David C. Chiara

Identification of a GABAA Receptor Anesthetic Binding Site at Subunit Interfaces by Photolabeling with an Etomidate Analog

General anesthetics, including etomidate, act by binding to and enhancing the function of GABA type A receptors (GABAARs), which mediate inhibitory neurotransmission in the brain. Here, we used a radiolabeled, photoreactive etomidate analog ([3H]azietomidate), which retains anesthetic potency in vivo and enhances GABAAR function in vitro, to identify directly, for the first time, amino acids that contribute to a GABAAR anesthetic binding site. For GABAARs purified by affinity chromatography from detergent extracts of bovine cortex, [3H]azietomidate photoincorporation was increased by GABA and inhibited by etomidate in a concentration-dependent manner (IC50 = 30 μm). Protein microsequencing of fragments isolated from proteolytic digests established photolabeling of two residues: one within the αM1 transmembrane helix at α1Met-236 (and/or the homologous methionines in α2,3,5), not previously implicated in etomidate function, and one within the βM3 transmembrane helix at β3Met-286 (and/or the homologous methionines in β1,2), an etomidate sensitivity determinant. The pharmacological specificity of labeling indicates that these methionines contribute to a single binding pocket for etomidate located in the transmembrane domain at the interface between β and α subunits, in what is predicted by structural models based on homology with the nicotinic acetylcholine receptor to be a water-filled pocket ∼50 Å below the GABA binding site. The localization of the etomidate binding site to an intersubunit, not an intrasubunit, binding pocket is a novel conclusion that suggests more generally that the localization of drug binding sites to subunit interfaces may be a feature not only for GABA and benzodiazepines but also for etomidate and other intravenous and volatile anesthetics.

Mapping general anesthetic binding site(s) in human α1β3 γ-aminobutyric acid type A receptors with [³H]TDBzl-etomidate, a photoreactive etomidate analogue.

The γ-aminobutyric acid type A receptor (GABA(A)R) is a target for general anesthetics of diverse chemical structures, which act as positive allosteric modulators at clinical doses. Previously, in a heterogeneous mixture of GABA(A)Rs purified from bovine brain, [³H]azietomidate photolabeling of αMet-236 and βMet-286 in the αM1 and βM3 transmembrane helices identified an etomidate binding site in the GABA(A)R transmembrane domain at the interface between the β and α subunits [Li, G. D., et.al. (2006) J. Neurosci. 26, 11599-11605]. To further define GABA(A)R etomidate binding sites, we now use [³H]TDBzl-etomidate, an aryl diazirine with broader amino acid side chain reactivity than azietomidate, to photolabel purified human FLAG-α1β3 GABA(A)Rs and more extensively identify photolabeled GABA(A)R amino acids. [³H]TDBzl-etomidate photolabeled in an etomidate-inhibitable manner β3Val-290, in the β3M3 transmembrane helix, as well as α1Met-236 in α1M1, a residue photolabeled by [³H]azietomidate, while no photolabeling of amino acids in the αM2 and βM2 helices that also border the etomidate binding site was detected. The location of these photolabeled amino acids in GABA(A)R homology models derived from the recently determined structures of prokaryote (GLIC) or invertebrate (GluCl) homologues and the results of computational docking studies predict the orientation of [³H]TDBzl-etomidate bound in that site and the other amino acids contributing to this GABA(A)R intersubunit etomidate binding site. Etomidate-inhibitable photolabeling of β3Met-227 in βM1 by [³H]TDBzl-etomidate and [³H]azietomidate also provides evidence of a homologous etomidate binding site at the β3-β3 subunit interface in the α1β3 GABA(A)R.

IDENTIFICATION OF AMINO ACIDS CONTRIBUTING TO HIGH AND LOW AFFINITY D-TUBOCURARINE SITES IN THE TORPEDO NICOTINIC ACETYLCHOLINE RECEPTOR

d-Tubocurarine (dTC) is a potent competitive antagonist of the Torpedo nicotinic acetylcholine receptor (nAChR) that binds non-equivalently to the two agonist sites (K d values of 30 nm and 8 μm). When nAChR-rich membranes equilibrated with [3H]dTC are irradiated with 254 nm UV light, [3H]dTC is covalently incorporated into the α-, γ-, and δ-subunits in a concentration-dependent and agonist-inhibitable manner, consistent with the localization of the high and low affinity dTC binding sites at the α-γ- and α-δ-subunit interfaces, respectively (Pedersen, S. E. and Cohen, J. B. (1990) Proc. Natl. Acad. Sci. U. S. A.87, 2785–2789). We report on the amino acids within α-, γ-, and δ-subunits that are the sites of specific photoincorporation of [3H]dTC. Subunits isolated from nAChR-rich membranes photolabeled with [3H]dTC were subjected to enzymatic digestion, and peptides containing 3H were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and/or reversed-phase high performance liquid chromatography. Isolated peptides were then subjected to NH2-terminal sequence analysis to identify specifically labeled residues. Within the α-subunit, 95% of specific incorporation was contained within a 20-kDa proteolytic fragment beginning at Ser-173, with αTyr-190 the primary site of [3H]dTC photoincorporation and αCys-192 and αTyr-198 labeled at lower efficiency. Within γ- and δ-subunits, specific labeling was contained within proteolytic fragments of 14 and 21 kDa, respectively, beginning at γAla-49 and δThr-51. γTrp-55 and δTrp-57 were identified as the sites of specific [3H]dTC photoincorporation. Sequence alignment studies reveal γTrp-55 and δTrp-57 to be homologous residues at whose position in receptor subunit primary structure a unique pattern of conservation exists in all nAChR (neuronal and muscle). Specifically, all subunits that associate with an α-subunit to form an agonist site contain a tryptophan homologous to γTrp-55/δTrp-57. This pattern of conservation may indicate a functional significance for tryptophan at that location in all nAChR agonist sites.

Specificity of Intersubunit General Anesthetic-binding Sites in the Transmembrane Domain of the Human α1β3γ2 γ-Aminobutyric Acid Type A (GABAA) Receptor

Background: General anesthetics of diverse chemical structure potentiate GABAA receptors by binding to unknown sites. Results: A photoreactive barbiturate identifies intersubunit-binding sites distinct from, but homologous to, sites identified by photoreactive etomidate analogs. Conclusion: Propofol, barbiturates, and etomidate analogs bind with variable selectivities to two classes of sites. Significance: This study helps define the diversity of GABAA receptor general anesthetic-binding sites. GABA type A receptors (GABAAR), the brains major inhibitory neurotransmitter receptors, are the targets for many general anesthetics, including volatile anesthetics, etomidate, propofol, and barbiturates. How such structurally diverse agents can act similarly as positive allosteric modulators of GABAARs remains unclear. Previously, photoreactive etomidate analogs identified two equivalent anesthetic-binding sites in the transmembrane domain at the β+-α− subunit interfaces, which also contain the GABA-binding sites in the extracellular domain. Here, we used R-[3H]5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl) barbituric acid (R-mTFD-MPAB), a potent stereospecific barbiturate anesthetic, to photolabel expressed human α1β3γ2 GABAARs. Protein microsequencing revealed that R-[3H]mTFD-MPAB did not photolabel the etomidate sites at the β+-α− subunit interfaces. Instead, it photolabeled sites at the α+-β− and γ+-β− subunit interfaces in the transmembrane domain. On the (+)-side, α1M3 was labeled at Ala-291 and Tyr-294 and γ2M3 at Ser-301, and on the (−)-side, β3M1 was labeled at Met-227. These residues, like those in the etomidate site, are located at subunit interfaces near the synaptic side of the transmembrane domain. The selectivity of R-etomidate for the β+-α− interface relative to the α+-β−/γ+-β− interfaces was >100-fold, whereas that of R-mTFD-MPAB for its sites was >50-fold. Each ligand could enhance photoincorporation of the other, demonstrating allosteric interactions between the sites. The structural heterogeneity of barbiturate, etomidate, and propofol derivatives is accommodated by varying selectivities for these two classes of sites. We hypothesize that binding at any of these homologous intersubunit sites is sufficient for anesthetic action and that this explains to some degree the puzzling structural heterogeneity of anesthetics.

Numerous Classes of General Anesthetics Inhibit Etomidate Binding to γ-Aminobutyric Acid Type A (GABAA) Receptors

Enhancement of γ-aminobutyric acid type A receptor (GABAAR)-mediated inhibition is a property of most general anesthetics and a candidate for a molecular mechanism of anesthesia. Intravenous anesthetics, including etomidate, propofol, barbiturates, and neuroactive steroids, as well as volatile anesthetics and long-chain alcohols, all enhance GABAAR function at anesthetic concentrations. The implied existence of a receptor site for anesthetics on the GABAAR protein was supported by identification, using photoaffinity labeling, of a binding site for etomidate within the GABAAR transmembrane domain at the β-α subunit interface; the etomidate analog [3H]azietomidate photolabeled in a pharmacologically specific manner two amino acids, α1Met-236 in the M1 helix and βMet-286 in the M3 helix (Li, G. D., Chiara, D. C., Sawyer, G. W., Husain, S. S., Olsen, R. W., and Cohen, J. B. (2006) J. Neurosci. 26, 11599–11605). Here, we use [3H]azietomidate photolabeling of bovine brain GABAARs to determine whether other structural classes of anesthetics interact with the etomidate binding site. Photolabeling was inhibited by anesthetic concentrations of propofol, barbiturates, and the volatile agent isoflurane, at low millimolar concentrations, but not by octanol or ethanol. Inhibition by barbiturates, which was pharmacologically specific and stereospecific, and by propofol was only partial, consistent with allosteric interactions, whereas isoflurane inhibition was nearly complete, apparently competitive. Protein sequencing showed that propofol inhibited to the same extent the photolabeling of α1Met-236 and βMet-286. These results indicate that several classes of general anesthetics modulate etomidate binding to the GABAAR: isoflurane binds directly to the site with millimolar affinity, whereas propofol and barbiturates inhibit binding but do not bind in a mutually exclusive manner with etomidate.

Multiple Propofol-binding Sites in a γ-Aminobutyric Acid Type A Receptor (GABAAR) Identified Using a Photoreactive Propofol Analog

Background: Propofol binding to GABAAR sites of uncertain location potentiates receptor function and produces anesthesia in vivo. Results: A photoreactive propofol analog identifies propofol-binding sites in α1β3 GABAARs. Conclusion: Propofol binds to each class of intersubunit sites in the GABAAR transmembrane domain. Significance: This study demonstrates that propofol binds to the same sites in a GABAAR as etomidate and barbiturates. Propofol acts as a positive allosteric modulator of γ-aminobutyric acid type A receptors (GABAARs), an interaction necessary for its anesthetic potency in vivo as a general anesthetic. Identifying the location of propofol-binding sites is necessary to understand its mechanism of GABAAR modulation. [3H]2-(3-Methyl-3H-diaziren-3-yl)ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate (azietomidate) and R-[3H]5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid (mTFD-MPAB), photoreactive analogs of 2-ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate (etomidate) and mephobarbital, respectively, have identified two homologous but pharmacologically distinct classes of intersubunit-binding sites for general anesthetics in the GABAAR transmembrane domain. Here, we use a photoreactive analog of propofol (2-isopropyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenol ([3H]AziPm)) to identify propofol-binding sites in heterologously expressed human α1β3 GABAARs. Propofol, AziPm, etomidate, and R-mTFD-MPAB each inhibited [3H]AziPm photoincorporation into GABAAR subunits maximally by ∼50%. When the amino acids photolabeled by [3H]AziPm were identified by protein microsequencing, we found propofol-inhibitable photolabeling of amino acids in the β3-α1 subunit interface (β3Met-286 in β3M3 and α1Met-236 in α1M1), previously photolabeled by [3H]azietomidate, and α1Ile-239, located one helical turn below α1Met-236. There was also propofol-inhibitable [3H]AziPm photolabeling of β3Met-227 in βM1, the amino acid in the α1-β3 subunit interface photolabeled by R-[3H]mTFD-MPAB. The propofol-inhibitable [3H]AziPm photolabeling in the GABAAR β3 subunit in conjunction with the concentration dependence of inhibition of that photolabeling by etomidate or R-mTFD-MPAB also establish that each anesthetic binds to the homologous site at the β3-β3 subunit interface. These results establish that AziPm as well as propofol bind to the homologous intersubunit sites in the GABAAR transmembrane domain that binds etomidate or R-mTFD-MPAB with high affinity.

Neurosteroids Allosterically Modulate Binding of the Anesthetic Etomidate to γ-Aminobutyric Acid Type A Receptors

Photoaffinity labeling of γ-aminobutyric acid type A (GABAA)-receptors (GABAAR) with an etomidate analog and mutational analyses of direct activation of GABAAR by neurosteroids have each led to the proposal that these structurally distinct general anesthetics bind to sites in GABAARs in the transmembrane domain at the interface between the β and α subunits. We tested whether the two ligand binding sites might overlap by examining whether neuroactive steroids inhibited etomidate analog photolabeling. We previously identified (Li, G. D., Chiara, D. C., Sawyer, G. W., Husain, S. S., Olsen, R. W., and Cohen, J. B. (2006) J. Neurosci. 26, 11599–11605) azietomidate photolabeling of GABAAR α1Met-236 and βMet-286 (in αM1 and βM3). Positioning these two photolabeled amino acids in a single type of binding site at the interface of β and α subunits (two copies per pentamer) is consistent with a GABAAR homology model based upon the structure of the nicotinic acetylcholine receptor and with recent αM1 to βM3 cross-linking data. Biologically active neurosteroids enhance rather than inhibit azietomidate photolabeling, as assayed at the level of GABAAR subunits on analytical SDS-PAGE, and protein microsequencing establishes that the GABAAR-modulating neurosteroids do not inhibit photolabeling of GABAAR α1Met-236 or βMet-286 but enhance labeling of α1Met-236. Thus modulatory steroids do not bind at the same site as etomidate, and neither of the amino acids identified as neurosteroid activation determinants (Hosie, A. M., Wilkins, M. E., da Silva, H. M., and Smart, T. G. (2006) Nature 444, 486–489) are located at the subunit interface defined by our etomidate site model.

Identification of Binding Sites in the Nicotinic Acetylcholine Receptor for TDBzl-etomidate, a Photoreactive Positive Allosteric Effector

Etomidate, one of the most potent general anesthetics used clinically, acts at micromolar concentrations as an anesthetic and positive allosteric modulator of γ-aminobutyric acid responses, whereas it inhibits muscle-type nicotinic acetylcholine receptors (nAChRs) at concentrations above 10 μm. We report here that TDBzl-etomidate, a photoreactive etomidate analog, acts as a positive allosteric nAChR modulator rather than an inhibitor, and we identify its binding sites by photoaffinity labeling. TDBzl-etomidate (>10 μm) increased the submaximal response to acetylcholine (10 μm) with a 2.5-fold increase at 60 μm. At higher concentrations, it inhibited the binding of the noncompetitive antagonists [3H]tetracaine and [3H]phencyclidine to Torpedo nAChR-rich membranes (IC50 values of 0. 8 mm). nAChR-rich membranes were photolabeled with [3H]TDBzl-etomidate, and labeled amino acids were identified by Edman degradation. For nAChRs photolabeled in the absence of agonist (resting state), there was tetracaine-inhibitable photolabeling of amino acids in the ion channel at positions M2-9 (δLeu-265) and M2-13 (αVal-255 and δVal-269), whereas labeling of αM2-10 (αSer-252) was not inhibited by tetracaine but was enhanced 10-fold by proadifen or phencyclidine. In addition, there was labeling in γM3 (γMet-299), a residue that contributes to the same pocket in the nAChR structure as αM2-10. The pharmacological specificity of labeling of residues, together with their locations in the nAChR structure, indicate that TDBzl-etomidate binds at two distinct sites: one within the lumen of the ion channel (labeling of M2-9 and -13), an inhibitory site, and another at the interface between the α and γ subunits (labeling of αM2-10 and γMet-299) likely to be a site for positive allosteric modulation.

Identification of tryptophan 55 as the primary site of [3H]nicotine photoincorporation in the γ-subunit of the Torpedo nicotinic acetylcholine receptor

[3H]nicotine has been used as a photoaffinity agonist to identify amino acids within the Torpedo nicotinic acetylcholine receptor (nAChR) γ‐subunit that contributes to the structure of the agonist binding site. UV irradiation (254 nm) of nAChR‐rich membranes equilibrated with [3H]nicotine results in covalent incorporation into α‐ and γ‐subunits that is inhibitable by agonists and competitive antagonists, but not by non‐competitive antagonists (Middleton, R.E. and Cohen, J.B. (1991) Biochemistry 30, 6887–6897). To identify sites of specific incorporation, SDS‐PAGE and reversed‐phase HPLC were used to isolate proteolytic fragments of [3H]nicotine‐labeled γ‐subunit. Amino‐terminal sequence analysis identified γTrp‐55 as the major site of [3H]nicotine photoincorporation in γ‐subunit. Thus γTrp‐55 is the first amino acid within a non‐α‐subunit to be identified by affinity labeling in direct contact with a bound agonist.

Gating-enhanced accessibility of hydrophobic sites within the transmembrane region of the nicotinic acetylcholine Receptor's δ-subunit : A time-resolved photolabeling study

General anesthetics often interact more strongly with sites on open than on closed states of ligand-gated ion channels. To seek such sites, Torpedo membranes enriched in nicotinic acetylcholine receptors (nAChRs) were preincubated with the hydrophobic probe 3-(trifluoromethyl)-3-(m-iodophenyl) diazirine ([125I]TID) and exposed to agonist for either 0 ms (closed state), 1.5 and 10 ms (activated states), 1 s (fast desensitized state), or ≥1 h (equilibrium or slow desensitized state) and then rapidly frozen (<1 ms) and photolabeled. Within 1.5 ms, the fractional change in photoincorporation relative to the closed state decreased to 0.7 in the β- and γ-subunits, whereas in the α-subunit, it changed little. The most dramatic change occurred in the δ-subunit, where it increased to 1.6 within 10 ms but fell to 0.7 during fast desensitization. Four residues in the δ-subunits transmembrane domain accounted for the enhanced photoincorporation induced by a 10-ms agonist exposure both when TID was added simultaneously with agonist and when it was preincubated with membranes. In the published closed state structure, two residues (δThr274 and δLeu278) are situated toward the extracellular end of helix M2, both contralateral to the ion channel and adjacent to the third residue (δPhe232) on M1. The fourth labeled residue (δIle288) is toward the end of the M2-M3 loop. Contact with these residues occurs on the time scale of a rapid phase of TID inhibition in Torpedo nAChRs, suggesting the formation of a transient hydrophobic pocket between M1, M2, and M3 in the δ-subunit during gating.