Effect of Halothane on Gα^i-3and Its Coupling to the M²Muscarinic Receptor

A plasmid encoding human Gα^i-3in the mammalian expression vector pcDNA3.1 was obtained from the University of Missouri-Rolla cDNA Resource Center (Rolla, Missouri).∥Gα^i-3was expressed in Escherichia coli  and purified by conventional chromatography as previously described,29with minimal modifications. Briefly, the Gα^i-3insert was excised from the pcDNA3.1:Gα^i-3construct and ligated to pET28a plasmid. pET28a:Gα^i-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.29Purified Gα^i-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).

Baculoviruses containing untagged G-protein β¹(β¹) and amino-terminally hexahistadine-tagged, Flag-tagged G-protein γ²(γ^2HF) were generous gifts from James C. Garrison, Ph.D. (Professor, Department of Pharmacology, University of Virginia, Charlottesville, Virginia). To generate a baculovirus containing Gα^i-3, the pcDNA3.1:Gα^i-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:Gα^i-3was digested with Pst  I and Xba  I, and the resulting Gα^i-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).

Sf  9 cells were coinfected with the recombinant baculoviruses encoding Gα^i-3, β¹, and γ^2HF. 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 Gα^i-3, β¹, and γ^2HF, 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.

Membrane preparation and protein purification were performed, with minor modifications, according to the methods of Kozasa.30Cell 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.

Gα^i-3was eluted from the β¹γ^2HFdimer 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 Gα^i-3β¹γ^2HFheterotrimer 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 Gα^i-3or Gα^i-3β¹γ^2HFwere 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.

One-dimensional nuclear magnetic resonance (¹H NMR) and saturation transfer difference (STD)31spectra 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.28This 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 Gαⁱactivity32and was used as a positive control for ligand–protein binding. Then the spectra of these compounds were recorded in the presence of 40 μm Gα^i-3.

Guanosine-5′-triphosphatase (GTPase) activity was measured using a standard phosphohydrolase assay with radioactive inorganic phosphate ([³²Pi])–labeled GTP33as previously described by our laboratory.27,34Gα^i-3or Gα^i-3β¹γ^2HF(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.

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 Gα^i-3cDNA were used, and for transfection of Gα^i-3alone, 5 μg Gα^i-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.

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.35The 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.

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).36,37Specific 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–Gα^i-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.

Gα^i-3guanosine nucleotide exchange was assayed in crude membrane prepared from M²–Gα^i-3cotransfected COS-7 cells using previously described methods.38Reaction 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 Gα^i-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 Gα^i-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 Gα^i-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.

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.14,26–28After 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).39In 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).

To determine the dependence of the Gα^i-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 Gα^i-3only, or both the M²muscarinic receptor and Gα^i-3. The crude membranes were incubated without (for basal Gα^i-3[³⁵S]GTPγS–GDP exchange measurements) or with (for acetylcholine-promoted Gα^i-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-Gα^i-3immunoglobulin G. These data were not subjected to nonspecific background subtraction.

These protocols were conducted to help guide the experimental approach of subsequent protocols that examined possible anesthetic effects on Gα^i-3[³⁵S]GTPγS–GDP exchange. To determine the time course for Gα^i-3[³⁵S]GTPγS–GDP exchange, crude membrane prepared from M²–Gα^i-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 Gα^i-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 Gα^i-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).

Crude membranes prepared from M²–Gα^i-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 Gα^i-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,14,26thereby allowing comparisons with these previous data. The reactions were terminated at a time determined to produce an approximately 70–80% increase in maximal Gα^i-3[³⁵S]GTPγS–GDP exchange, and then the samples were subjected to the immunoprecipitation step of the assay. Basal Gα^i-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 Gα^i-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).

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 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 Gα^i-3[³⁵S]GTPγS–GDP exchange were determined by nonlinear regression analysis as previously described (SigmaPlot 8; Systat Software, Point Richmond, CA).14,26,40Data 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.

Saturation transfer from Gα^i-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 Gα^i-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,31the 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.

Gα^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.41,42The presence of halothane in the assay tended to cause a slight increase in Gα^i-3GTPase activity, reaching statistical significance only at 0.75 and 3 mm halothane (fig. 3).

The GTPase activities of the Gα^i-3and Gα^i-3β¹γ^2HFheterotrimer expressed in and purified from membranes of Sf  9 insect cells were also comparable to those previously reported.43As expected, the GTPase activity of the heterotrimer was approximately fourfold greater than that of the Gα^i-3subunit (0.0158 ± 0.009 and 0.0619 ± 0.0141 pmol ³²Pi · pmol protein⁻¹· min⁻¹for Gα^i-3and Gα^i-3β¹γ^2HF, respectively).43The presence of 3 mm halothane in the assay had no significant effect on the GTPase activity of the Gα^i-3β¹γ^2HF, whereas it caused a small but significant inhibition of Gα^i-3GTPase activity (fig. 4).

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.44,45However, the membranes prepared from COS-7 cells cotransfected with the cDNA constructs encoding for the M²muscarinic receptor and Gα^i-3expressed the M²muscarinic receptor at 2–3.4 pmol/mg protein. In crude membranes prepared from Gα^i-3-only transfected cells or M²–Gα^i-3cotransfected cells, there was a significant increase in Gα^i-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 Gα^i-3did not promote an additional increase in this basal Gα^i-3[³⁵S]GTPγS–GDP exchange (fig. 5). These data indicate that the expressed M²muscarinic receptor does not possess constitutive activity, as described by others,38but rather reflects the constitutive activity of endogenously expressed receptors that are functionally coupled to the expressed Gα^i-3. This assertion is supported by the finding that adenosine-5′-diphosphate ribosylation of Gα^i-3by pertussis toxin in crude membranes prepared from Gα^i-3-alone transfected cells abolished the Gα^i-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–Gα^i-3cotransfected cells caused a further approximately twofold increase in the magnitude of the Gα^i-3-specific [³⁵S]GTPγS–GDP exchange (fig. 5). However, when the receptor was omitted from the transfection (i.e. , crude membranes prepared from Gα^i-3-alone transfected cells), acetylcholine did not promote Gα^i-3[³⁵S]GTPγS–GDP exchange above the basal exchange levels.

In the absence of acetylcholine in the assay, membranes prepared from M²–Gα^i-3cotransfected cells incorporated [³⁵S]GTPγS into Gα^i-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 Gα^i-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 Gα^i-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 Gα^i-3[³⁵S]GTPγS–GDP exchange were performed using 10 μm acetylcholine to optimize the ability to detect modest shifts in acetylcholine-promoted Gα^i-3[³⁵S]GTPγS–GDP exchange and were measured after 10 min of stimulation.

The presence of 0.5 or 1 mm halothane in the assay had no significant effect on basal Gα^i-3[³⁵S]GTPγS–GDP exchange measured in the absence of acetylcholine; however, 3 mm halothane inhibited basal Gα^i-3[³⁵S]GTPγS–GDP exchange by approximately 40% (fig. 7A). Halothane caused a concentration-dependent inhibition of Gα^i-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.

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 Gα^i-3without direct inhibition of the intrinsic GTPase activity of the Gα^i-3β¹γ^2HFheterotrimer. Although halothane binds to Gα^i-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.46In 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,47–49that increase Ca²⁺sensitivity in ASM.19,25Hydrolysis 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 Gα^i-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.16,17,19For example, in one study, the magnitude of relaxation caused by halothane was partially attenuated by adenosine-5′-diphosphate ribosylation of Gαⁱwith pertussis toxin.19Accordingly, 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.14Consequently, we used an experimental approach that investigated anesthetic effects on increasingly more complex protein models beginning with the recombinant Gα^i-3monomer and the Gα^i-3β¹γ^2HFheterotrimer (isoforms expressed in ASM) and ending with a well-established, mammalian protein expression model in which the M²muscarinic receptor and Gα^i-3heterotrimeric G protein are transiently expressed and are functionally coupled.38 

Halothane (0.5–1.0 mm) had no effect on basal Gα^i-3[³⁵S]GTPγS–GDP exchange in membranes prepared from the M²–Gα^i-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.27These 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 Gα^i-3monomer expressed in Sf  9 insect cells (studied at 3 mm), had no effect on the Gβ¹γ^2HF-regulated GTPase activity of Gα^i-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 Gα^i-3of the Gα^i-3β¹γ^2HFheterotrimeric protein.

Interestingly, we also observed that in contrast to the more clinically relevant halothane concentrations, very high concentrations (3 mm) inhibited basal Gα^i-3[³⁵S]GTPγS–GDP exchange in the M²–Gα^i-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 Gα^i-3. This assertion is based on the observations that the basal Gα^i-3guanosine exchange was not due to constitutive M²muscarinic receptor activity, because there was no difference in the magnitude of [³⁵S]GTPγS incorporation into Gα^i-3between membranes prepared from cells expressing Gα^i-3alone or Gα^i-3and the M²muscarinic receptor. In addition, treatment of membranes prepared from cells expressing Gα^i-3alone with pertussis toxin abolished basal Gα^i-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 Gα^i-3heterotrimer, we found that incubating the crude membrane prepared from the M²–Gα^i-3cotransfected cells with 0.5–1 mm halothane caused a concentration-dependent inhibition of Gα^i-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 Gα^q/11, but also had no effect on basal exchange.14,26A possible direct anesthetic effect on native, recombinant Gα^qsubfamily 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,28which is an NMR technique used to probe low-affinity interactions (K^d≈ 10⁻⁸to 10⁻³m) between small molecules and soluble proteins.51With 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.52The 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.28The 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 Gα^i-3to suramin, a positive control for the technique, and both halothane and hexanol, evidence of anesthetic binding to Gα^i-3. However, this binding was not associated with inhibition of the Gα^i-3heterotrimer activity. Halothane did not inhibit the GTPase activity of nonmyristoylated Gα^i-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 Gα^i-3purified from Sf  9 cells, there was no halothane effect on the GTPase activity of the Gα^i-3β¹γ^2HF, 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 Gα^i-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–Gα^i-3heterotrimeric complex in a manner that prevents acetylcholine-promoted guanosine nucleotide exchange the Gα^i-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 stimulated9and to cause direct bronchodilation of airways.5,6Halothane had no direct inhibitory effect on the intrinsic biochemical activity of the Gα^i-3b¹g^2HFheterotrimer, despite evidence of anesthetic binding to the Gα^i-3subunit.