Gating-enhanced Accessibility of Hydrophobic Sites within the Transmembrane Region of the Nicotinic Acetylcholine Receptor's δ-Subunit
2005; Elsevier BV; Volume: 280; Issue: 14 Linguagem: Inglês
10.1074/jbc.m413911200
ISSN1083-351X
AutoresEnrique Arevalo, David C. Chiara, Stuart A. Forman, Jonathan B. Cohen, Keith W. Miller,
Tópico(s)Ion channel regulation and function
ResumoGeneral 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-subunit's 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. 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-subunit's 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. Gating-enhanced accessibility of hydrophobic sites within the transmembrane region of the nicotinic acetylcholine receptor's δ subunit. A time-resolved photolabeling study. Vol. 280 (2005) 13631–13640Journal of Biological ChemistryVol. 280Issue 23PreviewPage 13638,Fig. 8: The original figure was determined to be of low resolution. See the revised Fig. 8 below. Full-Text PDF Open Access General anesthetics exert their primary actions on ion channels of neuronal plasma membranes, particularly at synapses. Among the most sensitive of these targets are the ligand-gated ion channel superfamily of receptors that include anion channels gated by γ-aminobutyric acid (GABA) 1The abbreviations used are: GABA, γ-aminobutyric acid; GABAA R, subtype A γ-aminobutyric acid receptor; ACh, acetylcholine; Carb, carbamylcholine; EndoLys-C, endoproteinase Lys-C; HPLC, high performance liquid chromatography; nAChR, nicotinic acetylcholine receptor; OPA, o-phthalaldehyde; TID, 3-(trifluoromethyl)-3-(m-iodophenyl) diazirine; V8 protease, S. aureus endopeptidase Glu-C; Tricine, N-[2-hydroxy1,1-bis(hydroxymethyl)ethyl]glycine. 1The abbreviations used are: GABA, γ-aminobutyric acid; GABAA R, subtype A γ-aminobutyric acid receptor; ACh, acetylcholine; Carb, carbamylcholine; EndoLys-C, endoproteinase Lys-C; HPLC, high performance liquid chromatography; nAChR, nicotinic acetylcholine receptor; OPA, o-phthalaldehyde; TID, 3-(trifluoromethyl)-3-(m-iodophenyl) diazirine; V8 protease, S. aureus endopeptidase Glu-C; Tricine, N-[2-hydroxy1,1-bis(hydroxymethyl)ethyl]glycine. and glycine and cation channels gated by acetylcholine (ACh) and serotonin (1.Krasowski M.D. Harrison N.L. Cell. Mol. Life Sci. 1999; 55: 1278-1303Crossref PubMed Scopus (340) Google Scholar, 2.Campagna J.A. Miller K.W. Forman S.A. N. Engl. J. Med. 2002; 348: 2110-2124Crossref Scopus (631) Google Scholar). In general, the action on anion channels is to shift the agonist concentration-response curve to the left (enhancement) (3.Harrison N.L. Kugler J.L. Jones M.V. Greenblatt E.P. Pritchett D.B. Mol. Pharmacol. 1993; 44: 628-632PubMed Google Scholar), probably as a result of stabilizing the open state (4.Ruesch D. Zhong H. Forman S.A. J. Biol. Chem. 2004; 279: 20982-20992Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). In cation channels, only a few of the smallest general anesthetics (e.g. urethane and ethanol) act similarly, whereas most are noncompetitive open channel inhibitors (5.Dilger J.P. Br. J. Anaesth. 2002; 89: 41-51Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). General anesthetics interact with these receptors in a conformationally sensitive manner, often having their highest affinity for those transient conformational states that occur immediately after the agonist binds to the closed state (5.Dilger J.P. Br. J. Anaesth. 2002; 89: 41-51Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Structural information is key to a complete understanding at the molecular level of anesthetic interactions with the open state, but, given its transitory nature, other techniques will be required for the foreseeable future. Currently, the structure of the transmembrane domain of the Torpedo nicotinic acetylcholine receptor (nAChR) is known to 4.0 Å in the closed state (6.Miyazawa A. Fujiyoshi Y. Unwin N. Nature. 2003; 424: 949-955Crossref Scopus (1078) Google Scholar). The nAChR consists of four subunits, α, β, δ, and γ with a stoichiometry 2:1:1:1, each having a bundle of four transmembrane helices (M1–M4, 27–34 residues in length). The five M2 helices are arranged about a central axis orthogonal to the membrane forming the channel lumen. A 9-Å structure of the open state (7.Unwin N. Nature. 1995; 373: 37-43Crossref PubMed Scopus (908) Google Scholar) is of too low a resolution to resolve secondary structure. Electrophysiological studies combined with site-directed mutagenesis provide kinetic evidence that general anesthetics interact with an enhancing site in anion channels that is located within the four-helix bundle of a given subunit (8.Mihic S.J. Ye Q. Wick M.J. Koltchine V.V. Krasowski M.D. Finn S.E. Mascia M.P. Valenzuela C.F. Hanson K.K. Greenblatt E.P. Harris R.A. Harrison N.L. Nature. 1997; 389: 385-389Crossref PubMed Scopus (1101) Google Scholar) and with an inhibitory site in cation channels located in the channel lumen (9.Forman S.A. Miller K.W. Yellen G. Mol. Pharmacol. 1995; 48: 574-581PubMed Google Scholar). Whereas these models of anesthetic action are self-consistent, much work is necessary to rule out alternative explanations. For example, the mutations might increase the affinity for a distant general anesthetic site by stabilizing a conformation that has high affinity for the anesthetic (5.Dilger J.P. Br. J. Anaesth. 2002; 89: 41-51Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Thus, a complementary approach that can provide structural information such as ligand-protein contact points is desirable. Photolabeling is an approach that has provided abundant information about agonist and antagonist sites on the nAChR, a member of the superfamily that is abundantly available from the electric tissue of Torpedo (10.Corringer P.J. Le Novere N. Changeux J.P. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 431-458Crossref PubMed Scopus (706) Google Scholar, 11.Arias H.R. Kem W.R. Trudell J.R. Blanton M.P. Int. Rev. Neurobiol. 2003; 54: 1-50Crossref PubMed Scopus (22) Google Scholar), and some, more difficult to achieve, information on GABAA receptors (GABAARs) from the brain (12.Sawyer G.W. Chiara D.C. Olsen R.W. Cohen J.B. J. Biol. Chem. 2002; 277: 50036-50045Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). With some exceptions (13.Heidmann T. Changeux J.P. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 1897-1901Crossref PubMed Scopus (63) Google Scholar, 14.Cox R.N. Kaldany R.R. DiPaola M. Karlin A. J. Biol. Chem. 1985; 260: 7186-7193Abstract Full Text PDF PubMed Google Scholar), these studies have been on the closed and desensitized states, conformations that exist at equilibrium, but an improved method of time-resolved photolabeling has now been introduced that has allowed nanomoles of nAChR to be efficiently photolabeled following exposure to agonists and other ligands for times as short as 1 ms (15.Addona G.H. Kloczewiak M.A. Miller K.W. Anal. Biochem. 1999; 267: 135-140Crossref PubMed Scopus (12) Google Scholar). The interaction of the lipophilic photoactivatable probe 3-(trifluoromethyl)-3-(m-iodophenyl) diazirine (TID) with every subunit of the nAChR in the closed and desensitized states has been thoroughly characterized (16.White B.H. Cohen J.B. J. Biol. Chem. 1992; 267: 15770-15783Abstract Full Text PDF PubMed Google Scholar, 17.White B.H. Cohen J.B. Biochemistry. 1988; 27: 8741-8751Crossref PubMed Scopus (85) Google Scholar, 18.Blanton M.P. Cohen J.B. Biochemistry. 1992; 31: 3738-3750Crossref PubMed Scopus (137) Google Scholar, 19.Blanton M.P. Cohen J.B. Biochemistry. 1994; 33: 2859-2872Crossref PubMed Scopus (211) Google Scholar), as has the ability of general anesthetics, such as barbiturates, to modulate its photoincorporation (20.Arias H.R. McCardy E.A. Gallagher M.J. Blanton M.P. Mol. Pharmacol. 2001; 60: 497-506PubMed Google Scholar). Preincubation of nAChRs with TID causes closed state inhibition, which develops on the same 100-ms timescale (21.Wu G. Raines D.E. Miller K.W. Biochemistry. 1994; 33: 15375-15381Crossref PubMed Scopus (19) Google Scholar) as the increase in photolabeling of the channel lumen, whereas equilibration with the lipid-protein interface is complete within a few milliseconds (22.Chiara D.C. Kloczewiak M.A. Addona G.H. Yu J.A. Cohen J.B. Miller K.W. Biochemistry. 2001; 40: 296-304Crossref PubMed Scopus (14) Google Scholar). In addition to the above closed state inhibition, rapid perfusion studies of mouse muscle nAChRs reveal a phase of inhibition that occurs on the 10-ms time scale immediately following agonist-induced activation (23.Forman S.A. Biochemistry. 1999; 38: 14559-14564Crossref PubMed Scopus (31) Google Scholar). In the present work, we find that during channel gating, TID interacts uniquely with the nAChR δ-subunit, where, within 10 ms after the addition of agonist, photoincorporation has increased about one and a half times but then decreases during fast desensitization. With reference to the nAChR closed state structure (6.Miyazawa A. Fujiyoshi Y. Unwin N. Nature. 2003; 424: 949-955Crossref Scopus (1078) Google Scholar), three of the additional activation-dependent amino acid residues photolabeled by TID are clustered together on M1 and M2, and the fourth is on the M2-M3 loop close to the start of M3, suggesting the formation of a transient hydrophobic pocket in that region during activation. Materials—3-(Trifluoromethyl)3-m-([125I]iodophenyl)-diazirine ([125I]TID; nominal specific activity of 10 Ci/mmol) was obtained from Amersham Biosciences. Endoproteinase Lys-C (EndoLys-C) was obtained from Roche Applied Science, and Staphylococcus aureus endopeptidase Glu-C (V8 protease) was from ICN. Sample buffer for gel electrophoresis was obtained from Invitrogen. All other chemicals were from Sigma or Aldrich. Torpedo physiological saline contained 250 mm NaCl, 5 mm KCl, 2 mm MgCl2, 5 mm sodium phosphate, pH 7.0, and 0.02% NaN3. Preparation of nAChR-rich Membranes—nAChR-rich membranes, prepared from the electric organs of Torpedo californica (Aquatic Research Consultants, San Pedro, CA) as described (24.Pedersen S.E. Sharp S.D. Liu W.S. Cohen J.B. J. Biol. Chem. 1992; 267: 10489-10499Abstract Full Text PDF PubMed Google Scholar), were stored at –80 °C in 38% sucrose, 0.02% NaN3. Protein concentrations were determined by the micro-BCA assay method (Pierce), and the ACh binding site concentration was determined by an [3H]ACh (PerkinElmer Life Sciences) binding assay. Time-resolved Photolabeling of nAChR-rich Membranes—The method was detailed previously (15.Addona G.H. Kloczewiak M.A. Miller K.W. Anal. Biochem. 1999; 267: 135-140Crossref PubMed Scopus (12) Google Scholar, 22.Chiara D.C. Kloczewiak M.A. Addona G.H. Yu J.A. Cohen J.B. Miller K.W. Biochemistry. 2001; 40: 296-304Crossref PubMed Scopus (14) Google Scholar). Briefly, the rapid mixing device was completely filled with buffer from the drive syringes to the two six-way sample valves, each of which contained a 0.5-ml sample loop loaded with appropriate reagents. The pneumatic ram delivered sufficient buffer both to force the reagents in the sample loops through the mixer in 3 h) or separated directly by reversed-phase HPLC. Enzymatic Digestion of [125I]TID-labeled nAChR Subunits—EndoLys-C digestions were performed in 25 mm Tris, 0.5 mm EDTA, 0.1% SDS, pH 8.6, for 2 weeks at 25 °C. For V8 protease digestion of material after HPLC, the fractions (∼350 μl) were first neutralized by the addition of 200 μl of 25 mm Tris, pH 8.6, containing 0.1% SDS and 0.5 mm EDTA. The acid-neutralized pool was then rotary-evaporated to remove most of the organic solvent, and V8 protease (100 μg) was added for 2 days at 25 °C. Reversed-phase HPLC Purification of [125I]TID-labeled Fragments— Purification was performed on an Agilent 1100 HPLC with an inline degasser, column heater, and external absorbance detector. Separations were achieved at 40 °C using a Brownlee Aquapore C-4 column (100 × 2.1 mm) with a C-2 guard column. All solvents were HPLC grade. The aqueous phase (solvent A) was 0.08% trifluoroacetic acid, and the organic phase (solvent B) was 60% acetonitrile, 40% 2-propanol, 0.05% trifluoroacetic acid. The flow rates were 0.2 ml/min, and fractions of 0.5 ml were collected. Amino-terminal Sequence Analysis of [125I]TID-labeled Fragments— Samples were sequenced on a Procise 492 protein sequencer (Applied Biosystems) modified such that one-sixth of each cycle was used for amino acid identification and quantification, and five-sixths were collected to measure 125I. HPLC fractions for sequencing were pooled and drop-loaded onto Biobrene-treated glass fiber filters (Applied Biosystems catalog no. 401111) at 45 °C. When samples in detergent were loaded onto the filters, the detergent was removed by a prewash consisting of 5 min of gas trifluoroacetic acid, followed by 5-min washes with ethyl acetate and then n-butylchloride. The cpm detected in each cycle of Edman degradation were corrected by subtraction of the background, which varied from 10 to 25 cpm, in each of the channels of a Packard Cobra B5005 γ-counter. All samples were sequenced between 60 and 100 days after the labeling and were back-corrected for 125I decay to the date of labeling. Phenylthiohydantoin-derivatized amino acids were quantified from chromatographic peak heights, and the corrected 125I cpm and pmol released are reported. Initial pmol (I0) and repetitive yields (R) were calculated from a nonlinear least squares fit (Sigma Plot, Jandel Scientific) of the equation, f(x) = I0·Rx, where f(x)is the pmol of the amino acid in cycle x. Arginines, serines, histidines, tryptophans, and cysteines were excluded from the fit because of known problems with their recovery. To quantify [125I]TID photoincorporation into specific residues, the increase in 125I cpm released at that cycle (i.e. cpmn – cpm(n - 1)) was divided by 5 times the pmol of the amino acid calculated from the values of I0 and R. For some samples, the sequencing run was interrupted, and the material on the filter was treated with o-phthalaldehyde (OPA) as described (26.Middleton R.E. Cohen J.B. Biochemistry. 1991; 30: 6987-6997Crossref PubMed Scopus (177) Google Scholar). OPA reacts with primary amines preferentially over secondary amines (i.e. proline), and it may be used at any sequencing cycle to block Edman degradation of peptides not containing an N-terminal proline (27.Brauer A.W. Oman C.L. Margolies M.N. Anal. Biochem. 1984; 137: 134-142Crossref PubMed Scopus (106) Google Scholar). Oocyte Expression of Torpedo nAChRs—Stage V and VI oocytes were harvested from anesthetized Xenopus frogs, in accordance with local and federal guidelines for animal care. Plasmids containing cDNAs for Torpedo α, β, γ, and δ nAChR subunits were linearized with restriction endonucleases and used as templates for in vitro mRNA transcription using commercial kits (mMessage Machine, Ambion, Austin, TX). Mixtures of mRNAs at 2α:1β:1γ:1δ stoichiometry were injected into oocytes (total = 20–50 ng) and incubated at 17 °C for 48–96 h in ND96 buffer (96 mm NaCl, 2 mm KCl, 1.8 mm CaCl2, 1 mm MgCl2, and 5 mm Hepes, pH 7.5) supplemented with penicillin/streptomycin (1% v/v). Oocytes were manually stripped of vitelline membranes for patch clamp electrophysiology. Patch Clamp Electrophysiology—Methods have been described previously (23.Forman S.A. Biochemistry. 1999; 38: 14559-14564Crossref PubMed Scopus (31) Google Scholar). All experiments were performed at 21–23 °C. Internal pipette and external buffers were K-100 (97 mm KCl, 1 mm MgCl2, 0.2 mm EGTA, and 5 mm Hepes, pH 7.5). Oocyte membrane patches were excised in the outside-out configuration on borosilicate pipettes (1.2–3 megaohms) and voltage-clamped at –50 mV. Patches were positioned in the outflow of a custom-built 2 × 2 quad-barrel superfusion pipette, coupled to two orthogonal piezo electric elements. Application of RC-damped high DC voltages to the piezo elements moved the superfusion pipette and resulted in switching between adjacent superfusion solutions in <1 ms (10–90% rise time of open pipette junction potential). In some experiments, two superfusion barrels were used: one containing K-100 buffer and an adjacent barrel containing ACh or ACh plus TID. If TID preincubation was required, three barrels were used: K-100, TID, and ACh plus TID. Patch currents stimulated by ACh were monitored and filtered (8-pole bessel, 1 kHz) with an Axopatch 200A amplifier (Axon Instruments, Foster City, CA). Recording of digitized data (1–2 kHz) and control of the piezo-driven superfusion device were achieved using a Digidata 1200 series interface and pClamp 7.0 software (both from Axon Instruments, Foster City, CA). Analysis of Electrophysiological Data—Current traces were analyzed offline. Base-line leak currents were subtracted digitally. Current decay rates were determined by fitting exponential decay functions to data points from the peak current to the point at which ACh superfusion ceased. Nonlinear least squares fitting was performed using Clampfit 7.0 (Axon Instruments, Foster City, CA). Overall Experimental Design Considerations—Our experimental design was based on knowledge of Torpedo nAChR kinetics. Agonist-induced channel activation occurs in ∼100 μs (28.Lingle C.J. Maconochie D. Steinbach J.H. J. Membr. Biol. 1992; 126: 195-217Crossref PubMed Scopus (39) Google Scholar), within the dead time of our apparatus, and peak Carb-induced cation flux in Torpedo vesicles is linear over at least 10 ms, being terminated by fast desensitization with a time constant of ∼200 ms (29.Forman S.A. Firestone L.L. Miller K.W. Biochemistry. 1987; 26: 2807-2814Crossref PubMed Scopus (31) Google Scholar, 30.Forman S.A. Miller K.W. Biophys. J. 1988; 54: 149-158Abstract Full Text PDF PubMed Scopus (31) Google Scholar). In addition, in the absence of agonist, ∼10% of receptors are in the desensitized rather than the closed state (31.Boyd N.D. Cohen J.B. Biochemistry. 1984; 23: 4023-4033Crossref PubMed Scopus (102) Google Scholar). Therefore, we chose agonist incubation times of 0 (resting states), 1.5 and 10 ms (activated states), 1 s (fast desensitized state), and ∼1 h (slow desensitized state). Initially, the membranes were preincubated with TID before being rapidly mixed with saturating concentrations of agonist so that nAChR-TID interaction kinetics with the closed state (see Introduction) (15.Addona G.H. Kloczewiak M.A. Miller K.W. Anal. Biochem. 1999; 267: 135-140Crossref PubMed Scopus (12) Google Scholar, 22.Chiara D.C. Kloczewiak M.A. Addona G.H. Yu J.A. Cohen J.B. Miller K.W. Biochemistry. 2001; 40: 296-304Crossref PubMed Scopus (14) Google Scholar) would not be superimposed upon agonist-induced changes. Subsequently, TID was added simultaneously with agonist to obtain a sense of the relative accessibility of the TID interaction sites. Time Dependence of Agonist Action on TID Photoincorporation into nAChR Subunits—Fig. 1A shows a typical phosphor image of an SDS-polyacrylamide gel of membranes preincubated with TID and exposed for 10 ms either to 10 mm Carb or to buffer before freeze quenching and photolabeling. In this experiment, Carb increased photoincorporation of [125I]TID into the δ-subunit markedly, whereas that into the γ-subunit decreased, and that into all other bands changed little. The combined results from several separate experiments are shown in Fig. 1B, where the data for each experiment have been normalized to the zero time (no Carb) control (see legend). The overall percentage S.D. (coefficient of variation) for the data set of ratios was 12%. The δ-subunit showed the most complex changes with exposure time to agonist. Photoincorporation increased 1.6-fold 10 ms after adding Carb, followed by an equally dramatic decrease first to just below control values at 1 s and then to much lower values similar to the β- and γ-subunits at equilibrium. The α-subunit did not decrease at early times but showed a slight decline at later times. The β- and γ-subunits were the only subunits to decline significantly at 1.5 ms. They declined further by 1 s but did not change thereafter. Modest photoincorporation into the non-nAChR polypeptides was always observed but did not change upon exposure to agonist. [125I]TID Photoincorporation into the Transmembrane Domain of the δ-Subunit—The δ-subunit was chosen for more detailed study because of its unique kinetics (Fig. 1). nAChR-rich membranes preincubated with [125I]TID were exposed to 10 mm Carb or to buffer (six replicates each) for 10 ms, freeze-quenched, and photolabeled. The membrane polypeptides were separated by SDS-PAGE, and the δ-subunits (visualized by Coomassie stain) were excised, eluted, concentrated, and acetone-precipitated. The resuspended δ-subunits were digested in solution with EndoLys-C, which produces subunit fragments of ∼21 kDa (δEKC-21, beginning at δHis20/δHis26 and containing most of the extracellular domain), ∼10 kDa (beginning at δMet257, the beginning of δM2), and ∼12 kDa (beginning at δPhe206 and containing a site of N-linked glycosylation (δAsn208) and δM1) (22.Chiara D.C. Kloczewiak M.A. Addona G.H. Yu J.A. Cohen J.B. Miller K.W. Biochemistry. 2001; 40: 296-304Crossref PubMed Scopus (14) Google Scholar, 32.Ziebell M.R. Nirthanan S. Husain S.S. Miller K.W. Cohen J.B. J. Biol. Chem. 2004; 279: 17640-17649Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). When the δ-subunit EndoLys-C digests were separated by Tricine gel SDS-PAGE, phosphorimaging revealed two major photolabeled bands of ∼10 kDa (δEKC-10) and ∼13 kDa (δEKC-13) (Fig. 2A). For both labeling conditions, the amount of 125I in bands between 20 and 24 kDa was less than 15% of that in δEKC-10 and δEKC-13, which sets an upper limit on the amount of [125I]TID incorporation in the δEKC-21. Using this image as a template, δEKC-10 and -13 were excised, eluted, concentrated, and further purified by reversed-phase HPLC (Fig. 2, B and C, respectively). Below, we first describe the analysis of purified δEKC-10, leading to the identification of photolabeled amino acids in δM2-δM3 (Figs. 3, 4, 5). Subsequently, we describe the analysis of fractions purified from δEKC-13 that identify the labeled amino acids in δM1 (Fig. 6).Fig. 3Identification of amino acids in δM2 and δM3 photolabeled by [125I]TID after 10-ms exposure to agonist. Shown is 125I (•, ○) and pmol (□) release when aliquots of fractions 26 and 27 from the HPLC fractionation of δEKC-10 (Fig. 2B) were sequenced. A, for the –Carb (○) and +Carb (•) samples, the only sequence detected began at δMet256 (+Carb (□), I0 = 8 pmol, R = 96% for cycles 1–18 and R = 91% thereafter; –Carb (not shown), I0 = 9 pmol, R = 94% for cycles 1–18 and 92% thereafter). For the +Carb and –Carb samples, 46,780 and 24,180 cpm were loaded on the filters, with 12,370 and 6,590 cpm remaining after 55 and 40 cycles of Edman degradation. The photolabeling efficiencies calculated from the 125I and pmol release are tabulated in Table I. Inset, a replot of the data on an expanded cpm scale to highlight the agonist-dependent release in cycles 18, 22, and 32. B, another aliquot of the +Carb sample was sequenced, with sequencing interrupted after cycle 29, when δPro286 was the N-terminal amino acid, and the filter was treated with OPA (↓) before resumption of sequencing (39,000 cpm loaded, 16,130 cpm remaining). The sequence beginning at δMet257 (□, I0 = 10 pmol, R = 92%) was identified for all 40 cycles of Edman degradation, and after OPA treatment, 125I release in cycle 32 was preserved. C, sequence analysis of a third aliquot of the +Carb sample after further digestion with V8 protease (see "Experimental Procedures"). Sequencing was interrupted after cycle 5 (↓) for treatment with OPA (35,190 cpm loaded, 5,425 cpm remaining after 30 cycles).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4Isolation of [125I]TID-labeled fragments from an EndoLys-C digest of δ-subunit
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