Identification of Binding Sites in the Nicotinic Acetylcholine Receptor for [H]Azietomidate, a Photoactivatable General Anesthetic

To identify binding domains in a ligand-gated ion channel for etomidate, an intravenous general anesthetic, we photolabeled nicotinic acetylcholine-receptor (nAChR)-rich membranes from Torpedo electric organ with a photoactivatable analog, [H]azietomidate. Based upon the inhibition of binding of the noncompetitive antagonist [H]phencyclidine, azietomidate and etomidate bind with ten-fold higher affinity to nAChRs in the desensitized state (IC50 = 70 M) than in the closed channel state. In addition, both drugs between 0.1 and 1 mM produced a concentration dependent enhancement of [H]ACh equilibrium binding affinity, but they inhibited binding at higher concentrations. UV irradiation resulted in preferential [H]azietomidate photoincorporation into the nAChR and subunits. Photolabeled amino acids in both subunits were identified in the ion channel domain and in the ACh binding sites by Edman degradation. Within the nAChR ion channel in the desensitized state, there was labeling of Glu-262 and Gln-276, at the extracellular end, and Ser-258 and Ser-262, towards the cytoplasmic end. Within the ACh binding sites, [H]azietomidate photolabeled Tyr-93, Tyr190, and Tyr-198 in the site at the interface and Asp-59 (but not the homologous position, Glu-57). Increasing [H]azietomidate concentration from 1.8 to 150 M increased the efficiency of incorporation into amino acids within the ion channel by 10-fold and in the ACh sites by 100-fold, consistent with higher affinity binding within the ion channel. The state dependence and subunit selectivity of [H]azietomidate photolabeling are discussed in terms of the structures of the nAChR transmembrane and extracellular domains. 3 by gest on O cber 1, 2017 hp://w w w .jb.org/ D ow nladed from INTRODUCTION At clinically relevant concentrations, most general anesthetics modulate the responses of ligand-gated ion channels in the “Cystine-loop” superfamily that includes nicotinic acetylcholine receptors (nAChRs) and serotonin 5-HT3 receptors, with cation selective channels, and the GABAA and glycine receptors with anion selective channels (1-3). General anesthetics enhance responses for submaximal concentrations of GABA and glycine, and at higher concentrations they can directly activate these receptors, while they noncompetitively inhibit nAChRs. Members of this superfamily contain five homologous subunits arranged about a central axis that is the ion channel (4, 5). Each subunit has a large N-terminal domain that contributes to the receptor extracellular domain and four transmembrane segments (M1-M4), organized as a four helix bundle, with M2 segments from each subunit contributing to the lumen of the ion channel (6). In the muscle-type nAChR, with a subunit stoichiometry 2 , the two agonist binding sites, which are in the extracellular domain at a distance 30 Å above the lipid bilayer, are at the interfaces between the and subunits. The crystal structure of the molluscan ACh binding protein (AChBP), a soluble, homopentameric homolog of the nAChR extracellular domain, provides a general description of the extracellular domain and transmitter binding sites of nAChRs and other members of this protein superfamily (7). R(+)-etomidate, one of the most potent general anesthetics used clinically, acts at micromolar concentrations both as an anesthetic and as a potentiator of the responses to submaximal concentrations of GABA, while at concentrations above 10 M, it inhibits GABA responses and directly activates GABAA receptors (8), and it inhibits nAChRs (9). GABAA receptors containing 2 or 3, but not 1, subunits are most sensitive to etomidate, and site directed mutagenesis has identified a single amino acid within the M2 segment that determines 4 by gest on O cber 1, 2017 hp://w w w .jb.org/ D ow nladed from etomidate sensitivity in GABAA receptors in vitro (10) and for etomidate anesthesia in vivo (11, 12). The position within M2 conferring etomidate sensitivity corresponds to that within M2 of GABAA receptor subunits associated with sensitivity to volatile anesthetics (13). In the absence of atomic resolution structures of these ligand-gated ion channels in the presence of anesthetics, it is difficult to distinguish whether the positions at which substitutions alter anesthetic potency contribute directly to anesthetic binding sites or are involved in the transduction mechanism and allosterically modulate anesthetic potency. Photoaffinity labeling provides a complementary approach to identifying amino acids contributing to drug binding sites (reviewed in (14, 15)). For the nAChR, available in high abundance from the electric organs of Torpedo, photoreactive agonist and antagonists have provided extensive identification of amino acids contributing to the transmitter binding sites and to the ion channel (16, 17). Amino acids photolabeled by 3-[H]azioctanol, a general anesthetic containing a photoreactive diazirine, have been identified at the extracellular end of the M2 ion channel domain, in the agonist binding site, and at the lipid interface (18). Azietomidate, a diazirine derivative of etomidate, has been recently developed as a photoreactive analog to identify etomidate binding sites in GABAA receptors and nAChRs (19). The two drugs are equipotent as anesthetics for tadpoles and as positive allosteric modulators of GABA responses, and azietomidate inhibits agonist activation of muscle type nAChRs with IC50 = 25 M. In addition, [H]azietomidate at 1 M was shown to photoincorporate preferentially into the Torpedo nAChR and subunits, with labeling of the subunit enhanced and the subunit inhibited in the presence of agonist. Within the subunit, the agonist-enhanced labeling was localized to a 20 kDa fragment containing the M1, M2, and M3 transmembrane segments, while labeling inhibited by agonist was localized to an 18 kDa fragment containing ACh binding 5 by gest on O cber 1, 2017 hp://w w w .jb.org/ D ow nladed from site Segments A and B. We now characterize the effects of azietomidate and etomidate on the binding of radiolableled drugs to the nAChR agonist sites and within the ion channel, and we use protein chemistry techniques to identify the amino acids contributing to [H]azietomidate binding sites in the nAChR. 6 by gest on O cber 1, 2017 hp://w w w .jb.org/ D ow nladed from EXPERIMENTAL PROCEDURES Materials. nAChR-enriched membranes were isolated from Torpedo californica electric organ (20). The final membrane suspensions were stored in 38% sucrose at -80 C under argon. The membranes used here contained 1-2 nmol of [H]acetylcholine (ACh) binding sites per milligram of protein. [H]Azietomidate (11 Ci/mmol) and nonradioactive R(+)-azietomidate were synthesized as described previously (19). [H]Azietomidate was stored at a concentration of 1.5 M at -80 C in ethanol, which was removed via evaporation immediately prior to the addition of membranes or Torpedo physiological saline (TPS: 250 mM NaCl, 5 mM KCl, 3 mM CaCl2, 2 mM MgCl2, 5 mM sodium phosphate, pH 7.0). For studies of photoincorporation at 150 M, [H]azietomidate stock at 11 Ci/mmol was isotopically diluted with an appropriate volume of a freshly made stock of nonradioactive azietomidate at 1 mg/mL in ethanol. This solution was then dried down prior to the addition of membranes as outlined above. R-(+) etomidate was gift from Dr. David Gemmell (Organon Labs, U.K.). [H]Phencyclidine (PCP, 27 Ci/mmol) was from New England Nuclear and [H]ACh (3.6 Ci/mmol) was synthesized from choline and [H]acetic anhydride. Staphylococcus aureus glutamylendopeptidase (V8 protease) was from ICN Biomedical Inc; endoproteinase Lys C (EndoLys-C) from Roche Molecular Biochemicals. Trifluoroacetic acid was from Pierce, 10% Genapol C-100 was from Calbiochem. Radioligand Binding Assays. The equilibrium of [H]ACh and [H]PCP in the presence or absence of 1 mM carbamylcholine (Carb) to Torpedo nAChR-rich membranes in TPS was measured by centrifugation in a TOMY MXT-150 microcentrifuge as described previously (21). Membrane suspensions were pretreated with diisopropylphosphofluoridate (~0.5 mM) for 15 min to inhibit acetylcholinesterase activity. For [H]ACh (20 nM), binding was measured using dilute membrane suspensions (1 mL, 84 g protein/mL, 40 nM ACh binding sites), whereas for 7 by gest on O cber 1, 2017 hp://w w w .jb.org/ D ow nladed from [H]PCP (6 nM), 200 L aliquots at 0.7 mg protein/mL were used. Membrane suspensions were equilibrated with [H]ACh and [H]PCP for 30 min and 2 h, respectively, prior to centrifugation. Non-specific binding of [H]ACh and [H]PCP were determined in the presence of, respectively, 1 mM Carb and 1 mM proadifen (+ Carb) or 1 mM tetracaine (Carb). Data Analysis. The concentration-dependence of azietomidate or etomidate inhibition of radioligand binding and d-tubocurarine(dTC) inhibition of [H]azietomidate labeling of nAChR subunits was fit to: Equation 1: fx = f0 / [1 + (x / IC50)] + fns where fx is the total [H]ACh or [H]PCP binding or [H]azietomidate subunit labeling in the presence of inhibitor concentration x; f0 is the specific radioligand binding or [H]azietomidate incorporation in the absence of inhibitor; fns is a fixed parameter defined by the non-specific binding or labeling; IC50 is the total inhibitor concentration associated with 50 % inhibition of radioligand binding or subunit photolabeling; n is the Hill coefficient. Photolabeling of nAChR-enriched Membranes with [H]Azietomidate. Freshly thawed nAChR-rich Torpedo membranes were diluted with TPS and pelleted, and the pellets were resuspended at 2 mg protein/ml (~1 M nAChR) in TPS supplemented with 1 mM oxidized glutathione to serve as an aqueous scavenger. Membrane aliquots were combined with [H]azietomidate and agitated for 5 min prior to the addition of other drugs as noted in the figure legends. Samples were incubated for 1 h in the dark at 4 C in polypropylene microfuge tubes after the addition of drugs. For photolabeling on an analytical scale, 200 g aliquots were placed in a 96 well polypropylene microtiter plate (Falcon #3911), while preparative photolabeling (10 mg protein) was performed in a 2 cm glass petri dish with a stir bar. The suspensions, on ice, were irradiated for 25 min in a horizontal photochemical chamber reactor (Rayonet RPR-200, 8 by gest on O cber 1, 2017 hp://w w w .jb.org/ D ow nladed from Southern New England Ultraviolet Company, Branford, CT) using RPR-3500 bulbs that emit ~24 watts with an intensity maxima at 365 nm. In analytical experiments the suspensions were then diluted with 4X sample loading buffer and submitted to SDS-PAGE, while preparative samples were pelleted and solubilized in 1X sample buffer. In control analytical photolabeling experiments, we assessed by SDS-PAGE the dependence upon UV irradiation of the H incorporation into membrane polypeptides. No covalent incorporation occurred when samples were incubated for 90 min in the absence of irradiation, and no significant “dark reaction” was detected when samples were incubated in the dark for 30 min after irradiation for 15 min. When membrane suspensions containing 1 Ci were electrophoresed after incubation for 60 90 min in the absence of irradiation, a time-independent background of ~100 cpm was associated with each gel slice, compared to the 1000 to 10000 cpm incorporated after irradiation in gel slices containing nAChR subunits or other membrane polypeptides. When after irradiation samples were incubated in the dark for a further 30 min before dilution in sample loading buffer, the counts in the gel slices were within 5% of the nonincubated samples. Gel Electrophoresis. SDS-PAGE was performed as described by Laemmli (22) with modifications (23). The polypeptides were resolved on a 1.5-mm thick 8% acrylamide gel and visualized by staining with Coomassie Blue (0.25% w/v in 45% methanol and 10% acetic acid). For autoradiography, the gels were impregnated with fluor (Amplify, Amersham Pharmacia Biotech) for 30 min, dried, and exposed at -80°C to Eastman Kodak X-OMAT film for various times (4-8 weeks). Incorporation of H into individual polypeptides was also quantified by scintillation counting of excised gel slices (20). For fragmentation of the nAChR subunit by “in gel” digestion with S. aureus V8 protease, following electrophoresis, the gels were briefly stained with Coomassie Blue and destained to allow visualization of the subunits. The nAChR 9 by gest on O cber 1, 2017 hp://w w w .jb.org/ D ow nladed from subunit from a preparative scale labeling was then excised and placed directly into individual wells of a 1.5 mm mapping gel, composed of a 5-cm long 4.5% acrylamide stacking gel, and a 15 cm 15% acrylamide separating gel (23, 24). S. aureus V8 protease (200 g) in overlay buffer (5% sucrose, 125 mM Tris-HCl, 0.1% SDS, pH 6.8) was added to each well. The gel was run at 150 V for 2 h, and then the current was turned off for 1 h. The gel was then run at constant current overnight until the dye front reached the end of the gel. The gel was stained with Coomassie Blue, and the proteolytic fragments of ~20 kDa ( V8-20), 18 kDa ( V8-18) and 10 kDa ( V8-10) were visualized and excised (23). The excised proteolytic fragments were isolated by passive elution into 15 mL 0.1 M NH4HCO3, 0.1% SDS, and 2.5 mM dithiotreitol. The eluate was concentrated using Vivaspin 15 Mr 5,000 concentrators (Vivascience Inc., Edgewood, NY). To remove excess SDS, acetone was added (70% final volume), and following incubation at -20 °C overnight, the polypeptides were pelleted. Proteolytic Digestions. For digestion with EndoLysC or V8 protease, acetoneprecipitated subunits or subunit fragments isolated from preparative scale labelings (10 mg protein) were resuspended in 200 L of 15 mM Tris, 0.5 mM EDTA, pH 8.1, 0.1% SDS. For digestion of V8-18, V8-20 or subunit, EndoLysC (0. 5 -1.5 units in 100 L water) was added, and after 14 days at room temperature, the V8-18 and V8-20 digests were fractionated by HPLC, while the subunit digest was fractionated on 1.5 mm thick, Tricine SDS-PAGE gels (25, 26). EKC-21 in resuspension buffer was digested with V8 protease (1 g) at room temperature for 3-4 days before separation of fragments by HPLC. HPLC Purification. Proteolytic fragments from the nAChR and subunit digests were purified by reverse-phase HPLC on an Agilent 1100 binary HPLC system, using a Brownlee C4-Aquapore column (100 x 2.1 mm, 7 M particle size) at 40 C. Solvent A was 10 by gest on O cber 1, 2017 hp://w w w .jb.org/ D ow nladed from 0.08% trifluoroacetic acid in water, and solvent B was 0.05% trifluoroacetic acid in 60% acetonitrile, 40% 2-propanol. A linear stepwise gradient at 0.2 mL/min was used (in % Solvent B): 0 min, 25%; 15 min, 28%; 30 min, 37%; 45 min, 52%; 60 min, 73%; 75 min 100%; 80 min, 100%; 85 min 25%; 90 min, 25%. Fractions were collected every 2 min (45 fractions/run) or 2.5 min (36 fractions/run). The elution of peptides was monitored by absorbance at 214 nm. Aliquots (2-4% by volume) from each fraction were taken to determine the H distribution by liquid scintillation counting. Sequence Analysis. Automated N-terminal sequence analysis was performed on an Applied Biosystems Model 494 protein sequencer with an in-line 140C PTH analyzer. Samples were usually applied directly to Biobrene-pretreated, micro TFA glass fiber disks (Applied Biosystems #401111). For samples resuspended in 0.1% SDS, after drying, the filters were treated with gas trifluoroacetic acid (5 min) followed by an ethyl acetate wash (4 min) to remove detergent. HPLC samples (400 L or 500 μl fractions) were loaded directly onto the glass fiber disks in 20 μl aliquots, allowing the solvent to evaporate at 40 °C between loads. Because sample loading under this condition results in cleavage on the N-terminal side of Trp residues (16), when fragments beginning at Thr-51, which contain a Trp in cycle 7, were sequenced (Figure 8B and Supplemental Figure 2B), the HPLC fractions were loaded directly onto the glass fiber disk, which was then dried and treated with Biobrene. Sequencing was performed using gas-phase 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, usually one-sixth, went to the PTH analyzer, while the remaining five-sixths were collected for scintillation counting. For sequencing of fragments containing M2 (Figure 7B), two-thirds went to the PTH analyzer and one-third was collected for scintillation counting. For these 11 by gest on O cber 1, 2017 hp://w w w .jb.org/ D ow nladed from samples, the plotted H release and PTH-amino acid release are those caclulated based for the usual 1/6 mass analysis and 5/6 H determination. Yield of PTH-amino acids was calculated from peak height compared with standards using the Model 610A Data Analysis Program Version 2.1A. Initial and repetitive yields were calculated by a nonlinear least squares regression to the equation M = I0 × R , where M is the observed release, I0 is the initial yield, R is the repetitive yield, and n is the cycle number. PTH-derivatives known to have poor recovery (Ser, Arg, Cys, and His) were omitted from the fit. Details concerning the values of I0 and R for each sequencing run as well as the cpm loaded on the filters and retained after sequencing are provided in the the Figure legends for Supplemental Figs. 1 and 2. H incorporation into nAChR residues was quantified based on the results of sequence analysis where the mass of that residue was calculated from the initial and repetitive yields. In most cases the increased H release in that cycle (cpmn-cpm(n-1)) was divided by five times the mass of that cycle, since five times more sample is measured for radioactivity than for mass. H release was divided by one-half the mass when two-thirds were analyzed for mass and one-third analyzed for H. In either of these calculations, the radioactivity released and the mass levels reflect only the sequenced material. In some cases, the sequencing run was interupted and the material on the filter treated with o-phthalaldehyde as described previously (20). oPhthalaldehyde reacts with primary amines preferentially over secondary amines (i.e. proline) and can be used at any sequencing cycle to block Edman degradation of peptides not containing an N-terminal proline at that cycle (27). 12 by gest on O cber 1, 2017 hp://w w w .jb.org/ D ow nladed from RESULTS Etomidate and Azietomidate Inhibition of [H]PCP and [H]ACh Binding. We tested etomidate and azietomidate as inhibitors of the binding to Torpedo nAChR-rich membranes of [H]phencyclidine ([H]PCP), a positively charged, aromatic amine noncompetitive antagonist. [H]PCP binds with high affinity (Keq = 1 M) to a single site per nAChR in the desensitized state and more weakly (Keq = 7 in the absence of agonist (28). In the absence or presence of agonist, azietomidate or etomidate at high concentrations inhibited the specific binding of [H]PCP by >95% (Fig. 1). For nAChRs in the desensitized state, the concentration dependence of inhibition was fit to a single site model with IC50 equal to 70 for azietomidate and 130 for etomidate. In the absence of agonist, both drugs inhibited [H]PCP binding with IC50 = 0.7 mM. We also characterized their effects on the equilibrium binding of [H]ACh at a concentration sufficient to occupy ~20 % of sites, a condition that makes the assay sensitive to drugs that either increase or decrease ACh binding affinity (29). At concentrations between 100 and 600 azietomidate increased [H]ACh binding by 130%, with inhibition seen only at concentrations above millimolar. Etomidate also produced a similar increase and then decrease of [H]ACh binding, but at three-fold higher concentrations (Fig. 1). In parallel assays, proadifen (100 M), a well characterized desensitizing aromatic amine noncompetitive antagonist (29), also produced a maximal increase of [H]ACh binding of 130 % (data not shown). Photoincorporation of [H]Azietomidate into nAChR-rich Membranes. The pattern of photoincorporation of [H]azietomidate into nAChR-rich membranes was determined by incubating membranes with [H]azietomidate at two concentrations: 1 M (11 Ci/mmol) and 150 M (0.07 Ci/mmol), with the isotope dilution for 150 M chosen so that similar amounts of 13 by gest on O cber 1, 2017 hp://w w w .jb.org/ D ow nladed from H were added to the membranes at both concentrations. Membranes were photolabeled with [H]azietomidate in four conditions: (i) in the absence of other drugs; (ii) in the presence of proadifen, which binds to the ion channel and stabilizes nAChRs in the desensitized state without occupying the agonist sites; (iii) in the presence of the agonist Carb, which occupies the ACh sites and stabilizes the nAChR in the desensitized state; or (iv) in the presence of Carb and proadifen. After irradiation, the samples were fractionated on 8% SDS-PAGE. Samples were prepared in triplicate, with one gel prepared for fluorography (Fig. 2A) and two stained and cut into slices for determination of the H distribution by liquid scintillation counting (Fig. 2B). At both concentrations [H]azietomidate was primarily photoincorporated within the nAChR in the and subunits, and also in a 34 kDa polypeptide that is a mitochondrial voltagedependent anion channel (VDAC) (30). Photolabeling of VDAC was inhibited by proadifen, but not by Carb. Comparison of the photolabeling at 1 M [H]azietomidate in the four conditions (Fig. 2A, lanes 2-5 and Fig. 2B, left) established that within the nAChR and subunits, there were two components of labeling: agonist inhibitable and proadifen inhibitable. However, either proadifen or Carb alone increased the labeling within the subunit by 1-1.5 fold compared to no drug addition, while labeling in the presence of both Carb and proadifen was at the same level as the no drug control. Within the subunit, proadifen increased incorporation by 80%, while Carb inhibited incorporation by 30% compared to no added drug. The proadifen inhibitable labeling in the subunit in the presence of agonist was ~20% the level of the agonist inhibitable labeling seen in the presence of proadifen. At 150 M [H]azietomidate (Fig. 2A, lanes 6-9 and Fig. 2B, right), H incorporation in the subunit was not altered by Carb or proadifen, but it was reduced by 30% in the presence of both drugs. In contrast, the subunit labeling was inhibited by ~60% 14 by gest on O cber 1, 2017 hp://w w w .jb.org/ D ow nladed from in the presence of Carb or Carb and proadifen. At either 1 M or 150 M [H]azietomidate, the total H incorporation in the and subunits was ~1/3 that in the subunit in the absence of drugs, with Carb or proadifen altering the labeling by <25%. The efficiency of incorporation into the nAChR and subunits was estimated by calculating the ratio between the pmol [H]azietomidate incorporated in each subunit band and the pmol of nAChR subunit loaded on the gel. At 2 M [H]azietomidate, the ~4000 cpm of Carb or proadifen inhibitable labeling in the subunit constituted specific labeling of ~0.3% of subunits, while at 150 M [H]azietomidate, the ~1000 cpm of specific labeling in the and subunits constituted labeling of 15% and 30% of subunits, respectively. Photoincorporation into nAChR subunits was also measured from 10 to 150 M [H]azietomidate at a constant radiochemical specific activity. Membranes were photolabeled in the absence of any additional drugs, in the presence of Carb, or with Carb and proadifen (Fig. 3). In the absence of Carb, photoincorporation in the subunit increased linearly, whereas in the presence of Carb incorporation increased up to ~75 M [H]azietomidate and then appeared to saturate. At 150 M, H incorporation into the subunit was the same in the absence and presence of Carb, and proadifen inhibited this incorporation by 50%. Incorporation into the subunit increased linearly in the absence of agonist, with labeling reduced by ~60 % in the presence of Carb. As seen at 1 M [H]azietomidate (Fig. 2), the proadifen inhibitable labeling in the subunit in the presence of Carb was <20 % the level of Carb inhibitable labeling. The H incorporation in the and subunits was less than 20% that of the subunit labeling in the absence of Carb at all concentrations. 15 by gest on O cber 1, 2017 hp://w w w .jb.org/ D ow nladed from Inhibition of Photoincorporation by d-Tubocurarine. At 1 M [H]azietomidate, there was agonist inhibitable labeling in the and subunits in the presence of proadifen, with any agonist inhibitable labeling in the subunit at less than 15 % the labeling in the subunit. While the agonist-inhibitable labeling within the subunit was likely to reflect binding to the agonist site at the interface, we wanted to determine whether the labeling in the subunit resulted from binding at that site or at the site at the interface. To address this, we took advantage of the fact that the competitive antagonist d-tubocurarine (dTC) binds with high affinity to the site at the interface (Keq = 50 nM) and with low affinity to the site (Keq = 4 M) (31). We determined the concentration dependence of dTC inhibition of [H]azietomidate photolabeling of nAChRs also equilibrated with proadifen (Fig. 4). After photolabeling and SDS-PAGE, bands containing the nAChR subunits were excised from the stained gels, and H photoincorporation was measured by scintillation counting. At high concentrations, dTC inhibited subunit labeling by the same extent as Carb, with the Carb-inhibitable labeling in the , , and subunits of 1850, 280, and 3630 cpm, respectively. The concentration dependence of dTC inhibition of subunit labeling was fit by a single site model with IC50 = 9 M, while for the and subunits, IC50 = 1 M (total concentration). Inhibition of Photoincorporation by Etomidate. We also characterized by SDS-PAGE and gel slice analysis the effects of 300 M (R+)-etomidate on the photoincorporation of 1 M [H]azietomidate into nAChRrich membranes (Fig. 5). As seen for proadifen (Fig. 2), in the absence of Carb, etomidate increased [H]azietomidate incorporation into the nAChR and subunits. In the presence of Carb, meproadifen, the quaternary ammonium analog of proadifen, inhibited subunit photolabeling by 60% (as did proadifen, not shown), and etomidate reduced incorporation by half that amount. VDAC photolabeling was inhibited by 75% by 16 by gest on O cber 1, 2017 hp://w w w .jb.org/ D ow nladed from nonradioactive etomidate, but not by meproadifen (in contrast to proadifen), and none of the drugs altered the photolabeling of the Na/K-ATPase subunit. Mapping [H]Azietomidate Photoincorporation into and Subunit Proteolytic Fragments. For nAChRs labeled on a preparative scale (10 mg protein) with 1.8 M [H]azietomidate in the absence and presence of Carb, the H distribution within the and subunits was first characterized by digestion of the isolated subunit with S. aureus V8 protease and the subunit with EndoLysC, followed by fractionation of the digests by SDS-PAGE (Fig. 6). Digestion of nAChR subunit in gel by V8 protease generates four non-overlapping subunit fragments referred to as: V8-4 (a 4 kDa fragment), containing the N-terminal 45 amino acids of subunit; V8-10, (a 10kDa fragment), beginning at Asn-338 and including the M4 transmembrane segment; V8-18 (a 18 kDa fragment), beginning at Val-46 and including segments A and B of agonist binding site; and V8-20 (a 20 kDa fragment), beginning at Ser173 and containing segment C of the binding site as well as the M1-M3 transmembrane segments (23). For nAChRs photolabeled in the presence of Carb, H incorporation in V8-20 was increased by 2.4 fold, while within V8-18 it was inhibited by 85%. H incorporation into V8-10 was the same in the absence or presence of Carb. A solution digest of subunit by endoproteinase Lys C (EndoLys-C) generates several large subunit fragments, including a 10kDa fragment ( EKC-10) (32), beginning at Met-257 at the N-terminus of M2 and including M3, and a 21 kDa fragment ( EKC-21), beginning at Glu-48 and containing ACh binding site segments D, E and F (33). H incorporation into EKC-10 was increased 2.6 fold in the presence of Carb, while incorporation into EKC-21 was