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Meeting Abstracts  |   November 2005
Effect of Halothane on Gαi-3and Its Coupling to the M2Muscarinic Receptor
Author Affiliations & Notes
  • Fang Jin, M.D.
    *
  • Shuyan Wang, M.D.
    *
  • Joshua D. Spencer
  • Sumedha G. Penheiter, Ph.D.
  • John H. Streiff, Ph.D.
    *
  • Alan R. Penheiter, Ph.D.
  • David O. Warner, M.D.
    §
  • Keith A. Jones, M.D.
    §
  • * Research Fellow, † Research Technologist, ‡ Assistant Professor, § Professor.
Article Information
Meeting Abstracts   |   November 2005
Effect of Halothane on Gαi-3and Its Coupling to the M2Muscarinic Receptor
Anesthesiology 11 2005, Vol.103, 1015-1025. doi:
Anesthesiology 11 2005, Vol.103, 1015-1025. doi:
VOLATILE anesthetics are potent bronchodilators that can effectively reverse severe perioperative bronchospasm in patients with hyperreactive airway diseases, such as asthma.1–4 This beneficial effect is due in significant part to a direct inhibitory effect on the airway smooth muscle (ASM) cell.5–7 Although the mechanisms of this direct effect is not fully known, considerable evidence indicates that volatile anesthetics inhibit the increase in cytoplasmic calcium (Ca2+) concentration ([Ca2+]c)8–13 and activation of the signaling proteins that regulate the amount of force at a given [Ca2+]c(i.e.  , Ca2+sensitivity)9,11,14–19 induced by physiologic agonists, including acetylcholine.
Agonists mediate ASM contractile state by activating signaling cascades via  their cognate heterotrimeric guanosine 5′-triphosphate (GTP) binding protein (G-protein)–coupled receptors. The GTP-bound form of the α subunit (Gα) of the heterotrimer activates the signaling pathways that mediate Ca2+sensitivity, whereas both Gα and the βγ dimer (Gβγ) activate signaling proteins that regulate [Ca2+]c.20,21 
Acetylcholine induces ASM contraction by reversibly binding and activating two of the five isoforms of the muscarinic receptor, the M2and M3muscarinic receptors.22 Studies of isolated, intact tracheal smooth muscle obtained from M2and M3muscarinic receptor knockout mice indicate that acetylcholine-induced ASM contraction is mediated approximately equally by each receptor.23,24 In porcine ASM, acetylcholine-induced increases in Ca2+sensitivity are mediated by the α subunit of both Giand Gqsubfamily proteins (Gαiand Gαq, respectively),19,25 which are thought to couple to M2and M3muscarinic receptors, respectively.
Our previous work showed that halothane and hexanol each inhibit acetylcholine-induced increases in Ca2+sensitivity mediated by both pertussis toxin–sensitive and pertussis toxin–insensitive heterotrimeric G proteins,17,19 such as Gαiand Gαqsubfamily proteins, respectively. Recent studies of crude membranes prepared from porcine ASM showed that these anesthetics inhibit acetylcholine-promoted guanosine nucleotide exchange at Gαq.14,26 In another study of recombinant Gαiisoform-1 (Gαi-1)– or Gαiheterotrimer–derived ASM membranes, halothane had no effect on either GTP binding or hydrolysis, suggesting that halothane does not directly interact with Gαisubunits in a functionally significant manner.27 These experiments did not examine preparations in which the M2muscarinic receptor was coupled to the Giheterotrimer. Therefore, it is unknown whether halothane affects the ability of the M2muscarinic receptor to promote guanosine nucleotide exchange at Gαi.
The current study tested the hypothesis that clinically relevant concentrations of halothane (≤ 1 mm) inhibit acetylcholine-promoted guanosine nucleotide exchange at Gαiisoform-3 (Gαi-3) but not the intrinsic, basal Gαi-3activity. This hypothesis was tested using recombinant Gαi-3and Gαi-3β1γ2heterotrimer (isoforms expressed in ASM) and crude membranes prepared from mammalian cells transfected to transiently coexpress the human M2muscarinic receptor and human Gαi-3. Based on these results and additional experiments using a novel technique to detect anesthetic–protein interactions,28 we make inferences regarding the salient mechanism for the anesthetic action on the M2–Gαi-3βγ heterotrimer complex.
Materials and Methods
Expression and Purification of Heterotrimeric G-protein Subunits
Expression and Purification of Gαi-3in E. coli  Bacteria.
A plasmid encoding human Gαi-3in the mammalian expression vector pcDNA3.1 was obtained from the University of Missouri-Rolla cDNA Resource Center (Rolla, Missouri).1i-3was expressed in Escherichia coli  and purified by conventional chromatography as previously described,29 with minimal modifications. Briefly, the Gαi-3insert was excised from the pcDNA3.1:Gαi-3construct and ligated to pET28a plasmid. pET28a:Gαi-3was then transformed into E. coli  BL21(DE3). After growing a single colony (4–5 h, 30°C) in 80 ml Luria-Bertani medium containing 50 μg/ml kanamycin, protein expression was induced with 30 μm isopropyl thiogalactoside (16 h, 30°C). The cells were pelleted (14,000g  , 15 min, 4°C) and then lysed in 40 ml ice-cold lysis buffer composed of 20 mm HEPES (pH 8.0), 1 mm EDTA, 6.25 mm MgCl2, 3 mm dithiothreitol, 10 μm guanosine 5′-diphosphate (GDP), 0.5 ml protease inhibitor cocktail P8849, 0.2 mg/ml lysozyme, and 0.15 mg/ml DNAse I. The lysate was centrifuged (35,000g  , 45 min, 4°C), filtered, and subjected to sequential chromatography with Q sepharose, hydroxyapatite, and then phenyl sepharose columns.29 Purified Gαi-3was dialyzed, concentrated, and stored (−70°C) in 50 mm HEPES (pH 8.0), 1 mm EDTA, and 2 mm dithiothreitol. The yield was approximately 4 mg/l of culture, with purity greater than 95% as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (fig. 1).
Fig. 1. Coomassie brilliant blue stains of sodium dodecyl sulfate polyacrylamide gels of the α isoform-3 G-protein subunit (Gαi-3) expressed in and purified from  Escherichia coli  bacteria (  lane 3  ) or  Spodoptera frugiperda  (S  f  9) insect cells (  lane 2  ).  Lane 1  shows the same for the heterotrimer composed of Gαi-3, and the untagged β isoform-1 (β1) and amino-terminally hexahistadine-tagged, Flag-tagged γ isoform-2 (γ2HF) G-protein subunits (Gαi-3β1γ2HF) expressed in and purified from S  f  9 insect cells. The molecular weight (MW) marker ladder is shown in the  left lane  . 
Fig. 1. Coomassie brilliant blue stains of sodium dodecyl sulfate polyacrylamide gels of the α isoform-3 G-protein subunit (Gαi-3) expressed in and purified from  Escherichia coli  bacteria (  lane 3  ) or  Spodoptera frugiperda  (S  f  9) insect cells (  lane 2  ).  Lane 1  shows the same for the heterotrimer composed of Gαi-3, and the untagged β isoform-1 (β1) and amino-terminally hexahistadine-tagged, Flag-tagged γ isoform-2 (γ2HF) G-protein subunits (Gαi-3β1γ2HF) expressed in and purified from S  f  9 insect cells. The molecular weight (MW) marker ladder is shown in the  left lane  . 
Fig. 1. Coomassie brilliant blue stains of sodium dodecyl sulfate polyacrylamide gels of the α isoform-3 G-protein subunit (Gαi-3) expressed in and purified from  Escherichia coli  bacteria (  lane 3  ) or  Spodoptera frugiperda  (S  f  9) insect cells (  lane 2  ).  Lane 1  shows the same for the heterotrimer composed of Gαi-3, and the untagged β isoform-1 (β1) and amino-terminally hexahistadine-tagged, Flag-tagged γ isoform-2 (γ2HF) G-protein subunits (Gαi-3β1γ2HF) expressed in and purified from S  f  9 insect cells. The molecular weight (MW) marker ladder is shown in the  left lane  . 
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Construction of Recombinant Baculoviruses.
Baculoviruses containing untagged G-protein β11) and amino-terminally hexahistadine-tagged, Flag-tagged G-protein γ22HF) were generous gifts from James C. Garrison, Ph.D. (Professor, Department of Pharmacology, University of Virginia, Charlottesville, Virginia). To generate a baculovirus containing Gαi-3, the pcDNA3.1:Gαi-3construct was amplified by polymerase chain reaction, and Pst  I and Xba  I restriction sites were incorporated into the 5′ and 3′ ends, respectively. The resulting polymerase chain reaction product was gel purified and subcloned into the pCR-BluntII TOPO vector. The pCR-BluntII:Gαi-3was digested with Pst  I and Xba  I, and the resulting Gαi-3insert was gel purified and ligated into the baculovirus transfer vector pVL1392. Recombinant baculoviruses were generated by cotransfection of Spodoptera frugiperda  (Sf  9) insect cells with the transfer vector and Sapphire baculoviral DNA. After amplification, the titer of recombinant baculovirus was determined by immunocytochemistry of infected Sf  9 cells with a monoclonal antibody directed against the baculovirus-encoded gp64 (FastPlax; Novagen, Madison, WI).
Expression of Gαi-3and Gαi-3β1γ2HFin Sf  9 Cells.
Sf  9 cells were coinfected with the recombinant baculoviruses encoding Gαi-3, β1, and γ2HF. Typically, cells were grown to a density of 2 × 106cells/ml in 250 ml Hinks TNM-FH media supplemented with 10% fetal bovine serum, 10 μg/ml gentamicin, and 0.1% Pluronic F-68. The cells were infected at a multiplicity of infection of 3, 1.5, and 1 for Gαi-3, β1, and γ2HF, respectively. Forty-eight hours after infection, the cells were harvested by centrifugation (400g  , 15 min), flash-frozen in liquid nitrogen, and stored at −70°C.
Purification of Gαi-3and Gαi-3β1γ2HFfrom Sf  9 Cell Membrane.
Membrane preparation and protein purification were performed, with minor modifications, according to the methods of Kozasa.30 Cell pellets were thawed and lysed by nitrogen cavitation (Parr Instruments, Moline, IL) in 37.5 ml ice-cold lysis buffer composed of 20 mm HEPES-NaOH (pH 8.0), 100 mm NaCl, 0.1 mm EDTA, 2 mm MgCl2, 9.8 mm 2-mercaptoethanol, 10 μm GDP, 1 mm phenylmethylsulphonylfluoride and the protease inhibitor cocktail P8849. The cell lysates were centrifuged (400g  , 10 min, 4°C) to remove intact cells and nuclei, and the supernatant was subjected to ultracentrifugation (100,000g  , 30 min, 4°C) to pellet cell membranes. The membranes were resuspended in 18.75 ml wash buffer composed of 20 mm HEPES-NaOH (pH 8.0), 100 mm NaCl, 1 mm MgCl2, 9.8 mm 2-mercaptoethanol, and 10 μm GDP, homogenized (8–10 strokes) with a Potter-Elvehjem homogenizer (Pierce Biotechnology, Rockford, IL) and ultracentrifuged (100,000g  , 30 min, 4°C). Washed membranes were resuspended to a concentration of 1–2 mg membrane protein/ml in wash buffer containing fresh protease inhibitors.
To extract the G protein from the membranes, sodium cholate was added to the preparation at a concentration of 1% and the mixture was stirred on ice for 1 h. After ultracentrifugation (100,000g  , 30 min, 4°C), the supernatant was diluted fourfold with nickel–nitrilotriacetic acid (Ni-NTA) loading buffer composed of 20 mm HEPES-NaOH (pH 8.0), 100 mm NaCl, 1 mm MgCl2, 9.8 mm 2-mercaptoethanol, 10 μm GDP, and 0.5% polyoxyethylene 10-laurylether and then loaded onto an Ni-NTA resin column preequilibrated with the Ni-NTA loading buffer (4°C). The column was washed with 20 column volumes of Ni-NTA salt wash composed of 20 mm HEPES-NaOH (pH 8.0), 300 mm NaCl, 3 mm MgCl2, 9.8 mm 2-mercaptoethanol, 10 μm GDP, 10 mm imidazole-HCl (pH 8.0), and 0.5% polyoxyethylene 10-laurylether followed by three column volumes of 0.2% cholate wash composed of 20 mm HEPES-NaOH (pH 8.0), 50 mm NaCl, 3 mm MgCl2, 9.8 mm 2-mercaptoethanol, 10 μm GDP, 10 mm imidazole-HCl (pH 8.0), and 0.2% sodium cholate. The column was warmed to room temperature and washed again with three column volumes of cholate wash.
i-3was eluted from the β1γ2HFdimer with Ni-NTA elution buffer composed of 20 mm HEPES-NaOH (pH 8.0), 50 mm NaCl, 50 mm MgCl2, 9.8 mm 2-mercaptoethanol, 10 μm GDP, and 1% sodium cholate. Alternatively, the intact Gαi-3β1γ2HFheterotrimer was eluted from the column with Ni-NTA cholate wash containing 150 mm imidazole-HCl (pH 8.0) and 1% sodium cholate. One-milliliter elution fractions were collected, and samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (10% acrylamide) to screen for protein (fig. 1). The fractions containing Gαi-3or Gαi-3β1γ2HFwere pooled and concentrated with Microcon-10 centrifugal concentrators (Millipore, Bedford, MA). Finally the buffer was exchanged into storage buffer composed of 20 mm HEPES-NaOH (pH 8.0), 100 mm NaCl, 2 mm MgCl2, 0.5 mm EDTA, 1 mm dithiothreitol, 11 mm CHAPS, and 1 μm GDP. The procedure typically yielded 150–200 μg purified G-protein subunit from a 10-g Sf  9 cell pellet.
Nuclear Magnetic Resonance Saturation Transfer Difference
One-dimensional nuclear magnetic resonance (1H NMR) and saturation transfer difference (STD)31 spectra were recorded with a 500-MHz Bruker BioSpin NMR (Billerica, MA) as previously described and validated by our laboratory for the purpose of detecting anesthetic–protein binding.28 This technique is based on the nuclear Overhauser effect between bound anesthetic protons and all protein protons. Gα subunit samples were prepared in deuterated water containing 20 mm phosphate buffer (pH 7.0) with 100 mm NaCl, 50 μm dithiothreitol, 2 mm MgSO4, and 0.5 μm GDP. Samples were placed in 5-mm NMR tubes (Wilmad Labglass, Buena, NJ) and sealed with a capillary stem insert tube containing 10 mm sodium acetate in deuterated water as an external standard. The external standard was used to quantify the aqueous concentration of ligands and also functioned as a negative control because it does not bind to protein. As a control, the 1H NMR and STD spectra of 1 mm halothane, 1 mm suramin, and 10 mm hexanol were recorded to confirm that the pulse sequence did not directly saturate the ligands. Suramin binds and inhibits Gαiactivity32 and was used as a positive control for ligand–protein binding. Then the spectra of these compounds were recorded in the presence of 40 μm Gαi-3.
i-3and Gαi-3β1γ2HFGTP Hydrolysis
Guanosine-5′-triphosphatase (GTPase) activity was measured using a standard phosphohydrolase assay with radioactive inorganic phosphate ([32Pi])–labeled GTP33 as previously described by our laboratory.27,34 Gαi-3or Gαi-3β1γ2HF(0.2 μm final concentration in the assay) was incubated (30°C, 5 min) with or without halothane (control) in an assay buffer composed of 25 mm HEPES (pH 7.8), 100 mm NaCl, 10 mm MgSO4, 1 mm EDTA, 1 mm dithiothreitol, 0.1 μm GDP, 5 μm GTP, and 0.5 mg/ml bovine serum albumin. After initiating the reactions 0.025 mCi [γ32Pi]GTP for 3, 6, 9, and 12 min, aliquots from each assay tube were quenched on ice with 7% (wt/vol) activated charcoal in 2N HCl and 0.35 M NaH2PO4and vigorously vortexed. The tubes were centrifuged (12,000g  , 10 min, room temperature) to pellet the charcoal and the amount of radioactivity (from 32Pi) in supernatant was determined by liquid scintillation counting.
Culture and Transfection of COS-7 Cells
COS-7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (10% DMEM) and penicillin and streptomycin (50 U/ml each). The day before transfection, confluent cells were trypsinized and seeded in 10-cm tissue culture plates to reach 90% confluence in 24 h (approximately 4.5 × 106cells per 10-cm plate). The cells were then transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) as per the manufacturer’s recommendation. Briefly, for each 10-cm plate transfection, the complementary DNA (cDNA) constructs were mixed (5 min, room temperature) with 1.5 ml Opt-MEM I in a 50-ml Falcon tube. For cotransfection, 10 μg M2cDNA plus 5 μg Gαi-3cDNA were used, and for transfection of Gαi-3alone, 5 μg Gαi-3cDNA plus 10 μg vector pcDNA3.1 were used. Lipofectamine 2000 was mixed in another Falcon tube with 1.5 ml Opt-MEM (2.5 μl/μg cDNA to be transfected). The two solutions were then mixed and allowed to stand for 20 min at room temperature to promote DNA–Lipofectamine complex formation. Three milliliters of the transfection mixture was added to each 10-cm plate with 5 ml DMEM, 10%, without penicillin or streptomycin. The transfection mixture was replaced with 7 ml fresh DMEM, 10%, plus penicillin and streptomycin after 12 h. Twenty-four hours after transfection, the cells were washed twice with phosphate-buffered saline (room temperature), scraped in ice-cold phosphate-buffered saline, transferred to 1.5-ml microfuge tubes, and pelleted by centrifugation (500g  , 2 min, 4°C). The cells were flash-frozen in liquid nitrogen and stored at −70°C until they were used to prepare crude membranes.
Crude Membrane Preparation
Frozen cells from three 10-cm plates were suspended for 15 min in ice-cold lysis buffer (500 μl/plate) composed of 20 mm HEPES (pH 8.0), 1 mm EDTA, 0.1 mm phenylmethysulfonyl fluoride, 10 μg/ml leupeptin, and 2 μg/ml aprotinin. Then the cells were gently homogenized on ice by repeated passage through a 27-gauge needle (approximately 10–12 times). The lysate was then subjected to low-speed centrifugation (400g  , 10 min, 4°C) to remove intact cells and nuclei, and then to ultracentrifugation (87,000g  , 30 min, 4°C) to pellet the crude membrane. The membrane pellet was washed with lysis buffer and then resuspended by repeated passage through a 27-gauge needle in assay buffer (100 μl/plate) composed of 10 mm HEPES (pH 8.0), 100 mm NaCl, 10 mm MgCl2, and 10 μm GDP. A portion of the crude membrane (50 μl) was solubilized in 12.5 μl NaOH, 0.1N, and boiled (3 min) to determine protein concentration.35 The crude membrane suspension was then diluted with assay buffer to a protein concentration of 2–3 mg/ml, frozen in liquid nitrogen, and stored at −70°C until used for the assay.
Quantification of M2Muscarinic Receptor Expression
M2muscarinic receptor was quantified in the crude membranes by saturation binding with the muscarinic receptor antagonist l  -[benzilic-4,4′-3H]-quinuclidinyl benzilate (3H-QNB).36,37 Specific binding was determined in triplicate assays with a single saturating concentration of 3H-QNB of 5 nm. Nonspecific binding was determined by performing triplicate assays in the presence of 5 μm atropine. Aliquots of crude membranes containing 20 μg protein from M2–Gαi-3cotransfected COS-7 cells, or untransfected COS-7 cells were incubated (90 min, room temperature) in 0.5 ml buffer containing 5 nm 3H-QNB, 50 mm Tris (pH 7.4), and 10 mm MgCl2, with and without 5 μm atropine. After the 90-min incubation period, reactions were applied to prewetted GF/B glass fiber filters sheets using a Brandel cell harvester (Brandel, Gaithersburg, MD) and washed three times with buffer containing 50 mm Tris (pH 7.4) and 10 mm MgCl2. Bound radioactivity on the filters was quantified by liquid scintillation counting.
i-3Guanosine Nucleotide Exchange Assay in COS-7 Cell Membranes
i-3guanosine nucleotide exchange was assayed in crude membrane prepared from M2–Gαi-3cotransfected COS-7 cells using previously described methods.38 Reaction mixtures containing 18–20 μg membrane protein, 10 mm HEPES, 100 mm NaCl, 10 mm MgCl2, and 10 μm GDP were incubated with and without halothane and with and without acetylcholine in a total volume of 62 μl for 5 min at 30°C. To minimize the loss of volatile anesthetics, reactions were performed in narrow 0.25-ml polypropylene tubes. The reactions were initiated by the addition of 5 μl of the radioactive, nonhydrolyzable form of GTP, [35S]GTPγS, to the assay mixture (1,250 Ci/mmol; 20 nm final concentration in assay). Reactions were terminated at times according to experimental design with 900 μl ice-cold assay buffer. The reaction tubes were then centrifuged (16,000g  , 10 min, 4°C), and the pellets were resuspended by vigorous vortexed mixing (2 min, room temperature) in 50 μl solubilization buffer composed of 100 mm Tris-HCl (pH 7.4), 1 mm EDTA, 200 mm NaCl, 1.25% (vol/vol) IGEPAL CA-630, and 0.2% sodium dodecyl sulfate. Then the samples were gently rocked (30 min, 4°C), diluted in 50 μl solubilization buffer without sodium dodecyl sulfate, vortex mixed, and centrifuged at (16,000g  , 10 min, 4°C). The supernatant (100 μl) containing detergent-solubilized [35S]GTPγS-bound Gαi-3was transferred to 1.5-ml microfuge tubes and incubated (1 h, 4°C) with rabbit polyclonal antibody raised against a peptide mapping at the carboxy terminus of rat Gαi-3(1:100 vol/vol dilution). Then 40 μl protein A-agarose beads was added to each sample for an additional hour (4°C). The beads were then washed three times by repeated pelleting (2,000g  , 1 min, 4°C) followed by resuspension in 0.5 ml solubilization buffer without sodium dodecyl sulfate and rotated on an orbital rocker (30 min, 4°C). Finally, the washed beads were placed in 4 ml Ultimate Gold scintillation cocktail (Packard Bioscience, Meriden, CT), and radioactivity was quantified by liquid scintillation counting.
Background radioactivity measurements were determined by performing tandem experiments with the same amount of protein, except that the assay was terminated immediately. The amount of background radioactivity was less than 40% of the radioactivity of the basal Gαi-3nucleotide exchange measurements. In preliminary work, halothane had no effect on this nonspecific background radioactivity. Data were normalized to the amount of membrane protein and the specific activity of the [35S]GTPγS in the assay, and each experimental condition was assayed in triplicate.
Preparation of Anesthetic Solutions
Saturated aqueous stocks of halothane were prepared by stirring 5 ml liquid anesthetic with 10 ml assay buffer for 30 min in an airtight, ground-glass flask as previously described.14,26–28 After the stirring had been stopped for 5 min, 5 ml of the saturated assay buffer was transfer to a 5-ml glass vial. Aliquots of the anesthetic-saturated stocks were added directly to the reaction tubes in volumes that produced the desired final anesthetic concentration. To account for any loss of volatile anesthetic in the assay tubes, tandem experiments were conducted under the same assay conditions, and anesthetic concentrations were measured after hexane extraction by gas chromatography using an electron capture detector (Hewlett-Packard, Waltham, MA; model 5880A).39 In preliminary studies, we found that after an initial loss of approximately 20% after transfer and mixing, the amount of volatile anesthetic in the reaction tubes was relatively stable, with less than 10% additional loss occurring during the longest assay time in this report (10 min).
Experimental Protocols
Characterization of the Gαi-3Guanosine Nucleotide Exchange Assay in COS-7 Cell Membranes.
To determine the dependence of the Gαi-3[35S]GTPγS–GDP exchange measurements on M2muscarinic receptor expression and activation by acetylcholine, assays were performed using crude membranes prepared from COS-7 cells transfected with the cDNA constructs encoding for Gαi-3only, or both the M2muscarinic receptor and Gαi-3. The crude membranes were incubated without (for basal Gαi-3[35S]GTPγS–GDP exchange measurements) or with (for acetylcholine-promoted Gαi-3[35S]GTPγS–GDP exchange measurements) 10 μm acetylcholine for 5 min, and then the reactions were initiated with [35S]GTPγS. The reactions were terminated after 10 min, and the samples were subjected to the immunoprecipitation step of the assay using either nonimmune rabbit serum (for nonspecific background measurements) or rabbit anti-Gαi-3immunoglobulin G. These data were not subjected to nonspecific background subtraction.
Effect of Acetylcholine on Gαi-3Guanosine Nucleotide Exchange in COS-7 Cell Membranes.
These protocols were conducted to help guide the experimental approach of subsequent protocols that examined possible anesthetic effects on Gαi-3[35S]GTPγS–GDP exchange. To determine the time course for Gαi-3[35S]GTPγS–GDP exchange, crude membrane prepared from M2–Gαi-3cotransfected COS-7 cells was incubated for 5 min with or without 10 μm acetylcholine, and then the reactions were initiated with [35S]GTPγS. The reactions were terminated after 1, 2, 5, or 10 min, and then the samples were subjected to the immunoprecipitation step of the assay.
To determine the acetylcholine concentrations that produced half-maximal and maximal promotion of Gαi-3[35S]GTPγS–GDP exchange, crude membrane was incubated with or without various concentrations of acetylcholine (0.001–100 μm) for 5 min. The reactions were then terminated 5 min after initiation with [35S]GTPγS, and the samples were subjected to the immunoprecipitation step of the assay. The acetylcholine-promoted Gαi-3[35S]GTPγS–GDP exchange was expressed as the percentage of the difference between the values measured in the absence of acetylcholine and that measured in the presence of the acetylcholine concentration that produced the maximal effect (i.e.  , percentage acetylcholine-promoted).
Effect of Halothane on Gαi-3Guanosine Nucleotide Exchange in COS-7 Cell Membranes.
Crude membranes prepared from M2–Gαi-3cotransfected COS-7 cells were incubated for 5 min with or without 0.5, 1, or 3 mm halothane and with or without the acetylcholine concentration determined to promote an increase in Gαi-3[35S]GTPγS–GDP exchange of approximately 80–90% of the maximum value. These concentrations of anesthetics include those studied in our previous work with crude membranes prepared from porcine tracheal smooth muscle,14,26 thereby allowing comparisons with these previous data. The reactions were terminated at a time determined to produce an approximately 70–80% increase in maximal Gαi-3[35S]GTPγS–GDP exchange, and then the samples were subjected to the immunoprecipitation step of the assay. Basal Gαi-3[35S]GTPγS–GDP exchange values are expressed as a percentage of the values measured in the absence of halothane (i.e.  , basal control). The acetylcholine-promoted Gαi-3[35S]GTPγS–GDP exchange values are expressed as a percentage of the difference between the basal control values (i.e.  , absence of acetylcholine or halothane in the assay) and the values measured in the presence of the chosen acetylcholine concentration but the absence of halothane in the assay (i.e.  , acetylcholine-promoted control).
Materials
The pET28a plasmid, BL21 (DE3) E. coli  bacteria, and the FastPlax Titer kit were purchased from Novagen (Madison, WI). The sepharose chromatography columns were purchased from Amersham Biosciences (Piscataway, NJ), and the hydroxyapatite CHT10-I column was purchased from Biocompare (San Francisco, CA). The Sapphire insect transfection kit and Sf  9 cells were obtained from Orbigen (San Diego, CA). COS-7 cells were purchased from American Type Culture Collection (Manassas, VA). Deuterated water was purchased from CDN Isotopes (Pointe-Claire, Quebec, Canada). All chemicals and supplies required for cell culture and cDNA transfection of COS-7 cells, pCDNA 3.1 plasmid, pCR-BluntII TOPO vector, and baculovirus transfer vector pVL1392 were purchased from Invitrogen (Carlsbad, CA). EndoFree maxi-prep kits used for cDNA preparation and Ni-NTA resin were purchased from Qiagen Science (Valencia, CA). Halothane was purchased from Ayerst Laboratories, Inc. (New York, NY). l-[Benzilic-4,4′-3H]-quinuclidinyl benzilate (42 Ci/mmol) and [35S]GTPγS (1,250 Ci/mmol) were purchased from Perkin-Elmer (Boston, MA). Rabbit nonimmune serum was purchased from Calbiochem (EMD Biosciences, Inc. Affiliate, San Diego, CA). Protein A-agarose beads and anti-rabbit immunoglobulin G were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Lowry protein assay kits were purchased from Bio-Rad Life Science Research Produces (Hercules, CA). All other chemicals were purchased from Sigma Chemical Company (St. Louis, MO). All drugs and chemicals were prepared in distilled, filtered water.
Data Analysis and Statistics
Data are reported as mean ± SE; n represents the number of independent times an assay was performed. All time- and concentration-dependent effects on acetylcholine on Gαi-3[35S]GTPγS–GDP exchange were determined by nonlinear regression analysis as previously described (SigmaPlot 8; Systat Software, Point Richmond, CA).14,26,40 Data were statistically compared by either repeated-measures analysis of variance with post hoc  testing performed using the Student-Newman-Keuls test or paired Student t  test as appropriate for experimental design. For all statistical comparisons, a value of P  < 0.05 was considered significant.
Results
Anesthetic Binding to Gαi-3by STD-1H NMR
Saturation transfer from Gαi-3to halothane, hexanol, and suramin was observed with STD (fig. 2A) and confirmed with 1H NMR (fig. 2B). The spectra showed intensity from resonances from all three molecules indicating that each binds Gαi-3. Halothane had a single peak at approximately 6.3 ppm, whereas the remaining resonances are from suramin and hexanol. Because the STD spectrum is dependent on the interaction geometry,31 the STD spectrum contains some but not all of the ligand-1H NMR peaks, and the relative intensities of these peaks are not identical to those in the 1H NMR spectra. The peak at approximately 4.8 ppm in the 1H NMR spectrum is from residual water, and its peak shape is due to suppression.
Fig. 2. Representative saturation transfer difference (  A  ) and one-dimensional nuclear magnetic resonance (  B  ) spectra recorded for a solution containing 1 mm suramin (S), 1 mm halothane (H), 10 mm hexanol (Hx), and the α isoform-3 G-protein subunit (Gαi-3). Gαi-3was expressed in and purified from  Spodoptera frugiperda  insect cells. The region of the saturation transfer difference spectra where deuterated water (HOD) absorbs (approximately 4.8 ppm) was deleted from the figure for clarity of presentation. 
Fig. 2. Representative saturation transfer difference (  A  ) and one-dimensional nuclear magnetic resonance (  B  ) spectra recorded for a solution containing 1 mm suramin (S), 1 mm halothane (H), 10 mm hexanol (Hx), and the α isoform-3 G-protein subunit (Gαi-3). Gαi-3was expressed in and purified from  Spodoptera frugiperda  insect cells. The region of the saturation transfer difference spectra where deuterated water (HOD) absorbs (approximately 4.8 ppm) was deleted from the figure for clarity of presentation. 
Fig. 2. Representative saturation transfer difference (  A  ) and one-dimensional nuclear magnetic resonance (  B  ) spectra recorded for a solution containing 1 mm suramin (S), 1 mm halothane (H), 10 mm hexanol (Hx), and the α isoform-3 G-protein subunit (Gαi-3). Gαi-3was expressed in and purified from  Spodoptera frugiperda  insect cells. The region of the saturation transfer difference spectra where deuterated water (HOD) absorbs (approximately 4.8 ppm) was deleted from the figure for clarity of presentation. 
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Effect of Halothane on Gαi-3and Gαi-3β1γ2GTPase Activity
i-3expressed in and purified from E. coli  exhibited an intrinsic GTPase activity of 0.0182 ± 0.0005 pmol 32Pi · pmol protein−1· min−1, which was similar to that reported by other investigators.41,42 The presence of halothane in the assay tended to cause a slight increase in Gαi-3GTPase activity, reaching statistical significance only at 0.75 and 3 mm halothane (fig. 3).
Fig. 3. Concentration-dependent effect of halothane on guanosine-5′-triphosphate (GTP) hydrolysis rate (GTPase activity) of the nonmyristoylated α isoform-3 G-protein subunit (Gαi-3) expressed in and purified from E coli. GTPase activity was determined using a standard phosphohydrolase assay with radiolabeled [γ32Pi]GTP as described in detail in the Materials and Methods. Assays were performed in the absence or presence of 0.5, 0.75, 1.0, and 3.0 mm halothane. Data are presented as mean ± SE of 3–14 assays. * Significant difference from values measured in the absence of halothane. 
Fig. 3. Concentration-dependent effect of halothane on guanosine-5′-triphosphate (GTP) hydrolysis rate (GTPase activity) of the nonmyristoylated α isoform-3 G-protein subunit (Gαi-3) expressed in and purified from E coli. GTPase activity was determined using a standard phosphohydrolase assay with radiolabeled [γ32Pi]GTP as described in detail in the Materials and Methods. Assays were performed in the absence or presence of 0.5, 0.75, 1.0, and 3.0 mm halothane. Data are presented as mean ± SE of 3–14 assays. * Significant difference from values measured in the absence of halothane. 
Fig. 3. Concentration-dependent effect of halothane on guanosine-5′-triphosphate (GTP) hydrolysis rate (GTPase activity) of the nonmyristoylated α isoform-3 G-protein subunit (Gαi-3) expressed in and purified from E coli. GTPase activity was determined using a standard phosphohydrolase assay with radiolabeled [γ32Pi]GTP as described in detail in the Materials and Methods. Assays were performed in the absence or presence of 0.5, 0.75, 1.0, and 3.0 mm halothane. Data are presented as mean ± SE of 3–14 assays. * Significant difference from values measured in the absence of halothane. 
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The GTPase activities of the Gαi-3and Gαi-3β1γ2HFheterotrimer expressed in and purified from membranes of Sf  9 insect cells were also comparable to those previously reported.43 As expected, the GTPase activity of the heterotrimer was approximately fourfold greater than that of the Gαi-3subunit (0.0158 ± 0.009 and 0.0619 ± 0.0141 pmol 32Pi · pmol protein−1· min−1for Gαi-3and Gαi-3β1γ2HF, respectively).43 The presence of 3 mm halothane in the assay had no significant effect on the GTPase activity of the Gαi-3β1γ2HF, whereas it caused a small but significant inhibition of Gαi-3GTPase activity (fig. 4).
Fig. 4. Effect of halothane on guanosine-5′-triphosphate (GTP) hydrolysis rate (GTPase activity) of the α isoform-3 G-protein subunit (Gαi-3) and the heterotrimer composed of Gαi-3, and the untagged β isoform-1 (β1) and amino-terminus hexahistadine-tagged, Flag-tagged γ isoform-2 G-protein subunits (Gαi-3β1γ2HF). Proteins were expressed in and purified from  Spodoptera frugiperda  insect cells. GTPase activity was determined using a standard phosphohydrolase assay with radiolabeled [g32Pi]GTP as described in detail in the Materials and Methods. Assays were performed in the absence (control) or presence of 3 mm halothane. Data are presented as mean ± SE of 9–12 assays. * Significant difference from values measured in the absence of halothane (control). 
Fig. 4. Effect of halothane on guanosine-5′-triphosphate (GTP) hydrolysis rate (GTPase activity) of the α isoform-3 G-protein subunit (Gαi-3) and the heterotrimer composed of Gαi-3, and the untagged β isoform-1 (β1) and amino-terminus hexahistadine-tagged, Flag-tagged γ isoform-2 G-protein subunits (Gαi-3β1γ2HF). Proteins were expressed in and purified from  Spodoptera frugiperda  insect cells. GTPase activity was determined using a standard phosphohydrolase assay with radiolabeled [g32Pi]GTP as described in detail in the Materials and Methods. Assays were performed in the absence (control) or presence of 3 mm halothane. Data are presented as mean ± SE of 9–12 assays. * Significant difference from values measured in the absence of halothane (control). 
Fig. 4. Effect of halothane on guanosine-5′-triphosphate (GTP) hydrolysis rate (GTPase activity) of the α isoform-3 G-protein subunit (Gαi-3) and the heterotrimer composed of Gαi-3, and the untagged β isoform-1 (β1) and amino-terminus hexahistadine-tagged, Flag-tagged γ isoform-2 G-protein subunits (Gαi-3β1γ2HF). Proteins were expressed in and purified from  Spodoptera frugiperda  insect cells. GTPase activity was determined using a standard phosphohydrolase assay with radiolabeled [g32Pi]GTP as described in detail in the Materials and Methods. Assays were performed in the absence (control) or presence of 3 mm halothane. Data are presented as mean ± SE of 9–12 assays. * Significant difference from values measured in the absence of halothane (control). 
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Characterization of Cotransfected COS-7 Cells
No muscarinic receptors could be detected above the nonspecific background measurements in crude membranes prepared from untransfected COS-7 cells as expected, because these cells do not express endogenous muscarinic receptors.44,45 However, the membranes prepared from COS-7 cells cotransfected with the cDNA constructs encoding for the M2muscarinic receptor and Gαi-3expressed the M2muscarinic receptor at 2–3.4 pmol/mg protein. In crude membranes prepared from Gαi-3-only transfected cells or M2–Gαi-3cotransfected cells, there was a significant increase in Gαi-3[35S]GTPγS–GDP exchange above that of the nonspecific background measurements even when acetylcholine was omitted from the assay (fig. 5). The coexpression of the M2muscarinic receptor with Gαi-3did not promote an additional increase in this basal Gαi-3[35S]GTPγS–GDP exchange (fig. 5). These data indicate that the expressed M2muscarinic receptor does not possess constitutive activity, as described by others,38 but rather reflects the constitutive activity of endogenously expressed receptors that are functionally coupled to the expressed Gαi-3. This assertion is supported by the finding that adenosine-5′-diphosphate ribosylation of Gαi-3by pertussis toxin in crude membranes prepared from Gαi-3-alone transfected cells abolished the Gαi-3-specific basal [35S]GTPγS–GDP exchange from 24.7 ± 1.1 (fig. 5) to 6.0 ± 0.8 fmol/mg protein. The inclusion of 10 μm acetylcholine in the assay buffer of membranes prepared from M2–Gαi-3cotransfected cells caused a further approximately twofold increase in the magnitude of the Gαi-3-specific [35S]GTPγS–GDP exchange (fig. 5). However, when the receptor was omitted from the transfection (i.e.  , crude membranes prepared from Gαi-3-alone transfected cells), acetylcholine did not promote Gαi-3[35S]GTPγS–GDP exchange above the basal exchange levels.
Fig. 5. Effect of acetylcholine on the exchange of the radioactive, nonhydrolyzable form of guanosine-5′-triphosphate, [35S]GTPγS, for guanosine-5′-diphosphate ([35S]GTPγS–GDP exchange) at the α isoform-3 G-protein subunit (Gαi-3). Assays were performed using crude membranes prepared from COS-7 cells transfected with the complementary DNA that encodes for human Gαi-3only, or human M2muscarinic receptor and human Gαi-3. Measurements were made in the absence (basal exchange) and presence of 10 μm acetylcholine and were terminated 10 min after initiation of the assays with [35S]GTPγS. The immunoprecipitation step of the assays was performed using nonimmune serum (for nonspecific background measurements) or antiserum specific for Gαi-3. Data are presented as mean ± SE of six assays. * Significant difference from background radioactivity. † Significant difference from basal Gα[35S]GTPγS–GDP exchange. 
Fig. 5. Effect of acetylcholine on the exchange of the radioactive, nonhydrolyzable form of guanosine-5′-triphosphate, [35S]GTPγS, for guanosine-5′-diphosphate ([35S]GTPγS–GDP exchange) at the α isoform-3 G-protein subunit (Gαi-3). Assays were performed using crude membranes prepared from COS-7 cells transfected with the complementary DNA that encodes for human Gαi-3only, or human M2muscarinic receptor and human Gαi-3. Measurements were made in the absence (basal exchange) and presence of 10 μm acetylcholine and were terminated 10 min after initiation of the assays with [35S]GTPγS. The immunoprecipitation step of the assays was performed using nonimmune serum (for nonspecific background measurements) or antiserum specific for Gαi-3. Data are presented as mean ± SE of six assays. * Significant difference from background radioactivity. † Significant difference from basal Gα[35S]GTPγS–GDP exchange. 
Fig. 5. Effect of acetylcholine on the exchange of the radioactive, nonhydrolyzable form of guanosine-5′-triphosphate, [35S]GTPγS, for guanosine-5′-diphosphate ([35S]GTPγS–GDP exchange) at the α isoform-3 G-protein subunit (Gαi-3). Assays were performed using crude membranes prepared from COS-7 cells transfected with the complementary DNA that encodes for human Gαi-3only, or human M2muscarinic receptor and human Gαi-3. Measurements were made in the absence (basal exchange) and presence of 10 μm acetylcholine and were terminated 10 min after initiation of the assays with [35S]GTPγS. The immunoprecipitation step of the assays was performed using nonimmune serum (for nonspecific background measurements) or antiserum specific for Gαi-3. Data are presented as mean ± SE of six assays. * Significant difference from background radioactivity. † Significant difference from basal Gα[35S]GTPγS–GDP exchange. 
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Effect of Acetylcholine on Gαi-3Guanosine Nucleotide Exchange
In the absence of acetylcholine in the assay, membranes prepared from M2–Gαi-3cotransfected cells incorporated [35S]GTPγS into Gαi-3in a time-dependent manner and could be adequately fit with to a monoexponential association equation with a K of 0.16 ± 0.06 min−1(fig. 6A). The presence of acetylcholine in the assay promoted a significant increase in the magnitude of Gαi-3[35S]GTPγS–GDP exchange at each time point but had no significant effect on K, which was 0.17 ± 0.05 min−1(P  = 0.8). The acetylcholine-promoted increase in Gαi-3[35S]GTPγS–GDP exchange above basal exchange levels was concentration dependent, with an EC50value of 0.72 ± 0.10 μm acetylcholine (fig. 6B). Based on these initial investigations, subsequent experiments examining the effect of halothane on Gαi-3[35S]GTPγS–GDP exchange were performed using 10 μm acetylcholine to optimize the ability to detect modest shifts in acetylcholine-promoted Gαi-3[35S]GTPγS–GDP exchange and were measured after 10 min of stimulation.
Fig. 6. (  A  ) Time-dependent change in the exchange of the radioactive, nonhydrolyzable form of guanosine-5′-triphosphate, [35S]GTPγS, for guanosine-5′-diphosphate ([35S]GTPγS–GDP exchange) at the α isoform-3 G-protein subunit (Gαi-3). Assays were performed using crude membranes prepared from COS-7 cells cotransfected with the complementary DNA constructs encoding for the human M2muscarinic receptor and human Gαi-3. Gαi-3[35S]GTPγS–GDP exchange was measured in the absence (basal exchange) or presence of 10 μm acetylcholine in the assay. The reactions were terminated 1, 2, 5, and 10 min after initiation of the assays with [35S]GTPγS. Data are presented as mean ± SE of six assays. (  B  ) Concentration-dependent effect of acetylcholine on Gαi-3[35S]GTPγS–GDP exchange measured in crude membranes prepared from M2–Gαi-3cotransfected COS-7 cells. Assays were performed in the absence (basal exchange) and presence of acetylcholine (ACh, 0.001–100 μm) in the assays and the reactions were terminated 10 min after initiation with [35S]GTPγS. Data are the percentage of the difference between the basal exchange values and those measured in the presence of the acetylcholine concentration that produced the maximal effect. Data are presented as mean ± SE of four assays. 
Fig. 6. (  A  ) Time-dependent change in the exchange of the radioactive, nonhydrolyzable form of guanosine-5′-triphosphate, [35S]GTPγS, for guanosine-5′-diphosphate ([35S]GTPγS–GDP exchange) at the α isoform-3 G-protein subunit (Gαi-3). Assays were performed using crude membranes prepared from COS-7 cells cotransfected with the complementary DNA constructs encoding for the human M2muscarinic receptor and human Gαi-3. Gαi-3[35S]GTPγS–GDP exchange was measured in the absence (basal exchange) or presence of 10 μm acetylcholine in the assay. The reactions were terminated 1, 2, 5, and 10 min after initiation of the assays with [35S]GTPγS. Data are presented as mean ± SE of six assays. (  B  ) Concentration-dependent effect of acetylcholine on Gαi-3[35S]GTPγS–GDP exchange measured in crude membranes prepared from M2–Gαi-3cotransfected COS-7 cells. Assays were performed in the absence (basal exchange) and presence of acetylcholine (ACh, 0.001–100 μm) in the assays and the reactions were terminated 10 min after initiation with [35S]GTPγS. Data are the percentage of the difference between the basal exchange values and those measured in the presence of the acetylcholine concentration that produced the maximal effect. Data are presented as mean ± SE of four assays. 
Fig. 6. (  A  ) Time-dependent change in the exchange of the radioactive, nonhydrolyzable form of guanosine-5′-triphosphate, [35S]GTPγS, for guanosine-5′-diphosphate ([35S]GTPγS–GDP exchange) at the α isoform-3 G-protein subunit (Gαi-3). Assays were performed using crude membranes prepared from COS-7 cells cotransfected with the complementary DNA constructs encoding for the human M2muscarinic receptor and human Gαi-3. Gαi-3[35S]GTPγS–GDP exchange was measured in the absence (basal exchange) or presence of 10 μm acetylcholine in the assay. The reactions were terminated 1, 2, 5, and 10 min after initiation of the assays with [35S]GTPγS. Data are presented as mean ± SE of six assays. (  B  ) Concentration-dependent effect of acetylcholine on Gαi-3[35S]GTPγS–GDP exchange measured in crude membranes prepared from M2–Gαi-3cotransfected COS-7 cells. Assays were performed in the absence (basal exchange) and presence of acetylcholine (ACh, 0.001–100 μm) in the assays and the reactions were terminated 10 min after initiation with [35S]GTPγS. Data are the percentage of the difference between the basal exchange values and those measured in the presence of the acetylcholine concentration that produced the maximal effect. Data are presented as mean ± SE of four assays. 
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Effect of Halothane on Gαi-3Nucleotide Exchange
The presence of 0.5 or 1 mm halothane in the assay had no significant effect on basal Gαi-3[35S]GTPγS–GDP exchange measured in the absence of acetylcholine; however, 3 mm halothane inhibited basal Gαi-3[35S]GTPγS–GDP exchange by approximately 40% (fig. 7A). Halothane caused a concentration-dependent inhibition of Gαi-3[35S]GTPγS–GDP exchange when 10 μm acetylcholine was included in the reactions (fig. 7B). The amounts of inhibition were 22.7 ± 8.2, 44.9 ± 6.5, and 86.3 ± 4.8% for 0.5, 1, and 3 mm halothane, respectively.
Fig. 7. Concentration-dependent effect of halothane on the exchange of the radioactive, nonhydrolyzable form of gua-nosine-5′-triphosphate, [35S]GTPγS, for guanosine-5′-diphosphate ([35S]GTPγS–GDP exchange) at the α isoform-3 G-protein subunit (Gαi-3). Assays were performed using crude membranes prepared from COS-7 cells cotransfected with the complementary DNA constructs encoding for the human M2muscarinic receptor and human Gαi-3. Measurements were made in the absence (basal ex-change,  A  ) or presence (  B  ) of 10 μm acetylcholine (ACh) in the assay, and the reactions were terminated 10 min after initiation with [35S]GTPγS. Basal Gαi-3[35S]GTPγS–GDP exchange values are expressed as the percentage of the values measured in the absence of halothane (basal control exchange), whereas the acetylcholine-promoted exchange values are expressed as a percentage of the difference between the basal control values and those measured in the presence of the 10 μm acetylcholine but in the absence of halothane in the assay (  i.e.  , percentage acetylcholine-promoted). Data are presented as mean ± SE of 9–42 assays. * Indicates significant difference from the values obtained in the absence of halothane. 
Fig. 7. Concentration-dependent effect of halothane on the exchange of the radioactive, nonhydrolyzable form of gua-nosine-5′-triphosphate, [35S]GTPγS, for guanosine-5′-diphosphate ([35S]GTPγS–GDP exchange) at the α isoform-3 G-protein subunit (Gαi-3). Assays were performed using crude membranes prepared from COS-7 cells cotransfected with the complementary DNA constructs encoding for the human M2muscarinic receptor and human Gαi-3. Measurements were made in the absence (basal ex-change,  A  ) or presence (  B  ) of 10 μm acetylcholine (ACh) in the assay, and the reactions were terminated 10 min after initiation with [35S]GTPγS. Basal Gαi-3[35S]GTPγS–GDP exchange values are expressed as the percentage of the values measured in the absence of halothane (basal control exchange), whereas the acetylcholine-promoted exchange values are expressed as a percentage of the difference between the basal control values and those measured in the presence of the 10 μm acetylcholine but in the absence of halothane in the assay (  i.e.  , percentage acetylcholine-promoted). Data are presented as mean ± SE of 9–42 assays. * Indicates significant difference from the values obtained in the absence of halothane. 
Fig. 7. Concentration-dependent effect of halothane on the exchange of the radioactive, nonhydrolyzable form of gua-nosine-5′-triphosphate, [35S]GTPγS, for guanosine-5′-diphosphate ([35S]GTPγS–GDP exchange) at the α isoform-3 G-protein subunit (Gαi-3). Assays were performed using crude membranes prepared from COS-7 cells cotransfected with the complementary DNA constructs encoding for the human M2muscarinic receptor and human Gαi-3. Measurements were made in the absence (basal ex-change,  A  ) or presence (  B  ) of 10 μm acetylcholine (ACh) in the assay, and the reactions were terminated 10 min after initiation with [35S]GTPγS. Basal Gαi-3[35S]GTPγS–GDP exchange values are expressed as the percentage of the values measured in the absence of halothane (basal control exchange), whereas the acetylcholine-promoted exchange values are expressed as a percentage of the difference between the basal control values and those measured in the presence of the 10 μm acetylcholine but in the absence of halothane in the assay (  i.e.  , percentage acetylcholine-promoted). Data are presented as mean ± SE of 9–42 assays. * Indicates significant difference from the values obtained in the absence of halothane. 
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Discussion
The major new finding of this study is that halothane, at concentrations typically achieved when administered to patients to treat acute, severe bronchospasm (0.5–1 mm), inhibits the biochemical coupling between the M2muscarinic receptor and Gαi-3without direct inhibition of the intrinsic GTPase activity of the Gαi-3β1γ2HFheterotrimer. Although halothane binds to Gαi-3, this binding either has biochemically significant consequences on the heterotrimer only when it is regulated by the M2muscarinic receptor or has no consequences at all for heterotrimer function.
In the simplest two-state model for heterotrimeric G protein–coupled receptors, the M2muscarinic receptor exists as two conformations, active and inactive.46 In the absence of ligand binding, these two forms are in dynamic equilibrium, with the inactive form being predominant. Receptor agonists induce or select for the active conformation, which promotes (or stabilizes) the nucleotide-free Gαisubunit. The high intracellular ratio of GTP to GDP ensures that activated receptor promotes GTP binding to Gαi, which ultimately reduces its affinity for the receptor and the Gβγ dimer. This results in its dissociation from the receptor–ligand complex. The GTP-bound Gαiis thought to couple to the downstream signaling proteins, such as RhoA,47–49 that increase Ca2+sensitivity in ASM.19,25 Hydrolysis of the bound GTP by the intrinsic GTPase activity of Gαipermits reassociation of the subunits into a heterotrimer and terminates the activation of the signaling cascade that promote Ca2+sensitivity and ASM contraction. Our findings are consistent with this two-state receptor model. Acetylcholine increased the fraction of receptors in the active conformation, as demonstrated by a concentration-dependent increase in the magnitude, but not the rate, of Gαi-3[35S]GTPγS–GDP exchange.
The rationale for conducting the current study was derived from a series of physiologic studies of ASM indicating that the mechanism by which halothane inhibits the acetylcholine-induced increase in Ca2+sensitivity is due, at least in part, to a direct inhibitory effect on the M2muscarinic receptor–Gαiheterotrimeric G-protein complex.16,17,19 For example, in one study, the magnitude of relaxation caused by halothane was partially attenuated by adenosine-5′-diphosphate ribosylation of Gαiwith pertussis toxin.19 Accordingly, the original goal of the current study was to determine, through direct biochemical assessment, whether halothane inhibits guanosine nucleotide exchange at Gαiin porcine ASM and, if so, whether this inhibition depended on whether the Gαiheterotrimer was regulated by the M2muscarinic receptor. However, in a previous study of crude membrane prepared from this tissue, we could not reliably measure acetylcholine-promoted Gαiguanosine nucleotide exchange using the techniques presented in the current study.14 Consequently, we used an experimental approach that investigated anesthetic effects on increasingly more complex protein models beginning with the recombinant Gαi-3monomer and the Gαi-3β1γ2HFheterotrimer (isoforms expressed in ASM) and ending with a well-established, mammalian protein expression model in which the M2muscarinic receptor and Gαi-3heterotrimeric G protein are transiently expressed and are functionally coupled.38 
Halothane (0.5–1.0 mm) had no effect on basal Gαi-3[35S]GTPγS–GDP exchange in membranes prepared from the M2–Gαi-3cotransfected COS-7 cells. This result is consistent with our previous findings that halothane, at similar concentrations, had no effect on the Gαiheterotrimer extracted from porcine ASM membrane in a preparation without functional receptor coupling.27 These data are also consistent with the findings of the current study that halothane, while binding to and causing a small but statistically significant inhibition of the intrinsic GTPase activity of the isolated Gαi-3monomer expressed in Sf  9 insect cells (studied at 3 mm), had no effect on the Gβ1γ2HF-regulated GTPase activity of Gαi-3(i.e.  , the heterotrimer), the biologically relevant protein complex. When interpreted in aggregate, these data suggest that halothane does not directly inhibit guanosine nucleotide exchange at Gαi-3of the Gαi-3β1γ2HFheterotrimeric protein.
Interestingly, we also observed that in contrast to the more clinically relevant halothane concentrations, very high concentrations (3 mm) inhibited basal Gαi-3[35S]GTPγS–GDP exchange in the M2–Gαi-3coexpressing membranes. It is likely that this effect was due to inhibition of the constitutive coupling between endogenous receptors in COS-7 cells and the transiently expressed Gαi-3. This assertion is based on the observations that the basal Gαi-3guanosine exchange was not due to constitutive M2muscarinic receptor activity, because there was no difference in the magnitude of [35S]GTPγS incorporation into Gαi-3between membranes prepared from cells expressing Gαi-3alone or Gαi-3and the M2muscarinic receptor. In addition, treatment of membranes prepared from cells expressing Gαi-3alone with pertussis toxin abolished basal Gαi-3[35S]GTPγS–GDP exchange, implying that the observed basal activity was promoted by constitutively active endogenous receptors.
In contrast to the lack of a direct effect on the intrinsic, receptor-unregulated GTPase activity of the Gαi-3heterotrimer, we found that incubating the crude membrane prepared from the M2–Gαi-3cotransfected cells with 0.5–1 mm halothane caused a concentration-dependent inhibition of Gαi-3[35S]GTPγS–GDP when promoted by acetylcholine binding to the M2muscarinic receptor. These data are similar to our previous studies of crude membrane prepared from differentiated porcine ASM, where halothane and the anesthetic hexanol inhibited acetylcholine-promoted guanosine nucleotide exchange at Gαq/11, but also had no effect on basal exchange.14,26 A possible direct anesthetic effect on native, recombinant Gαqsubfamily protein activity could not be examined in that study because of the essentially unmeasurable intrinsic GTPase activity of these proteins.50 
Protein–anesthetic binding can be determined with STD,28 which is an NMR technique used to probe low-affinity interactions (Kd≈ 10−8to 10−3m) between small molecules and soluble proteins.51 With this technique, samples are alternately irradiated at a frequency that only certain protons within the protein absorb (on-resonance) and a frequency far from resonance with any component of the protein (off-resonance). The absorbed on-resonance radiation (i.e.  , saturation) rapidly distributes throughout the network of protein protons by spin diffusion.52 The saturation is also transferred from the protein protons to the protons of bound molecules (ligands) with a rate that depends on the protein mobility, ligand–protein complex lifetime, and geometry.28 The transfer of saturation to the ligand is detected from the difference spectrum, which is calculated by subtracting the spectrum recorded with on-resonance irradiation from the spectrum collected with off-resonance irradiation. Because the chemical shifts of small molecules are distinctive in high-resolution spectroscopy, it is possible to screen several molecules for binding to a single protein simultaneously.51 
Using STD spectroscopy, we observed saturation transfer from Gαi-3to suramin, a positive control for the technique, and both halothane and hexanol, evidence of anesthetic binding to Gαi-3. However, this binding was not associated with inhibition of the Gαi-3heterotrimer activity. Halothane did not inhibit the GTPase activity of nonmyristoylated Gαi-3purified from E. coli  ; in fact, halothane tended to cause a slight activation at the higher concentrations. In addition, although 3 mm halothane (approximately 15 minimum alveolar concentration) caused a small but statistically significant inhibition of the intrinsic GTPase activity of myristoylated Gαi-3purified from Sf  9 cells, there was no halothane effect on the GTPase activity of the Gαi-3β1γ2HF, the more physiologically and biochemically relevant form. Therefore, halothane binding either has biochemically significant consequences on the heterotrimer only when it is regulated by the M2muscarinic receptor or has no consequences at all for heterotrimer function. When interpreted in aggregate, these data suggest that in this receptor–heterotrimer complex, the salient, biochemically relevant protein target might be M2muscarinic receptor in contrast to the Gαi-3heterotrimer. This assertion is consistent with previous studies showing photoaffinity labeling of [14C]halothane to a specific tryptophan of the prototypical G protein–coupled receptor, rhodopsin.53,54 
In summary, this study demonstrated for the first time through direct biochemical assessment that halothane, in concentrations used clinically to treat severe bronchospasm, interacts with the M2muscarinic receptor–Gαi-3heterotrimeric complex in a manner that prevents acetylcholine-promoted guanosine nucleotide exchange the Gαi-3. This novel effect of halothane was observed at near maximal activation of the M2muscarinic receptor (10 μm acetylcholine), a finding consistent with the ability of halothane to inhibit isolated ASM contraction even when maximally stimulated9 and to cause direct bronchodilation of airways.5,6 Halothane had no direct inhibitory effect on the intrinsic biochemical activity of the Gαi-3b1g2HFheterotrimer, despite evidence of anesthetic binding to the Gαi-3subunit.
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Fig. 1. Coomassie brilliant blue stains of sodium dodecyl sulfate polyacrylamide gels of the α isoform-3 G-protein subunit (Gαi-3) expressed in and purified from  Escherichia coli  bacteria (  lane 3  ) or  Spodoptera frugiperda  (S  f  9) insect cells (  lane 2  ).  Lane 1  shows the same for the heterotrimer composed of Gαi-3, and the untagged β isoform-1 (β1) and amino-terminally hexahistadine-tagged, Flag-tagged γ isoform-2 (γ2HF) G-protein subunits (Gαi-3β1γ2HF) expressed in and purified from S  f  9 insect cells. The molecular weight (MW) marker ladder is shown in the  left lane  . 
Fig. 1. Coomassie brilliant blue stains of sodium dodecyl sulfate polyacrylamide gels of the α isoform-3 G-protein subunit (Gαi-3) expressed in and purified from  Escherichia coli  bacteria (  lane 3  ) or  Spodoptera frugiperda  (S  f  9) insect cells (  lane 2  ).  Lane 1  shows the same for the heterotrimer composed of Gαi-3, and the untagged β isoform-1 (β1) and amino-terminally hexahistadine-tagged, Flag-tagged γ isoform-2 (γ2HF) G-protein subunits (Gαi-3β1γ2HF) expressed in and purified from S  f  9 insect cells. The molecular weight (MW) marker ladder is shown in the  left lane  . 
Fig. 1. Coomassie brilliant blue stains of sodium dodecyl sulfate polyacrylamide gels of the α isoform-3 G-protein subunit (Gαi-3) expressed in and purified from  Escherichia coli  bacteria (  lane 3  ) or  Spodoptera frugiperda  (S  f  9) insect cells (  lane 2  ).  Lane 1  shows the same for the heterotrimer composed of Gαi-3, and the untagged β isoform-1 (β1) and amino-terminally hexahistadine-tagged, Flag-tagged γ isoform-2 (γ2HF) G-protein subunits (Gαi-3β1γ2HF) expressed in and purified from S  f  9 insect cells. The molecular weight (MW) marker ladder is shown in the  left lane  . 
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Fig. 2. Representative saturation transfer difference (  A  ) and one-dimensional nuclear magnetic resonance (  B  ) spectra recorded for a solution containing 1 mm suramin (S), 1 mm halothane (H), 10 mm hexanol (Hx), and the α isoform-3 G-protein subunit (Gαi-3). Gαi-3was expressed in and purified from  Spodoptera frugiperda  insect cells. The region of the saturation transfer difference spectra where deuterated water (HOD) absorbs (approximately 4.8 ppm) was deleted from the figure for clarity of presentation. 
Fig. 2. Representative saturation transfer difference (  A  ) and one-dimensional nuclear magnetic resonance (  B  ) spectra recorded for a solution containing 1 mm suramin (S), 1 mm halothane (H), 10 mm hexanol (Hx), and the α isoform-3 G-protein subunit (Gαi-3). Gαi-3was expressed in and purified from  Spodoptera frugiperda  insect cells. The region of the saturation transfer difference spectra where deuterated water (HOD) absorbs (approximately 4.8 ppm) was deleted from the figure for clarity of presentation. 
Fig. 2. Representative saturation transfer difference (  A  ) and one-dimensional nuclear magnetic resonance (  B  ) spectra recorded for a solution containing 1 mm suramin (S), 1 mm halothane (H), 10 mm hexanol (Hx), and the α isoform-3 G-protein subunit (Gαi-3). Gαi-3was expressed in and purified from  Spodoptera frugiperda  insect cells. The region of the saturation transfer difference spectra where deuterated water (HOD) absorbs (approximately 4.8 ppm) was deleted from the figure for clarity of presentation. 
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Fig. 3. Concentration-dependent effect of halothane on guanosine-5′-triphosphate (GTP) hydrolysis rate (GTPase activity) of the nonmyristoylated α isoform-3 G-protein subunit (Gαi-3) expressed in and purified from E coli. GTPase activity was determined using a standard phosphohydrolase assay with radiolabeled [γ32Pi]GTP as described in detail in the Materials and Methods. Assays were performed in the absence or presence of 0.5, 0.75, 1.0, and 3.0 mm halothane. Data are presented as mean ± SE of 3–14 assays. * Significant difference from values measured in the absence of halothane. 
Fig. 3. Concentration-dependent effect of halothane on guanosine-5′-triphosphate (GTP) hydrolysis rate (GTPase activity) of the nonmyristoylated α isoform-3 G-protein subunit (Gαi-3) expressed in and purified from E coli. GTPase activity was determined using a standard phosphohydrolase assay with radiolabeled [γ32Pi]GTP as described in detail in the Materials and Methods. Assays were performed in the absence or presence of 0.5, 0.75, 1.0, and 3.0 mm halothane. Data are presented as mean ± SE of 3–14 assays. * Significant difference from values measured in the absence of halothane. 
Fig. 3. Concentration-dependent effect of halothane on guanosine-5′-triphosphate (GTP) hydrolysis rate (GTPase activity) of the nonmyristoylated α isoform-3 G-protein subunit (Gαi-3) expressed in and purified from E coli. GTPase activity was determined using a standard phosphohydrolase assay with radiolabeled [γ32Pi]GTP as described in detail in the Materials and Methods. Assays were performed in the absence or presence of 0.5, 0.75, 1.0, and 3.0 mm halothane. Data are presented as mean ± SE of 3–14 assays. * Significant difference from values measured in the absence of halothane. 
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Fig. 4. Effect of halothane on guanosine-5′-triphosphate (GTP) hydrolysis rate (GTPase activity) of the α isoform-3 G-protein subunit (Gαi-3) and the heterotrimer composed of Gαi-3, and the untagged β isoform-1 (β1) and amino-terminus hexahistadine-tagged, Flag-tagged γ isoform-2 G-protein subunits (Gαi-3β1γ2HF). Proteins were expressed in and purified from  Spodoptera frugiperda  insect cells. GTPase activity was determined using a standard phosphohydrolase assay with radiolabeled [g32Pi]GTP as described in detail in the Materials and Methods. Assays were performed in the absence (control) or presence of 3 mm halothane. Data are presented as mean ± SE of 9–12 assays. * Significant difference from values measured in the absence of halothane (control). 
Fig. 4. Effect of halothane on guanosine-5′-triphosphate (GTP) hydrolysis rate (GTPase activity) of the α isoform-3 G-protein subunit (Gαi-3) and the heterotrimer composed of Gαi-3, and the untagged β isoform-1 (β1) and amino-terminus hexahistadine-tagged, Flag-tagged γ isoform-2 G-protein subunits (Gαi-3β1γ2HF). Proteins were expressed in and purified from  Spodoptera frugiperda  insect cells. GTPase activity was determined using a standard phosphohydrolase assay with radiolabeled [g32Pi]GTP as described in detail in the Materials and Methods. Assays were performed in the absence (control) or presence of 3 mm halothane. Data are presented as mean ± SE of 9–12 assays. * Significant difference from values measured in the absence of halothane (control). 
Fig. 4. Effect of halothane on guanosine-5′-triphosphate (GTP) hydrolysis rate (GTPase activity) of the α isoform-3 G-protein subunit (Gαi-3) and the heterotrimer composed of Gαi-3, and the untagged β isoform-1 (β1) and amino-terminus hexahistadine-tagged, Flag-tagged γ isoform-2 G-protein subunits (Gαi-3β1γ2HF). Proteins were expressed in and purified from  Spodoptera frugiperda  insect cells. GTPase activity was determined using a standard phosphohydrolase assay with radiolabeled [g32Pi]GTP as described in detail in the Materials and Methods. Assays were performed in the absence (control) or presence of 3 mm halothane. Data are presented as mean ± SE of 9–12 assays. * Significant difference from values measured in the absence of halothane (control). 
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Fig. 5. Effect of acetylcholine on the exchange of the radioactive, nonhydrolyzable form of guanosine-5′-triphosphate, [35S]GTPγS, for guanosine-5′-diphosphate ([35S]GTPγS–GDP exchange) at the α isoform-3 G-protein subunit (Gαi-3). Assays were performed using crude membranes prepared from COS-7 cells transfected with the complementary DNA that encodes for human Gαi-3only, or human M2muscarinic receptor and human Gαi-3. Measurements were made in the absence (basal exchange) and presence of 10 μm acetylcholine and were terminated 10 min after initiation of the assays with [35S]GTPγS. The immunoprecipitation step of the assays was performed using nonimmune serum (for nonspecific background measurements) or antiserum specific for Gαi-3. Data are presented as mean ± SE of six assays. * Significant difference from background radioactivity. † Significant difference from basal Gα[35S]GTPγS–GDP exchange. 
Fig. 5. Effect of acetylcholine on the exchange of the radioactive, nonhydrolyzable form of guanosine-5′-triphosphate, [35S]GTPγS, for guanosine-5′-diphosphate ([35S]GTPγS–GDP exchange) at the α isoform-3 G-protein subunit (Gαi-3). Assays were performed using crude membranes prepared from COS-7 cells transfected with the complementary DNA that encodes for human Gαi-3only, or human M2muscarinic receptor and human Gαi-3. Measurements were made in the absence (basal exchange) and presence of 10 μm acetylcholine and were terminated 10 min after initiation of the assays with [35S]GTPγS. The immunoprecipitation step of the assays was performed using nonimmune serum (for nonspecific background measurements) or antiserum specific for Gαi-3. Data are presented as mean ± SE of six assays. * Significant difference from background radioactivity. † Significant difference from basal Gα[35S]GTPγS–GDP exchange. 
Fig. 5. Effect of acetylcholine on the exchange of the radioactive, nonhydrolyzable form of guanosine-5′-triphosphate, [35S]GTPγS, for guanosine-5′-diphosphate ([35S]GTPγS–GDP exchange) at the α isoform-3 G-protein subunit (Gαi-3). Assays were performed using crude membranes prepared from COS-7 cells transfected with the complementary DNA that encodes for human Gαi-3only, or human M2muscarinic receptor and human Gαi-3. Measurements were made in the absence (basal exchange) and presence of 10 μm acetylcholine and were terminated 10 min after initiation of the assays with [35S]GTPγS. The immunoprecipitation step of the assays was performed using nonimmune serum (for nonspecific background measurements) or antiserum specific for Gαi-3. Data are presented as mean ± SE of six assays. * Significant difference from background radioactivity. † Significant difference from basal Gα[35S]GTPγS–GDP exchange. 
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Fig. 6. (  A  ) Time-dependent change in the exchange of the radioactive, nonhydrolyzable form of guanosine-5′-triphosphate, [35S]GTPγS, for guanosine-5′-diphosphate ([35S]GTPγS–GDP exchange) at the α isoform-3 G-protein subunit (Gαi-3). Assays were performed using crude membranes prepared from COS-7 cells cotransfected with the complementary DNA constructs encoding for the human M2muscarinic receptor and human Gαi-3. Gαi-3[35S]GTPγS–GDP exchange was measured in the absence (basal exchange) or presence of 10 μm acetylcholine in the assay. The reactions were terminated 1, 2, 5, and 10 min after initiation of the assays with [35S]GTPγS. Data are presented as mean ± SE of six assays. (  B  ) Concentration-dependent effect of acetylcholine on Gαi-3[35S]GTPγS–GDP exchange measured in crude membranes prepared from M2–Gαi-3cotransfected COS-7 cells. Assays were performed in the absence (basal exchange) and presence of acetylcholine (ACh, 0.001–100 μm) in the assays and the reactions were terminated 10 min after initiation with [35S]GTPγS. Data are the percentage of the difference between the basal exchange values and those measured in the presence of the acetylcholine concentration that produced the maximal effect. Data are presented as mean ± SE of four assays. 
Fig. 6. (  A  ) Time-dependent change in the exchange of the radioactive, nonhydrolyzable form of guanosine-5′-triphosphate, [35S]GTPγS, for guanosine-5′-diphosphate ([35S]GTPγS–GDP exchange) at the α isoform-3 G-protein subunit (Gαi-3). Assays were performed using crude membranes prepared from COS-7 cells cotransfected with the complementary DNA constructs encoding for the human M2muscarinic receptor and human Gαi-3. Gαi-3[35S]GTPγS–GDP exchange was measured in the absence (basal exchange) or presence of 10 μm acetylcholine in the assay. The reactions were terminated 1, 2, 5, and 10 min after initiation of the assays with [35S]GTPγS. Data are presented as mean ± SE of six assays. (  B  ) Concentration-dependent effect of acetylcholine on Gαi-3[35S]GTPγS–GDP exchange measured in crude membranes prepared from M2–Gαi-3cotransfected COS-7 cells. Assays were performed in the absence (basal exchange) and presence of acetylcholine (ACh, 0.001–100 μm) in the assays and the reactions were terminated 10 min after initiation with [35S]GTPγS. Data are the percentage of the difference between the basal exchange values and those measured in the presence of the acetylcholine concentration that produced the maximal effect. Data are presented as mean ± SE of four assays. 
Fig. 6. (  A  ) Time-dependent change in the exchange of the radioactive, nonhydrolyzable form of guanosine-5′-triphosphate, [35S]GTPγS, for guanosine-5′-diphosphate ([35S]GTPγS–GDP exchange) at the α isoform-3 G-protein subunit (Gαi-3). Assays were performed using crude membranes prepared from COS-7 cells cotransfected with the complementary DNA constructs encoding for the human M2muscarinic receptor and human Gαi-3. Gαi-3[35S]GTPγS–GDP exchange was measured in the absence (basal exchange) or presence of 10 μm acetylcholine in the assay. The reactions were terminated 1, 2, 5, and 10 min after initiation of the assays with [35S]GTPγS. Data are presented as mean ± SE of six assays. (  B  ) Concentration-dependent effect of acetylcholine on Gαi-3[35S]GTPγS–GDP exchange measured in crude membranes prepared from M2–Gαi-3cotransfected COS-7 cells. Assays were performed in the absence (basal exchange) and presence of acetylcholine (ACh, 0.001–100 μm) in the assays and the reactions were terminated 10 min after initiation with [35S]GTPγS. Data are the percentage of the difference between the basal exchange values and those measured in the presence of the acetylcholine concentration that produced the maximal effect. Data are presented as mean ± SE of four assays. 
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Fig. 7. Concentration-dependent effect of halothane on the exchange of the radioactive, nonhydrolyzable form of gua-nosine-5′-triphosphate, [35S]GTPγS, for guanosine-5′-diphosphate ([35S]GTPγS–GDP exchange) at the α isoform-3 G-protein subunit (Gαi-3). Assays were performed using crude membranes prepared from COS-7 cells cotransfected with the complementary DNA constructs encoding for the human M2muscarinic receptor and human Gαi-3. Measurements were made in the absence (basal ex-change,  A  ) or presence (  B  ) of 10 μm acetylcholine (ACh) in the assay, and the reactions were terminated 10 min after initiation with [35S]GTPγS. Basal Gαi-3[35S]GTPγS–GDP exchange values are expressed as the percentage of the values measured in the absence of halothane (basal control exchange), whereas the acetylcholine-promoted exchange values are expressed as a percentage of the difference between the basal control values and those measured in the presence of the 10 μm acetylcholine but in the absence of halothane in the assay (  i.e.  , percentage acetylcholine-promoted). Data are presented as mean ± SE of 9–42 assays. * Indicates significant difference from the values obtained in the absence of halothane. 
Fig. 7. Concentration-dependent effect of halothane on the exchange of the radioactive, nonhydrolyzable form of gua-nosine-5′-triphosphate, [35S]GTPγS, for guanosine-5′-diphosphate ([35S]GTPγS–GDP exchange) at the α isoform-3 G-protein subunit (Gαi-3). Assays were performed using crude membranes prepared from COS-7 cells cotransfected with the complementary DNA constructs encoding for the human M2muscarinic receptor and human Gαi-3. Measurements were made in the absence (basal ex-change,  A  ) or presence (  B  ) of 10 μm acetylcholine (ACh) in the assay, and the reactions were terminated 10 min after initiation with [35S]GTPγS. Basal Gαi-3[35S]GTPγS–GDP exchange values are expressed as the percentage of the values measured in the absence of halothane (basal control exchange), whereas the acetylcholine-promoted exchange values are expressed as a percentage of the difference between the basal control values and those measured in the presence of the 10 μm acetylcholine but in the absence of halothane in the assay (  i.e.  , percentage acetylcholine-promoted). Data are presented as mean ± SE of 9–42 assays. * Indicates significant difference from the values obtained in the absence of halothane. 
Fig. 7. Concentration-dependent effect of halothane on the exchange of the radioactive, nonhydrolyzable form of gua-nosine-5′-triphosphate, [35S]GTPγS, for guanosine-5′-diphosphate ([35S]GTPγS–GDP exchange) at the α isoform-3 G-protein subunit (Gαi-3). Assays were performed using crude membranes prepared from COS-7 cells cotransfected with the complementary DNA constructs encoding for the human M2muscarinic receptor and human Gαi-3. Measurements were made in the absence (basal ex-change,  A  ) or presence (  B  ) of 10 μm acetylcholine (ACh) in the assay, and the reactions were terminated 10 min after initiation with [35S]GTPγS. Basal Gαi-3[35S]GTPγS–GDP exchange values are expressed as the percentage of the values measured in the absence of halothane (basal control exchange), whereas the acetylcholine-promoted exchange values are expressed as a percentage of the difference between the basal control values and those measured in the presence of the 10 μm acetylcholine but in the absence of halothane in the assay (  i.e.  , percentage acetylcholine-promoted). Data are presented as mean ± SE of 9–42 assays. * Indicates significant difference from the values obtained in the absence of halothane. 
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