ACCELERATED COMMUNICATION G Protein-Coupled Receptors as Direct Targets of Inhaled Anesthetics

The molecular pharmacology of inhalational anesthetics remains poorly understood. Despite accumulating evidence suggesting that neuronal membrane proteins are potential targets of inhaled anesthetics, most currently favored membrane protein targets lack any direct evidence for anesthetic binding. We report herein the location of the binding site for the inhaled anesthetic halothane at the amino acid residue level of resolution in the ligand binding cavity in a prototypical G proteincoupled receptor, bovine rhodopsin. Tryptophan fluorescence quenching and direct photoaffinity labeling with [C]halothane suggested an interhelical location of halothane with a stoichiometry of 1 (halothane/rhodopsin molar ratio). Radiosequence analysis of [C]halothane-labeled rhodopsin revealed that halothane contacts an amino acid residue (Trp265) lining the ligand binding cavity in the transmembrane core of the receptor. The predicted functional consequence, competition between halothane and the ligand retinal, was shown here by spectroscopy and is known to exist in vivo. These data suggest that competition with endogenous ligands may be a general mechanism of the action of halothane at this large family of signaling proteins. The mechanisms of general anesthetic action at the molecular level remain poorly understood, despite their use in millions of patients each year. Understanding the molecular mechanisms by which inhaled anesthetics produce behavioral effects, such as loss of consciousness and analgesia, is thus an important goal with therapeutic implications. Accumulating evidence suggests that these drugs act at multiple neuronal membrane proteins that function as ion channels and neurotransmitter receptors (Franks and Lieb, 1994). However, classification as an anesthetic target requires evidence of direct binding, and most currently favored targets lack any direct evidence for anesthetic binding. One of the major difficulties in demonstrating direct binding is the weak binding energetics of the inhaled anesthetic (Eckenhoff and Johansson, 1997). Weak binding, although consistent with the relatively featureless molecules and the high aqueous EC50 for general anesthesia in mammals (0.2–1.0 mM) (Franks and Lieb, 1994), essentially precludes conventional radioligand binding studies. Furthermore, there have been few good model systems for studying the actions of inhaled anesthetics in biological membranes (Forman et al., 1997). In addition to plausible roles in central nervous system signaling, it is important for potential model proteins to be available in sufficient abundance and purity to permit direct binding and high-resolution structural studies. A large superfamily of G protein-coupled receptors (GPCRs) modulates most signaling in central and peripheral nervous systems. In particular, the rhodopsin family of GPCRs includes many neurotransmitter receptors, such as muscarinic acetylcholine, noradrenaline, dopamine, adenosine, and opioid receptors (Baldwin et al., 1997). These receptors have highly conserved regions in the transmembrane portion (Baldwin et al., 1997), and the ligand-receptor interactions in the core formed by the seven -helices are thought to be similar in the GPCRs of this family (Strader et al., 1994; Ji et al., 1998). Functionally, cholinergic neurotransmission is known to influence awareness, sleep, and learning and memory (Durieux, 1996). The 2-adrenergic receptor seems to play a role in antinociceptive responses as well as in the This work was supported by National Institutes of Health grants GM51595 and EY00012. ABBREVIATIONS: GPCR, G protein-coupled receptor; RDM, rod disk membranes; MOPS, 3-(N-morpholino)propanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; PTH, phenylthiohydantoin; NATA, N-acetyl-tryptophan-amide; PKC, protein kinase C. 0026-895X/02/6105-945–952$7.00 MOLECULAR PHARMACOLOGY Vol. 61, No. 5 Copyright © 2002 The American Society for Pharmacology and Experimental Therapeutics 1502/979256 Mol Pharmacol 61:945–952, 2002 Printed in U.S.A. 945 at A PE T Jornals on A ril 4, 2017 m oharm .aspeurnals.org D ow nladed from state of arousal (Bol et al., 1999). In fact, agonists and/or antagonists that work through these GPCRs have been reported to significantly alter anesthetic requirements in humans and animals (Segal et al., 1988; Seitz et al., 1990; Glass et al., 1997; Ishizawa et al., 2000a). Although this might include unrelated, parallel effects on the central nervous system, recent studies show that inhaled anesthetics can interfere with GPCR signaling in vitro (Durieux, 1995; Honemann et al., 1998; Schotten et al., 1998), suggesting direct anesthetic effects. Halothane, a clinically used volatile anesthetic, has two features that allow monitoring of binding. First, the photolabile carbon-bromine bond allows photolabeling (Eckenhoff and Johansson, 1997); second, the bromine atom can quench intrinsic protein fluorescence if it is near the fluorophore (Johansson et al., 1995). Both features allow determination of location of the anesthetic within the protein matrix. Because the abundance of rhodopsin in native retinal membrane preparation facilitates direct binding approaches, we used bovine rhodopsin as a structural homolog for other neuronal GPCRs to characterize the binding domain for this inhaled anesthetic. We reported previously that halothane binds to rhodopsin but not to its associated G protein (Ishizawa et al., 2000b). In this study, using a higher resolution approach, we provide evidence for halothane binding to the endogenous ligand binding site in rhodopsin. Materials and Methods Rod Disk Membranes Preparation. Fresh bovine retinas were dissected in room light. Rod disk membranes (RDM) were prepared by sucrose flotation in isotonic buffer (20 mM MOPS, 100 mM KCl, 6 mM MgCl2, pH 7.0). Peripheral proteins were stripped by washing in hypotonic buffer (10 mM MOPS, 2 mM MgCl2, 100 M GTP, pH 7.0) (Panico et al., 1990). Estimated molar ratio of the protein in the RDM was 24:1 (rhodopsin/transducin) based on the relative mass in SDSPAGE using reflective density (GS-710; Bio-Rad Laboratories, Hercules, CA). Molar ratio of tryptophan residues was then 12:1 (rhodopsin/transducin). Tryptophan residues in cyclic GMP phosphodiesterase and arrestin were estimated to be lower than 3% and 0.5% of the total residues in RDM, respectively. The RDM were regenerated using 3-fold molar excess of 9-cis-retinal (Sigma Chemical Co., St. Louis, MO) for 12 h on ice in the dark followed by a further 1 h incubation at room temperature, as reported previously, to provide almost 100% of chromophore regeneration (Gibson et al., 1998). Steady-State Fluorescence and Absorption Spectra. All fluorescence measurements were performed with a spectrofluorophotometer (RF-5301PC; Shimadzu Scientific Instruments, Inc. Columbia, MD) using a 10-mm-pathlength quartz cell at 25°C. Bleached and regenerated RDM samples were equilibrated in sodium phosphate buffer (130 mM NaCl and 20 mM Na2HPO4, pH 7.0) at a rhodopsin concentration of 0.5 M with increasing concentrations of halothane (0–15 mM) in a gas-tight 4.0-ml cell. RDM samples were excited at 295 nm to probe tryptophan fluorescence. For the fluorescence time-based measurements, the RDM samples were equilibrated in sodium phosphate buffer at a rhodopsin concentration of 0.5 M with increasing concentrations of halothane (0–6.0 mM). Then the RDM were excited with 295 nm light and 3-fold molar excess of 9-cis-retinal (1.5 l) was added to the sample cell (4.0 ml) with continuous stirring. Data were recorded at 330 nm at 30-s intervals for 60 min, with the excitation shutter closed between acquisitions to minimize exposure to the excitation beam. Halothane and 9-cis-retinal, the two principle ligands in this study, do not absorb appreciably at the excitation (295 nm) or emission ( 330 nm) wavelength for tryptophan, so inner filter corrections were not performed. All UV/visible spectra were measured with a spectrophotometer (Cary300Bio; Varian Instruments, Walnut Creek, CA) using a 10mm-pathlength 1.8-ml quartz cell at 25°C. RDM samples were equilibrated in sodium phosphate buffer at a rhodopsin concentration of 5.0 M with increasing concentrations of halothane (0–4.0 mM). The time course of the increase in absorbance at 487 nm after addition of 9-cis-retinal (3-fold molar excess) was measured for 180 min. Photoaffinity Labeling of [C]Halothane. Bleached and regenerated RDM samples were incubated with [C]halothane (2bromo-2-chloro-1,1,1-[1-C]trifluoroethane; specific activity, 51 mCi/mmol; PerkinElmer Life Sciences, Boston, MA) and with increasing concentrations of unlabeled halothane in isotonic MOPS buffer in 2-ml quartz cuvettes at 25°C. The samples were exposed to 254-nm light at a distance of 5 mm for 30 s with continuous stirring. The final concentrations in the photolabeling solution were 1.5 M rhodopsin, 9.7 M [C]halothane, and 0 to 8.5 mM unlabeled halothane. SDS-PAGE of the labeled membranes was performed in modified Laemmli gels, and the gels were stained with Coomassie Brilliant Blue. The relative protein mass in the rhodopsin bands on SDS-PAGE was determined by reflective density. Then rhodopsin bands in the dried gels were excised and dissolved by incubating with 30% hydrogen peroxide at 60°C for 5 h. Stoichiometry of label incorporation into opsin or isorhodopsin was determined in the dissolved gel slices by scintillation counting, and disintegrations per minute were normalized to relative mass of rhodopsin. For proteolytic digestion and radiosequence analysis, the bleached and regenerated RDM samples were incubated with [C]halothane at 0.75 mM in isotonic MOPS buffer. The samples were exposed to 254-nm light for 40 s. The labeled samples were washed with the buffer, and the pellets were then used for proteolytic digestion. Proteolysis and Radiosequence Analysis. The pellets of the photolabeled RDM were resuspended in 15 mM Tris, 0.1% SDS, pH 8.1. For digestion with Staphylococcus aureus glutamyl endopeptidase (V8 protease; ICN Biomedicals Inc., Aurora, OH), V8 protease was added to the final concentration of 1:1 (w/w) protease/rhodopsin into the sample solution and incubated at 37°C for 3 h. For endoproteinase Lys-C (EndoLysC) digestion (Roche Applied Science, Indianapolis, IN), EndoLysC was added at 2 mU:1 g (protease: rhodopsin), and incubated at 37°C for 6 to 8 hours. The suspension of proteolytic fragments from enzymatic digestion was diluted in sample loading buffer. SDS/PAGE was performed in the modified Laemmli gels, and the gel was subsequently electroblotted to a polyvinylidene difluoride membrane (Problott Membranes; Applied Biosystems, Foster City, CA). Automated N-terminal sequence analysis was performed on an Applied Biosystems model 473A protein sequencer (Foster City, CA) with an in-line 120A PTH analyzer. Blotted samples were directly loaded onto the chamber, and sequencing was performed using gasphase trifluoroacetic acid to minimize possible hydrolysis. After conversion of the released amino acids to PTH-amino acids, the suspension was divided into two parts. One portion, approximately 30%, went to the PTH analyzer, whereas the remaining 70% was collected for scintillation counting. Yield of PTH amino acids was calculated from peak height compared with standards using the model 610A Data Analysis Program. Cysteine was not included in the standards. The analysis was done at least twice for each fragment.