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Molecular Determinants for G Protein βγ Modulation of Ionotropic Glycine Receptors
2006; Elsevier BV; Volume: 281; Issue: 51 Linguagem: Inglês
10.1074/jbc.m608272200
ISSN1083-351X
AutoresGonzalo E. Yévenes, Gustavo Moraga‐Cid, Leonardo Guzmán, Svenja Haeger, Laerte Oliveira, Juan Olate, Günther Schmalzing, Luis G. Aguayo,
Tópico(s)Ion channel regulation and function
ResumoThe ligand-gated ion channel superfamily plays a critical role in neuronal excitability. The functions of glycine receptor (GlyR) and nicotinic acetylcholine receptor are modulated by G protein βγ subunits. The molecular determinants for this functional modulation, however, are still unknown. Studying mutant receptors, we identified two basic amino acid motifs within the large intracellular loop of the GlyR α1 subunit that are critical for binding and functional modulation by Gβγ. Mutations within these sequences demonstrated that all of the residues detected are important for Gβγ modulation, although both motifs are necessary for full binding. Molecular modeling predicts that these sites are α-helixes near transmembrane domains 3 and 4, near to the lipid bilayer and highly electropositive. Our results demonstrate for the first time the sites for G protein βγ subunit modulation on GlyRs and provide a new framework regarding the ligand-gated ion channel superfamily regulation by intracellular signaling. The ligand-gated ion channel superfamily plays a critical role in neuronal excitability. The functions of glycine receptor (GlyR) and nicotinic acetylcholine receptor are modulated by G protein βγ subunits. The molecular determinants for this functional modulation, however, are still unknown. Studying mutant receptors, we identified two basic amino acid motifs within the large intracellular loop of the GlyR α1 subunit that are critical for binding and functional modulation by Gβγ. Mutations within these sequences demonstrated that all of the residues detected are important for Gβγ modulation, although both motifs are necessary for full binding. Molecular modeling predicts that these sites are α-helixes near transmembrane domains 3 and 4, near to the lipid bilayer and highly electropositive. Our results demonstrate for the first time the sites for G protein βγ subunit modulation on GlyRs and provide a new framework regarding the ligand-gated ion channel superfamily regulation by intracellular signaling. The ionotropic glycine receptors (GlyRs) 2The abbreviations used are: GlyR, glycine receptor; LGIC, ligand-gated ion channel; nAChR, nicotinic acetylcholine receptor; GTPγS, guanosine 5′-O-(3-thiotriphosphate); GST, glutathione S-transferase; ANOVA, analysis of variance; TM, transmembrane domain; GIRK, G protein-gated inwardly rectifying potassium channel; 5 HT3, 5 hydroxytryptamine type 3. are members of the ligand-gated ion receptor superfamily, which includes inhibitory γ-aminobutyric acid type A receptors and excitatory nAChR and 5-HT3 receptors. These homologous receptors mediate fast synaptic transmission in the central nervous system (1Kandel E.R. Schwartz J.H. Jessell T.M. Principles of Neural Science. McGraw-Hill Book Co., New York2000: 207-228Google Scholar, 2Sine S.M. Engel A.G. Nature. 2006; 440: 448-455Crossref PubMed Scopus (432) Google Scholar). Inhibitory GlyRs are critical for the control of excitability in the mammalian spinal cord and brain stem. Binding of glycine to the extracellular region induces a rapid increase in Cl- ion conductance, generating a hyperpolarization of the cell membrane (3Legendre P. Cell Mol. Life Sci. 2001; 58: 760-793Crossref PubMed Scopus (443) Google Scholar, 4Aguayo L.G. van Zundert B. Tapia J.C. Carrasco M.A. Alvarez F.J. Brain Res. Brain Res. Rev. 2004; 47: 33-45Crossref PubMed Scopus (57) Google Scholar, 5Laube B. Maksay G. Schemm R. Betz H. Trends Pharmacol. Sci. 2002; 23: 519-527Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). In neurons, the inhibitory action of GlyRs regulate several important physiological functions, like pain transmission, respiratory rhythms, motor coordination, and development (3Legendre P. Cell Mol. Life Sci. 2001; 58: 760-793Crossref PubMed Scopus (443) Google Scholar, 4Aguayo L.G. van Zundert B. Tapia J.C. Carrasco M.A. Alvarez F.J. Brain Res. Brain Res. Rev. 2004; 47: 33-45Crossref PubMed Scopus (57) Google Scholar, 5Laube B. Maksay G. Schemm R. Betz H. Trends Pharmacol. Sci. 2002; 23: 519-527Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 6Harvey R.J. Depner U.B. Wassle H. Ahmadi S. Heindl C. Reinold H. Smart T.G. Harvey K. Schutz B. Abo-Salem O.M. Zimmer A. Poisbeau P. Welzl H. Wolfer D.P. Betz H. Zeilhofer H.U. Muller U. Science. 2004; 304: 884-887Crossref PubMed Scopus (506) Google Scholar, 7Sebe J.Y. van Brederode J.F. Berger A.J. J. Neurophysiol. 2006; 96: 391-403Crossref PubMed Scopus (24) Google Scholar). Like all members of the LGIC superfamily, GlyRs are pentamers composed of five subunits in which each subunit possesses four transmembrane domains arranged to form the ion pore. In this structure, the individual subunits provide extracellular and intracellular domains that play roles in ligand binding and intracellular modulation, respectively (1Kandel E.R. Schwartz J.H. Jessell T.M. Principles of Neural Science. McGraw-Hill Book Co., New York2000: 207-228Google Scholar, 2Sine S.M. Engel A.G. Nature. 2006; 440: 448-455Crossref PubMed Scopus (432) Google Scholar, 3Legendre P. Cell Mol. Life Sci. 2001; 58: 760-793Crossref PubMed Scopus (443) Google Scholar, 5Laube B. Maksay G. Schemm R. Betz H. Trends Pharmacol. Sci. 2002; 23: 519-527Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). The function of GlyRs can be effectively modulated by extracellularly acting compounds like strychnine, picrotoxin, zinc ions, and ethanol (3Legendre P. Cell Mol. Life Sci. 2001; 58: 760-793Crossref PubMed Scopus (443) Google Scholar, 4Aguayo L.G. van Zundert B. Tapia J.C. Carrasco M.A. Alvarez F.J. Brain Res. Brain Res. Rev. 2004; 47: 33-45Crossref PubMed Scopus (57) Google Scholar, 5Laube B. Maksay G. Schemm R. Betz H. Trends Pharmacol. Sci. 2002; 23: 519-527Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 8Harris R.A. Alcohol Clin. Exp. Res. 1999; 23: 1563-1570PubMed Google Scholar, 9Smart T.G. Hosie A.M. Miller P.S. Neuroscientist. 2004; 10: 432-442Crossref PubMed Scopus (198) Google Scholar). Furthermore, the receptor can also be modulated by intracellular signaling. One of the most studied and recognized pathways involved in regulation of ligand-gated ion channel function are phosphorylation processes through protein kinases. Indeed, GlyR and other members of the LGIC superfamily are modulated by activation of cAMP-dependent kinases and protein kinase C (10Smart T.G. Curr. Opin. Neurobiol. 1997; 7: 358-367Crossref PubMed Scopus (164) Google Scholar). This involves specific serine residues in the loop between transmembrane domains 3 and 4 (6Harvey R.J. Depner U.B. Wassle H. Ahmadi S. Heindl C. Reinold H. Smart T.G. Harvey K. Schutz B. Abo-Salem O.M. Zimmer A. Poisbeau P. Welzl H. Wolfer D.P. Betz H. Zeilhofer H.U. Muller U. Science. 2004; 304: 884-887Crossref PubMed Scopus (506) Google Scholar, 10Smart T.G. Curr. Opin. Neurobiol. 1997; 7: 358-367Crossref PubMed Scopus (164) Google Scholar, 11Song Y. Huang L.Y. Nature. 1990; 348: 242-245Crossref PubMed Scopus (100) Google Scholar, 12Vaello M.L. Ruiz-Gomez A. Lerma J. Mayor F. J. Biol. Chem. 1994; 269: 2002-2008Abstract Full Text PDF PubMed Google Scholar, 13Ruiz-Gomez A. Vaello M.L. Valdivieso F. Mayor F. J. Biol. Chem. 1991; 266: 559-566Abstract Full Text PDF PubMed Google Scholar, 14Guo X. Wecker L. J. Neurochem. 2002; 82: 439-447Crossref PubMed Scopus (21) Google Scholar). On the other hand, recent reports have shown that the activity of GlyRs and nAChRs can be modulated by G protein βγ subunits in a phosphorylation-independent manner (15Fischer H. Liu D.M. Lee A. Harries J.C. Adams D.J. J. Neurosci. 2005; 25: 3571-3577Crossref PubMed Scopus (32) Google Scholar, 16Yevenes G.E. Peoples R.W. Tapia J.C. Parodi J. Soto X. Olate J. Aguayo L.G. Nat. Neurosci. 2003; 6: 819-824Crossref PubMed Scopus (77) Google Scholar). In both cases, activation of G proteins, using nonhydrolyzable GTP analogs or by application of purified Gβγ dimers, generates a strong enhancement in the agonist-evoked current, related to a shift to the left in the agonist concentration-response curve and an increased open channel probability (15Fischer H. Liu D.M. Lee A. Harries J.C. Adams D.J. J. Neurosci. 2005; 25: 3571-3577Crossref PubMed Scopus (32) Google Scholar, 16Yevenes G.E. Peoples R.W. Tapia J.C. Parodi J. Soto X. Olate J. Aguayo L.G. Nat. Neurosci. 2003; 6: 819-824Crossref PubMed Scopus (77) Google Scholar). However, the molecular determinants involved for this modulation are unresolved. On the other hand, some molecular characteristics for binding and modulation of Gβγ to other effector proteins are better understood (17Clapham D.E. Neer E.J. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 167-203Crossref PubMed Scopus (704) Google Scholar, 18Hamm H.E. J. Biol. Chem. 1998; 273: 669-672Abstract Full Text Full Text PDF PubMed Scopus (943) Google Scholar, 19Dolphin A.C. Pharmacol. Rev. 2003; 55: 607-627Crossref PubMed Scopus (240) Google Scholar). In fact, previous studies implicated the presence of basic amino acids, such as lysine and arginine, in the modulation of voltage-gated calcium channels, phospholipase Cβ, and β-adrenergic receptor kinases (20De Waard M. Liu H. Walker D. Scott V.E. Gurnett C.A. Campbell K.P. Nature. 1997; 385: 446-450Crossref PubMed Scopus (374) Google Scholar, 21Barr A.J. Ali H. Haribabu B. Snyderman R. Smrcka A.V. Biochemistry. 2000; 39: 1800-1806Crossref PubMed Scopus (39) Google Scholar, 22Touhara K. Koch W.J. Hawes B.E. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 17000-170005Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 23Canti C. Page K.M. Stephens G.J. Dolphin A.C. J. Neurosci. 1999; 19: 6855-6864Crossref PubMed Google Scholar). For example, mutations in basic residues reduced Gβγ binding to β-adrenergic receptor kinase 1 and phospholipase Cβ3 (21Barr A.J. Ali H. Haribabu B. Snyderman R. Smrcka A.V. Biochemistry. 2000; 39: 1800-1806Crossref PubMed Scopus (39) Google Scholar, 22Touhara K. Koch W.J. Hawes B.E. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 17000-170005Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Furthermore, similar results were obtained with mutations in the N-terminal region and in the cytoplasmic linker between the first and second transmembrane repeats of voltage-gated calcium channels (19Dolphin A.C. Pharmacol. Rev. 2003; 55: 607-627Crossref PubMed Scopus (240) Google Scholar, 20De Waard M. Liu H. Walker D. Scott V.E. Gurnett C.A. Campbell K.P. Nature. 1997; 385: 446-450Crossref PubMed Scopus (374) Google Scholar, 23Canti C. Page K.M. Stephens G.J. Dolphin A.C. J. Neurosci. 1999; 19: 6855-6864Crossref PubMed Google Scholar). In the present study, we have examined the molecular determinants for G protein modulation of the human GlyR α1 subunit. The present study shows that most of the large intracellular loop is not involved in the modulation. We identified two basic amino acid motifs within the loop of the GlyR as critical for binding and functional modulation by Gβγ dimers. Acidic residues, on the other hand, are not important for this regulation. These results provide novel information about the Gβγ modulatory sites in the GlyR, a member of the LGIC superfamily, and also demonstrate a new role for specific residues in the intracellular loop for the GlyR function. cDNA Constructs—The cDNA construct encoding the human glycine receptor α1 subunit with a C-terminal hexahistidyl tag has been described previously (25Sadtler S. Laube B. Lashub A. Nicke A. Betz H. Schmalzing G. J. Biol. Chem. 2003; 278: 16782-16790Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). This construct was first subcloned in a pCI vector (Promega) for expression in HEK 293 cells. Mutations were inserted using the QuikChange™ site-directed mutagenesis kit (Stratagene). All of the constructions were confirmed by sequencing. The glycine receptor amino acids were numbered according to their position in the mature protein sequence. pEYFP-Gβ1 and pECFP-Gγ2 expression vectors were kindly provided by Stephen R. Ikeda (National Institutes of Health) (27Ruiz-Velasco V. Ikeda S.R. J. Physiol. 2001; 537: 679-692Crossref PubMed Scopus (39) Google Scholar). G protein β1-FLAG and G protein γ2 were purchased from UMR cDNA resource center. Cell Culture and Transfection—HEK 293 cells were cultured using standard methodologies. HEK 293 cells were transfected using Lipofectamine 2000 (Invitrogen) with 2 μg of DNA for each plasmid studied per well. Expression of GFP was used as a marker of positively transfected cells, and recordings were made after 18-36 h. Electrophysiology—Whole cell recordings were performed as previously described (16Yevenes G.E. Peoples R.W. Tapia J.C. Parodi J. Soto X. Olate J. Aguayo L.G. Nat. Neurosci. 2003; 6: 819-824Crossref PubMed Scopus (77) Google Scholar, 38Van Zundert B. Alvarez F.J. Tapia J.C. Yeh H.H. Diaz E. Aguayo L.G. J. Neurophysiol. 2004; 91: 1036-1049Crossref PubMed Scopus (35) Google Scholar). A holding potential of -60 mV was used. Patch electrodes were filled with 140 mm CsCl, 10 mm 1,2-bis-(2-aminophenoxy)-ethane-N,N,N,N-tetraacetic acid, 10 mm HEPES (pH 7.4), 4 mm MgCl2, 2 mm ATP, and 0.5 mm GTP. The external solution contained 150 mm NaCl, 10 mm KCl, 2.0 mm CaCl2, 1.0 mm MgCl2, 10 mm HEPES (pH 7.4), and 10 mm glucose. For G protein activation experiments, GTPγS (0.5 mm) was added directly to the internal solution, replacing GTP. The amplitude of the glycine current was assayed using a short (1-2 s) pulse of 30-40 μm glycine every 60 s as previously described (16Yevenes G.E. Peoples R.W. Tapia J.C. Parodi J. Soto X. Olate J. Aguayo L.G. Nat. Neurosci. 2003; 6: 819-824Crossref PubMed Scopus (77) Google Scholar). Strychnine (1 μm) blocked all of the current elicited by wild type and mutant glycine receptors (not shown). Construction of Glutathione S-Transferase Fusion Proteins and GST Pull-down Assays—DNA fragments encoding wild type and mutant GlyR intracellular loops were subcloned in the GST fusion vector pGEX-5X3 (GE Healthcare). GST fusion proteins were generated in Escherichia coli BL21 using 50 μm isopropyl β-d-thiogalactopyranoside. After 6 h, the cells were collected and sonicated in lysis buffer (1× phosphate buffer, 1% Triton X-100, and protease inhibitor mixture set II (Calbiochem)). Subsequently, the proteins were purified using a glutathione resin (Novagen). Normalized amounts of GST fusion proteins were incubated with purified Gβγ protein (10 ng; Calbiochem). Incubations were done in 800 μl of binding buffer (200 mm NaCl, 10 mm EDTA, 10 mm Tris, pH 7.4, 0.1% Triton X-100, and protease inhibitor mixture set II) at 4 °C for 1 h. The beads were washed five times in binding buffer, and bound proteins were separated on 12% SDS-polyacrylamide gels. Bound Gβγ was detected using a Gβ antibody (1:1000; Santa Cruz Biotechnology) and a chemiluminiscence kit (PerkinElmer Life Sciences). Finally, the relative amounts of Gβγ were quantified by densitometry. Similar conditions have been used to study the interaction between Gβγ and other effector proteins (15Fischer H. Liu D.M. Lee A. Harries J.C. Adams D.J. J. Neurosci. 2005; 25: 3571-3577Crossref PubMed Scopus (32) Google Scholar, 20De Waard M. Liu H. Walker D. Scott V.E. Gurnett C.A. Campbell K.P. Nature. 1997; 385: 446-450Crossref PubMed Scopus (374) Google Scholar, 21Barr A.J. Ali H. Haribabu B. Snyderman R. Smrcka A.V. Biochemistry. 2000; 39: 1800-1806Crossref PubMed Scopus (39) Google Scholar, 22Touhara K. Koch W.J. Hawes B.E. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 17000-170005Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 35He C. Yan X. Zhang H. Mirshahi T. Jin T. Huang A. Logothetis D.E. J. Biol. Chem. 2002; 277: 6088-6096Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 36Huang C.L. Slesinger P.A. Casey P.J. Jan Y.N. Jan L.Y. Neuron. 1995; 15: 1133-1143Abstract Full Text PDF PubMed Scopus (287) Google Scholar). Immunofluorescence, Visualization, and Analysis—HEK 293 cells were first fixed for 30 min with 4% paraformaldehyde (0.1 m phosphate buffer, pH 7.4) and were then permeabilized (0.3% Triton X-100, 30 min) and blocked (10% normal horse serum, 60 min). Subsequently, all night incubation with a monoclonal FLAG (Stratagene) and polyclonal hexahistidine antibodies (His-Tag; U.S. Biological) was carried out. Epitope visualization was performed incubating the sample with two secondary antibodies conjugated to fluorescein isothiocyanate and Cy3 (1:600; Jackson ImmunoResearch Laboratories). Finally, the cells were coverslipped using fluorescence mounting medium (Dako Cytomation; Dako). For quantitative analysis, the cells were chosen randomly for imaging using a Nikon confocal microscopy. Stacks of optical sections in the z axis were acquired, and dual color immunofluorescent images were captured in simultaneous two-channel mode. Colocalization was studied by superimposing both color channels. The cross-correlation coefficient (r) between both fluorescence channels was measured using computer software (Metamorph; Universal Imaging Corp.) starting from separate immunoreactivity to GlyR-His and Gβ1-FLAG in the same cell (39Agnati L.F. Fuxe K. Torvinen M. Genedani S. Franco R. Watson S. Nussdorfer G.G. Leo G. Guidolin D. J. Histochem. Cytochem. 2005; 53: 941-953Crossref PubMed Scopus (39) Google Scholar). The theoretical maximum for (r) is 1 for identical images, and a value close to 0 implies a complete different localization of the labels. Subsequently, the obtained data were compiled, analyzed, and plotted. Molecular Modeling—The GlyR model was constructed by homology using coordinates from the Torpedo nAchR at 4 A° resolution (28Miyazawa A. Fujiyoshi Y. Unwin N. Nature. 2003; 423: 949-955Crossref PubMed Scopus (1087) Google Scholar, 29Unwin N. J. Mol. Biol. 2005; 346: 967-989Crossref PubMed Scopus (1425) Google Scholar) (Protein Data Bank code 2BG9) using the program WHAT IF (40Vriend G. J. Mol. Graph. 1990; 8: 52-56Crossref PubMed Scopus (3377) Google Scholar). The ligand-binding segment of the GlyR was modeled from the acetylcholine-binding protein structure (30Celie P.H. van Rossum-Fikkert S.E. van Dijk W.J. Brejc K. Smit A.B. Sixma T.K. Neuron. 2004; 41: 907-914Abstract Full Text Full Text PDF PubMed Scopus (719) Google Scholar) (Protein Data Bank code 1UV6). Electrostatic surface potentials were calculated using APBS (41Baker N.A. Sept D. Joseph S. Holst M.J. McCammon J.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10037-10041Crossref PubMed Scopus (5904) Google Scholar). The individual charges were assigned using pdb2pqr software (42Dolinsky T.J. Nielsen J.E. McCammon J.A. Baker N.A. Nucleic Acids Res. 2004; 32: W665-W667Crossref PubMed Scopus (2553) Google Scholar) with the AMBER force field (43Cornell W.D. Cieplak P. Bayly C.I. Gould I.R. Merz K.M. Ferguson D.M. Spellmeyer D.C. Fox T. Caldwell J.W. Kollman P. J. Am. Chem. Soc. 1995; 117: 5179-5197Crossref Scopus (11639) Google Scholar). The final images were generated with Pymol (44DeLano W.L. The PyMOL Molecular Graphics System. DeLano Scientific, San Carlos, CA2002Google Scholar). Data Analysis—Statistical analyses were performed using ANOVA and are expressed as the arithmetic means ± S.E.; values of p < 0.05 were considered statistically significant. For all of the statistical analysis and plots, the Origin 6.0 (MicroCal) software was used. The normalized values were obtained by dividing the current amplitude obtained with time of dialysis by the current at minute one. The control GTPγS percentage potentiation is the average for all of the single experiments. Effects of G Protein Activation on Wild Type and Truncated Human α1 Glycine Receptor Subunits—The currently accepted topology of the LGIC superfamily predicts that the large intracellular loop between transmembrane domains 3 and 4 is the region for interaction and regulation by intracellular pathways, including receptor phosphorylation (10Smart T.G. Curr. Opin. Neurobiol. 1997; 7: 358-367Crossref PubMed Scopus (164) Google Scholar, 11Song Y. Huang L.Y. Nature. 1990; 348: 242-245Crossref PubMed Scopus (100) Google Scholar, 12Vaello M.L. Ruiz-Gomez A. Lerma J. Mayor F. J. Biol. Chem. 1994; 269: 2002-2008Abstract Full Text PDF PubMed Google Scholar, 13Ruiz-Gomez A. Vaello M.L. Valdivieso F. Mayor F. J. Biol. Chem. 1991; 266: 559-566Abstract Full Text PDF PubMed Google Scholar, 14Guo X. Wecker L. J. Neurochem. 2002; 82: 439-447Crossref PubMed Scopus (21) Google Scholar). Noteworthy, Ser391 was the first residue in this sequence reported to be modulated by phosphorylation by protein kinase C (13Ruiz-Gomez A. Vaello M.L. Valdivieso F. Mayor F. J. Biol. Chem. 1991; 266: 559-566Abstract Full Text PDF PubMed Google Scholar). The cytosolic loop polypeptide contains ≈84 amino acids (Fig. 1A), which can present alternative splicing. Therefore, we examined the sensitivity of a recently identified α1 splicing variant, in which the sequence between residues Glu326 and Lys355 was deleted (24Inoue K. Ueno S. Yamada J. Fukuda A. Biochem. Biophys. Res. Commun. 2005; 327: 300-305Crossref PubMed Scopus (5) Google Scholar), to Gβγ modulation (Fig. 1A). In addition, we constructed a truncated GlyR, in which the whole sequence between residues Glu326 and Gln382 was deleted (Fig. 1A). As previously reported (16Yevenes G.E. Peoples R.W. Tapia J.C. Parodi J. Soto X. Olate J. Aguayo L.G. Nat. Neurosci. 2003; 6: 819-824Crossref PubMed Scopus (77) Google Scholar), the amplitude of the glycine-activated current in human wild type GlyRs was strongly enhanced above control (80 ± 10%, n = 18) after 15 min of intracellular dialysis with GTPγS (Fig. 1, B and C). Interestingly, similar effects were found on Δ326-355 and Δ326-382 truncated GlyRs (Fig. 1, B and C), and no statistical differences were detected (Fig. 1C). Thus, these data indicate that the whole deleted sequence between residues 326 and 382 is not a critical determinant for Gβγ modulation and directs further analysis toward other regions in the intracellular loop. Two Basic Motifs Are Critical for G Protein βγ Functional Modulation of Glycine Receptors—Studies on the molecular determinants involved in the binding and modulation by G protein βγ to several proteins have shown the existence of key basic amino acids (19Dolphin A.C. Pharmacol. Rev. 2003; 55: 607-627Crossref PubMed Scopus (240) Google Scholar, 20De Waard M. Liu H. Walker D. Scott V.E. Gurnett C.A. Campbell K.P. Nature. 1997; 385: 446-450Crossref PubMed Scopus (374) Google Scholar, 21Barr A.J. Ali H. Haribabu B. Snyderman R. Smrcka A.V. Biochemistry. 2000; 39: 1800-1806Crossref PubMed Scopus (39) Google Scholar, 22Touhara K. Koch W.J. Hawes B.E. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 17000-170005Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 23Canti C. Page K.M. Stephens G.J. Dolphin A.C. J. Neurosci. 1999; 19: 6855-6864Crossref PubMed Google Scholar). Interestingly, the large intracellular loop of the GlyR has a high density of residues, such as lysine and arginine (23 of 84), with several in a cluster near the transmembrane domain 3 (TM3), 316RFRRKRR (Fig. 2A). This cluster was found to be important for correct membrane topology of GlyRs (25Sadtler S. Laube B. Lashub A. Nicke A. Betz H. Schmalzing G. J. Biol. Chem. 2003; 278: 16782-16790Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). To investigate the importance of this motif on the G protein modulation, we studied mutant GlyRs in which these basic residues, together with a phenylalanine, were replaced by alanines. The data shows that GTPγS-mediated current enhancement was significantly attenuated in the 316-320A (11 ± 9%, n = 10) and the 316-322,325A (8 ± 11%, n = 8) mutants (Fig. 2, B-D). On the other hand, the data in Fig. 2 (B-D) show that mutations in an acidic motif (326EDE) near this basic cluster did not affect the receptor sensitivity to modulation (70 ± 13%, n = 8). The finding that GTPγS affected the 316-320A and 316-322,325A mutants in a similar fashion (Fig. 2D) suggests that residues 316-320 are the main determinants for Gβγ modulation. Nevertheless, the GlyR intracellular loop has two other pairs of basic residues near the C-terminal (377RK385KK; Figs. 1A and 2A), and we studied the functional importance of those residues on receptor modulation. Interestingly, the alanine replacement of these four residues also significantly attenuated the effect of G protein activation (-5 ± 7%, n = 8), in a similar way to the 316-320A mutant (Fig. 2, B-D). It is known that Gβγ overexpression can induce tonic modulation of Ca+2, GIRK channels, and GlyRs (16Yevenes G.E. Peoples R.W. Tapia J.C. Parodi J. Soto X. Olate J. Aguayo L.G. Nat. Neurosci. 2003; 6: 819-824Crossref PubMed Scopus (77) Google Scholar, 26Ikeda S.R. Nature. 1996; 380: 255-258Crossref PubMed Scopus (710) Google Scholar, 27Ruiz-Velasco V. Ikeda S.R. J. Physiol. 2001; 537: 679-692Crossref PubMed Scopus (39) Google Scholar). Therefore, we examined the ability of wild and mutant GlyRs to display this phenomenon. In these experiments, the concentration-response relationship for the wild type α1 GlyRs was shifted to the left after Gβγ dimers were coexpressed, as reflected by a significant reduction in its EC50 (-32 ± 4%) with respect to control cells (Fig. 3). Tonic modulation was absent in the 316-320A; 377-378, 385-386A (9 ± 12% and 12 ± 14%, respectively), which is in line with the loss of glycine-activated current potentiation after GTPγS dialysis. Other constructs where the basic residues were retained displayed normal tonic modulation after Gβγ coexpression (Fig. 3). Regarding the cell surface expression of these functional constructs, we found that with the exception of the 316-320A (25Sadtler S. Laube B. Lashub A. Nicke A. Betz H. Schmalzing G. J. Biol. Chem. 2003; 278: 16782-16790Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), all of the mutants studied displayed very similar maximal current values. For example, the amplitude of the maximal current (Imax) elicited by 1 mm glycine was 3339 ± 460 pA (n = 13) for the 377-378, 385-386A mutant, which was not significantly different from wild type GlyRs (3647 ± 394 pA, n = 23). On the other hand, although the truncated GlyRs showed lower Imax relative to wild type (Δ326-382, 1584 ± 210 pA, n = 11), the G protein modulation was unchanged. Thus, these data suggest that the elimination of G protein βγ modulation is independent of changes on cell surface expression. Thus, all of the above shows the importance of the basic residues in the functional G protein βγ modulation of GlyRs. To identify critical amino acids involved in the G protein modulation, we carried out a mutagenesis scanning in these two regions found to be important for modulation. For the 316RFRRK cluster, all single point mutations showed marked attenuations in the potentiating effect produced by the GTP analog, compared with the wild type GlyRs (Fig. 4A). For example, the potentiation for R316A was 40 ± 17% (n = 5), whereas the effect for K320A was only 29 ± 13% (n = 8). Interestingly, the mutation F317Q also altered the modulation (29 ± 14%, n = 5) implicating hydrophobic residues, in addition to basic ones, in the modulatory effects. Double mutants RK319-320A and RR316,318A were also less affected than the wild receptor (27 ± 8%, n = 6 and 33 ± 14%, n = 7, respectively) but were no different from the point mutants (Fig. 4, A and B). On the other hand, for the basic residues detected near the C-terminal region, no potentiation was detected in the double mutant 385-386A (2 ± 6%, n = 9), suggesting a critical role for the 385KK residues (Fig. 4, C and D). Subsequent studies with the point mutations K385A and K386A showed significant attenuations (3 ± 4%, n = 6 and 32 ± 16%, n = 5, respectively), confirming an important role for the 385KK motif for this intracellular regulation. Altogether, the data show the main importance of the basic motifs 316RFRRK and 385KK in the Gβγ modulation of GlyRs, suggesting that all of these residues are necessary for the functional effect. Basic Motifs Are Involved in the Protein Interaction between Gβγ and Glycine Receptor—To determine whether the intracellular motifs identified are necessary for the binding of Gβγ to the GlyR, we constructed GST fusion proteins encoding wild type and mutated forms of the GlyR intracellular loop in which the functional G protein modulation was abolished. GST fusion proteins were first expressed and purified (Fig. 5A), and then in vitro binding assays were performed using purified Gβγ, under conditions that warrant the native conformation of the heterodimer (15Fischer H. Liu D.M. Lee A. Harries J.C. Adams D.J. J. Neurosci. 2005; 25: 3571-3577Crossref PubMed Scopus (32) Google Scholar, 17Clapham D.E. Neer E.J. Annu. Rev. Pharmacol. 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