 1–85, Priority EP 99–1074549 A2.Google Scholar). [3H]Quisqualate (specific activity, 32–35 Ci/mmol; TRK 1070) was synthesized at Amersham Biosciences. 2-MPEP was obtained from Sigma. The position of each amino acid residue in the 7TMD of mGlu receptor is identified both by its sequence number (including the signal peptide) and by the generic numbering system proposed by Ballesteros and Weinstein (24Ballesteros J.A. Weinstein H. Methods Neurosci. 1995; 25: 366-428Google Scholar) which is shown as superscript. In this numbering system, amino acid residues in the 7TMD are given two numbers; the first refers to the TM number, and the second indicates its position relative to a highly conserved residue of family 1 GPCRs in that TM which is arbitrarily assigned 50. The amino acids in the extracellular loop EC2 are labeled 45 to indicate their location between the helix 4 and 5. The highly conserved cysteine, thought to be disulfide-bonded, is given the index number 45.50 (SWISS-PROT: opsd_bovin C187), and the residues within the EC2 are then indexed relative to the "50" position. An alignment of the seven-transmembrane helices of rmGlu1 toward the transmembrane helices of bovine rhodopsin (Protein Data Bank code 1f88) were obtained with help of our in-house program Xsae, 2C. Broger, unpublished work. using a modified version of ClustalV (25Higgins D.C. Bleasby A.J. Fuch R. Comput. Appl. Biosci. 1992; 8: 189-191Google Scholar). Sequences were obtained from SWISS-PROT: rmGlu1, P23385. The sequence of bovine rhodopsin was read directly from the rhodopsin structure (Protein Data Bank code 1f88). All modeling calculations were made on a Silicon Graphics Octane with a single R12000 processor using our in-house modeling package Moloc (26Gerber P.R. Muller K. J. Comput. Aided Mol. Des. 1995; 9: 251-268Google Scholar, 27Gerber P.R. J. Comput. Aided Mol. Des. 1998; 12: 37-51Google Scholar) (available on the World Wide Web at www.Moloc.ch). An initial C-α model of rat mGlu1 was built by fitting the aligned rmGlu1 sequence lacking the extracellular domain on the bovine rhodopsin template C-α structure. Loops were optimized with the Moloc C-α force field. In a next step, a full atom model was generated. φ and ψ angles were obtained for aligned amino acids from the rhodopsin template. χ angles were also adopted from the bovine rhodopsin structure where possible or in case of nonidentical amino acids generated by using the most probable value applying the Ponder-Richards method (28Ponder J.W. Richards F.M. J. Mol. Biol. 1987; 193: 775-791Google Scholar). An energy calculation of the initial full peptide structure revealed regions with bad van der Waals contacts of amino acid side chains that were subsequently improved by manually adjusting the relevant χ angles. Repulsive van der Waals interactions were removed manually where necessary. Refinement of the model was done by keeping all backbone atoms in fixed positions and allowing only the side chains to move. In a following optimization step, only C-α atoms were kept in a fixed position while all other atoms were allowed to move. In a third round of optimization, no atoms were kept stationary, but constraints were applied to C-α atoms. The quality of the model was then checked with Moloc internal programs. EM-TBPC was manually docked into the 7TMD region taking cis-retinal as template for location. Where necessary, nonconserved amino acid side chains in the rmGlu1 model were rotated such that no van der Waals conflicts with the antagonist occurred. All amino acid side chains reaching within a 6-Å distance of the antagonist were subsequently included in a round of optimization. cDNAs encoding the rmGlu1a and rmGlu5a receptors in pBlueScript II were obtained from Prof. S. Nakanishi (Kyoto, Japan). hmGlu1a receptor cDNA was amplified from a human fetal brain cDNA library in pCMV.SPORT2 (Invitrogen) using primers derived from the hmGlu1a sequence (AC:U31216). All point mutants were constructed using the QuikChange site-directed mutagenesis kit (Stratagene). The entire coding regions of all point mutants were sequenced from both strands using an automated cycle sequencer (Applied Biosystems). HEK-293 cells were transfected as previously described (29Malherbe P. Knoflach F. Broger C. Ohresser S. Kratzeisen C. Adam G. Stadler H. Kemp J.A. Mutel V. Mol. Pharmacol. 2001; 60: 944-954Google Scholar). 48 h posttransfection, the cells were harvested and washed three times with cold PBS and frozen at −80 °C. The pellet was suspended in cold 20 mm HEPES-NaOH buffer containing 10 mm EDTA (pH 7.4) and homogenized with a Polytron homogenizer (Kinematica, AG) for 10 s at 10,000 rpm. After centrifugation at 48,000 ×g for 30 min at 4 °C, the pellet was resuspended in cold 20 mm HEPES-NaOH buffer containing 0.1 mm EDTA (pH 7.4), homogenized, and respun as above. The pellet was resuspended in a smaller volume of a cold 20 mm HEPES-NaOH buffer containing 0.1 mm EDTA (pH 7.4). After homogenization for 10 s at 10,000 rpm, the protein content was measured using the BCA method (Pierce) with bovine serum albumin as the standard. The membrane homogenate was frozen at −80 °C before use. After thawing, the membrane homogenates were centrifuged at 48,000 × g for 10 min at 4 °C, the pellets were resuspended in the 20 mm HEPES-NaOH (pH 7.4) binding buffer to a final assay concentration of 20 μg of protein/ml. Saturation isotherms were determined by the addition of various [3H]EM-TBPC concentrations (0.3, 1, 3, 10, 30, 100, and 300 nm) to these membranes for 1 h at room temperature (equilibrium binding conditions determined in kinetic experiments). At the end of the incubation, membranes were filtered onto Filtermate (unitfilter Packard: 96-well white microplate with bonded GF/B filter preincubated 1 h in 0.1% polyethyleneimine) and washed three times with cold binding buffer. Nonspecific binding was measured in the presence of 100 μm CFBTE-TPC, a noncompetitive, subtype-selective antagonist of mGlu1 with K i = 390 nm, which is a compound from the same class as EM-TBPC, Fig. 1 (21.Adam, G., Binggeli, A., Maerki, H. P., Mutel, V., Wilhelm, M., and Wostl, W. (2001) European Patent Application, pp. 1–85, Priority EP 99–1074549 A2.Google Scholar). The radioactivity on the filter was counted on a Packard Top-count microplate scintillation counter with quenching correction after the addition of 40 μl of microscint 40 (Canberra Packard S.A.). Saturation experiments were analyzed by Prism 3.0 (GraphPad Software, San Diego, CA) using the rectangular hyperbolic equation derived from the equation of a bimolecular reaction and the law of mass action,B = (B max * [F])/(K D + [F]), where B is the amount of ligand bound at equilibrium, B max is the maximum number of binding sites, [F] is the concentration of free ligand, and K D is the ligand dissociation constant. The experiments were performed three times in triplicate, and the mean ± S.D. of the individual K D values were calculated and are reported in Table I. [3H]Quisqualate binding was performed as previously described (30Mutel V. Ellis G.J. Adam G. Chaboz S. Nilly A. Messer J. Bleuel Z. Metzler V. Malherbe P. Schlaeger E.-J. Roughley B.S. Faull R.L.M. Richards J.G. J. Neurochem. 2000; 75: 2590-2601Google Scholar).Table IBinding properties of WT mGlu1 and mutantsReceptorPosition in the 7TMD[3H]EM-TBPC K D ± S.D.[3H]QuisqualateK D± S.D.B maxnmnmpmol/mgrmGlu1αWT6.6 ± 0.530.0 ± 0.611.0 ± 0.05A669V3.373.0 ± 0.732.5 ± 119.0 ± 0.2Y672V3.4032.0 ± 5.431.5 ± 2.77.2 ± 0.7Y672F3.408.9 ± 2.3T723A4.566.1 ± 0.7N747A45.518.1 ± 1.446.0 ± 2410.5 ± 3.5S749T45.538.4 ± 1.232.0 ± 1310.1 ± 2.6S749A45.5310 ± 4N750A45.546.8 ± 1.331.0 ± 186.0 ± 1N750Q45.546.9 ± 1.1V757L5.4784 ± 39V757A5.4785 ± 1937.5 ± 157.2 ± 1.7W798F6.480.7 ± 0.236.0 ± 0.37.0 ± 0.3W798Y6.481.3 ± 0.232.0 ± 148.0 ± 2F801A6.51No binding35.0 ± 75.1 ± 1V802M6.522.8 ± 0.9Y805A6.55No binding44.0 ± 74.4 ± 0.4T815M7.39No binding36.0 ± 0.37.0 ± 0.3A818S7.425.3 ± 0.2A818I7.428.6 ± 2.3hmGlu1αWTNo bindingL757V5.479.9 ± 2.9 Open table in a new tab HEK-293 cells were plated at 5 × 104 cells on glass coverslips (diameter 15 mm) coated with 100 μg/ml poly-d-lysine. After 24 h, the cells were co-transfected with a 2:1 (w/w) mixture of mGlu/enhanced green fluorescent protein plasmids using LipofectAMINE 2000 (Invitrogen). 48 h later, the transfected cells were incubated with 20 μm fura-2 acetoxymethyl ester plus 0.5% Pluronic F-127 (Molecular Probes, Inc., Eugene, OR) for 40 min at room temperature with 20-min postincubation in balanced salt solution. Cells were stimulated at room temperature in artificial cerebrospinal fluid with drug as indicated. Glutamate applications (drug + 30 μm glutamate, 30-s exposure) were separated by 10-min intervals (3-min wash, 7-min antagonist incubation). Imaging measurements were made on an inverted microscope with a long distance ×40 objective (Axiovert 405M; Zeiss). A cooled CCD camera (CH-250; Photometrics) was used to acquire image pairs at 340- and 380-nm excitation wavelengths (with dark correction) to a computer. Exposure times were 400 ms. The intrinsic fluorescence in cells not dye-loaded was less than 5% and did not contribute a significant error to the measurements. Fluorescence ratio values were calculated as previously described (31Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Google Scholar). Inhibition curves were fitted according to the Hill equation: y = 100/(1 + (x/IC50)nH), wheren H represents slope factor. A Chinese hamster ovary (CHO) cell line stably expressing human GIRK1-GIRK2c dimer was co-transfected with a 1:1 (w/w) mixture of mGlu/enhanced green fluorescent protein plasmids using LipofectAMINE 2000. GIRK channel currents were recorded 24–96 h after transfection using the whole-cell configuration of the patch clamp technique as described previously (19Knoflach F. Mutel V. Jolidon S. Kew J.N.C. Malherbe P. Vieira E. Wichmann J. Kemp J.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13402-13407Google Scholar). Briefly, for GIRK current recordings, the pipette solution contained 130 mm KCl, 1 mmMgCl2, 10 mm HEPES, 5 mmK4BAPTA, 3 mm Na2ATP, 0.3 mm Na2GTP, adjusted to pH 7.2 with KOH, and osmolarity was adjusted to 310 mosm with sucrose. The drug was applied locally to the cell by fast perfusion from a double-barreled pipette assembly. The rate of solution exchange was around 20 ms. The CHO cells were held at −70 mV, and the recordings were made under conditions in which K+ currents would be inward ([K+]i = 150 mm, [K+]o = 30 mm). Concentration-response curves were obtained by applying 20-s pulses of varying concentrations of the compound every 90 s to the cells. [3H]EM-TBPC, 1-ethyl-2-methyl-6-oxo-4-(1,2,4,5-tetrahydro-benzo[d]azepin-3-yl)-1,6-dihydro-pyrimidine-5-carbonitrile (Fig. 1) is a highly potent, subtype-selective, noncompetitive antagonist that binds only to rat mGlu1 (K D = 6.6 ± 0.5 nm,K i = 11 ± 2 nm). It has a low affinity for human mGlu1 and none for the rat mGlu5 (22Knoflach F. Mutel V. Kew J.N.C. Waselle L. Vieira E. Jolidon S. Wichmann J. Malherbe P. Kemp J.A. Soc. Neurosci. Abstr. 2001; 705: 13Google Scholar). Using a series of chimeric rmGlu1/5a and rmGlu5/1α receptors (19Knoflach F. Mutel V. Jolidon S. Kew J.N.C. Malherbe P. Vieira E. Wichmann J. Kemp J.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13402-13407Google Scholar), the binding pocket of EM-TBPC has been localized to the 7TMD of mGlu1 (data not shown). To elucidate the binding mode of EM-TBPC further, an alignment of the seven-transmembrane helices of the whole rmGlu family toward the transmembrane helices of bovine rhodopsin (Protein Data Bank code 1f88) was made. The inverse agonist of rhodopsin,cis-retinal, was employed as a template for the location of EM-TBPC. Amino acids in mGlu1 located within 6.0 Å away from retinal in the x-ray crystal structure of rhodopsin (23Palczewski K. Kumasaka T. Hori T. Behnke C.A. Motoshima H. Fox B.A. Le Trong I. Teller D.C. Okada T. Stenkamp R.E. Yamamoto M. Miyano M. Science. 2000; 289: 739-745Google Scholar, 32Teller D.C. Okada T. Behnke C.A. Palczewski K. Stenkamp R.E. Biochemistry. 2001; 40: 7761-7772Google Scholar) were considered as likely candidates to affect binding of EM-TBPC. In Fig.2, the alignment of these amino acids of the rmGlu1 and the rmGlu family with rhodopsin is shown. From this preselection, amino acids in the TM3, -4, -5, -6, and -7 and EC2 regions (Fig. 2, boldface type) were chosen for the mutational studies. 20 mutations (19 in rmGlu1α and 1 in hmGlu1α) were accordingly introduced in the 7TMD region by site-directed mutagenesis. Saturation binding analyses were performed on membranes isolated from the HEK-293 transfected with the WT and mutated receptors using 0.3–300 nm of [3H]EM-TBPC. The dissociation constants (K D) derived from the saturation isotherms are given in Table I. Eleven mutations, A699V, Y672F, T723, N747A, S749T, S749A, N750A, N750Q, V802M, A818S, and A818I, did not significantly affect the [3H]EM-TBPC affinity compared with the WT rmGlu1α receptor (Table I). Three mutations, F801A, Y805A, and T815M, completely abolished [3H]EM-TBPC binding, and the mutation Y672V led to a 5-fold decrease in affinity (K D = 32 ± 5.4 nm). [3H]EM-TBPC does not bind to hmGlu1 under our experimental conditions (Table I). Interestingly, only one amino acid differs between the rat and human receptor in the TM domain; it is a valine at position 757 in the rat and a leucine in the human receptor. As expected, the replacement of the leucine residue at position 757 of the hmGlu1 receptor by a valine (L757V) gave to the mutant receptor a binding affinity for [3H]EM-TBPC comparable with that of WT rmGlu1α (K D = 9.9 ± 2.9 nm for the hmGlu1α L757V versus6.6 ± 0.5 nm rmGlu1α). Conversely, the replacement of the valine 757 with a leucine (V757L) or an alanine (V757A) led to a dramatic, although not complete, reduction in [3H]EM-TBPC affinity by 13-fold (K D = 84 ± 39 nm and K D = 85 ± 19 nm), respectively, in both mutants. Finally, the conversion of the tryptophan 798 to a phenylalanine (W798F) or a tyrosine (W798Y) significantly increased the binding affinity of the radioligand by 10- and 5-fold (K D = 0.7 ± 0.2 nm and K D = 1.3 ± 0.2 nm), respectively. In order to examine effects of the mutations on the glutamate binding pocket and to check for receptor expression, saturation analyses were performed on membranes from selected mutated receptors using [3H]quisqualate. The K D values and maximum number of binding sites (B max) derived from the saturation isotherms are given in Table I. As seen, the mutations have no significant effect on quisqualate affinity, and the level of receptor expression was comparable even with mutants that completely lost the antagonist binding (Table I). In HEK-293 cells transiently transfected with rmGlu1α receptors, glutamate elicited a concentration-dependent increase in intracellular free calcium [Ca2+]i, as assayed by single-cell fura-2 imaging (EC50 = 6.8 μm). All mutated receptors elicit an increase in [Ca2+]i response upon application of 30 μm glutamate, indicating the presence of functional receptors. However, co-application of EM-TBPC at various concentrations with 30 μm glutamate in the cells expressing WT rmGlu1α resulted in a concentration-dependent inhibition of glutamate-evoked [Ca2+]i response with IC50 = 128 nm (Fig. 3). The concentration-dependent inhibition of glutamate-evoked increases in [Ca2+]i by EM-TBPC in the cells expressing various mutated receptors is shown in Fig. 3, Aand B, and their derived IC50 andn H values are shown in TableII.Table IIEffect of mutations on inhibition of glutamate-induced [Ca2+]i response by EM-TBPCReceptorPosition in the 7TMDIC50 ± S.E.n HnmrmGlu1αWT128 ± 141.2N747A45.5131 ± 50.8N750A45.5410 ± 40.6V757L5.471160 ± 1301.9W798F6.484 ± 0.41.3Y805A6.55>10,000T815M7.39>10,000A818S7.42101 ± 281IC50 and Hill coefficient (n H) values for the inhibition by EM-TBPC of glutamate-evoked [Ca2+]i response in the HEK-293 cells transiently transfected with the mGlu1α WT and mutated receptors. Data are means ± S.E. of four separate experiments with 15–20 cells/experiment. Open table in a new tab IC50 and Hill coefficient (n H) values for the inhibition by EM-TBPC of glutamate-evoked [Ca2+]i response in the HEK-293 cells transiently transfected with the mGlu1α WT and mutated receptors. Data are means ± S.E. of four separate experiments with 15–20 cells/experiment. As expected from the binding studies, the antagonist inhibition of glutamate-induced [Ca2+]i response was not affected by the mutation A818S. In cells expressing the mutants Y805A and T815M, which did not bind [3H]EM-TBPC, EM-TBPC was not able to efficiently inhibit glutamate-evoked [Ca2+]i response and thus resulted in the large increases in IC50 values (>10,000 nm for Y805A and T815M). In agreement with the binding ex