INTRODUCTION

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The nicotinic acetylcholine receptor (AChR)¹ isolated from the electric organ of the marine elasmobranch Torpedo californica is the best characterized member of a family of ligand-gated ion channels which includes the -aminobutyric acid, glycine, and serotonin 5-HT₃ receptors (for recent reviews, see Refs. 1-3). The AChR is composed of four homologous subunits (₂) arranged quasi-symmetrically around a central cation-selective ion channel (4). The subunits each have a characteristic topology as follows: a large hydrophilic N-terminal domain containing the agonist binding sites, followed in the primary structure by three hydrophobic membrane-spanning segments (M1-M3), a cytoplasmic domain, a fourth hydrophobic transmembrane segment (M4), and a short extracellular C-terminal tail.

Noncompetitive antagonists (NCAs) are agents that block the AChR permeability response without binding to the agonist site. These compounds are structurally diverse and include many aromatic amines but also general anesthetics, steroids, and even the neuropeptide substance P (reviewed in Ref. 5). A number of NCAs have been instrumental in identifying regions of the AChR which contribute to the formation of the pore of the ion channel. Photoaffinity labeling studies with [³H]chlorpromazine (6-9) and [³H]triphenylmethylphosphonium (10) as well as reaction with [³H]meproadifen mustard (11) have all identified labeled residues within the M2 segments that would all lie on a common side of an -helix. These results, in conjunction with the observed functional properties of AChRs with mutations in the M2 segments (1, 2), provide the basis for a model of the ion channel comprised of M2 segments of each subunit arranged as transmembrane -helices around the central axis, a model consistent with studies of AChR three-dimensional structure derived from electron micrographic image analysis (4, 12).

The sites of [³H]chlorpromazine, [³H]triphenylmethylphosphonium, and [³H]meproadifen mustard incorporation were all identified in the presence of agonist under conditions where the AChR is expected to be in the desensitized state. More recently, 3-(trifluoromethyl)-3-(m-[¹²⁵I]iodo-phenyl)diazirine ([¹²⁵I]TID),a small, uncharged photoactivable probe, was used to identify residues in the channel lining M2 region both in the absence and presence of agonist, i.e. for AChRs predominantly in the resting state or desensitized state, respectively (13). [¹²⁵I]TID is a potent NCA (14, 15) that reacts nonspecifically with AChR amino acids in M3 and M4 hydrophobic segments at the lipid-protein interface (14-17) and specifically with amino acids in M2 segments of each AChR subunit (13). In the absence of agonist (resting state), [¹²⁵I]TID labeled Leu-265 and Val-269 and the homologous residues in the other subunits that are located 9 and 13 amino acids to the C-terminal side of the conserved lysine residue at the N terminus of the M2 region (positions 9 and 13). In the desensitized state, the pattern of [¹²⁵I]TID-labeled residues broadened to include homologous serine residues at positions 2 and 6 (i.e. Ser-258 and Ser-262). These results provided the first direct evidence of an agonist-induced structural rearrangement of the channel lining M2 helices. They further suggested that in the closed ion channel the aliphatic residues at positions 9 and 13 form a permeability barrier to the passage of ions.

To examine further the structure of the channel in different functional states, we examined the incorporation into the AChR of another lipophilic photoactivable probe, [³H]diazofluorene ([³H]DAF), both in the absence and presence of agonist. [³H]DAF has been used to probe the hydrophobic core of erythrocyte membranes (18) as well as the sites of lipid exposure of Staphylococcus aureus -toxin (19). Whereas incorporation of [¹²⁵I]TID and [³H]DAF both proceed through a UV-induced reactive carbene, the two compounds are structurally distinct, and TID produces a singlet carbene (20) whereas DAF produces a carbene with substantial triplet character (21, 22). We report here that like [¹²⁵I]TID, [³H]DAF not only incorporates into residues situated at the lipid-protein interface but also into residues in the channel lining M2 segments. We identify the amino acids within the M4 segments that are labeled nonspecifically as well as the pattern of specific photoincorporation within the M2 segments. The subunit selectivity of photolabeling within the M2 domain in conjunction with the agonist-dependent redistribution of labeling provide further information about the change in structure of the AChR ion channel domain between resting and desensitized states.

	EXPERIMENTAL PROCEDURES

Top Abstract Introduction Procedures Results Discussion References

Materials-- AChR-rich membranes were isolated from T. californica electric organ (17). 2-[³H]Diazofluorene ([³H]DAF) of specific activities ranging from 0.67 to 1.4 Ci/mmol was prepared from 2-[³H]fluorenone according to the procedure described by Pradhan and Lala (18), repurified, and stored at 20 °C in ethanol. [³H]Phencyclidine (43 Ci/mmol) was from NEN Life Science Products, and [³H]tetracaine (47 Ci/mmol) was prepared at NEN Life Science Products by catalytic tritiation of 3,3-dibromotetracaine. 1-Azidopyrene was purchased from Molecular Probes. Carbamylcholine and tetracaine were from Sigma, and phencyclidine (PCP) was from Alltech Associates. L-1-Tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin was purchased from Worthington and endoproteinase Lys-C from Boehringer Mannheim. Genapol C-100 (10%) was purchased from Calbiochem. Prestained low molecular weight gel standards were purchased from Life Technologies, Inc.

Photolabeling AchR-rich Membranes with [³H]DAF-- For analytical labeling experiments, Torpedo membranes (2 mg/ml) in Torpedo physiological saline (TPS, 250 mM NaCl, 5 mM KCl, 3 mM CaCl₂, 2 mM MgCl₂, 5 mM sodium phosphate, pH 7.0) were incubated with [³H]DAF at a final concentration of 5 µM in the absence or presence of 100 µM carbamylcholine and in the absence or presence of additional ligands. After a 30-min incubation, suspensions were irradiated for 5 min at a distance of less than 1 cm with a 365-nm lamp (EN-Spectroline). Following irradiation, each sample was pelleted (15,000 × g), the pellet solubilized in sample loading buffer (23), and then submitted to SDS-PAGE. Preparative photolabelings (12-15 mg per condition) were carried out at 10 µM [³H]DAF (±100 µM tetracaine) and in the presence of carbamylcholine (±100 µM phencyclidine). After 1 h incubation, sequential photoincorporation of [³H]DAF and then 1-azidopyrene (1-AP) was carried out as described previously for [¹²⁵I]TID (17), except that irradiation of suspensions with 1-AP was limited to 5 min. Samples were then pelleted and solubilized in electrophoresis sample loading buffer and submitted to SDS-PAGE.

SDS-Polyacrylamide Gel Electrophoresis-- SDS-PAGE was performed as described by Laemmli (23) using either 1.0-mm (analytical) or 1.5-mm (preparative scale) thick 8% polyacrylamide gels with 0.33% bis(acrylamide). For analytical gels, polypeptides were visualized by staining with Coomassie Blue R-250 (0.25% w/v in 45% methanol and 10% acetic acid) and destaining in 25% methanol, 10% acetic acid. The gels were then impregnated with fluor (Amplify, Amersham Pharmacia Biotech) for 20 min with rapid shaking, dried, and exposed at 80 °C to Kodak X-OMAT LS film for various times (3-12 weeks). Incorporation of ³H into individual polypeptides was quantified by scintillation counting of excised gel pieces as described (24). For preparative scale gels, polypeptides incorporating 1-AP were visualized from their associated fluorescence when the gels were illuminated at 365 nm on a UV-light box. Bands corresponding to AChR subunits were excised, and in some cases the gel pieces were transferred to the wells of individual 15% mapping gels (25, 26). Mapping gels were composed of a 4.5% acrylamide stacking gel and a 15% acrylamide separating gel. The gel pieces were overlaid with 350 µl of buffer (5% sucrose, 125 mM Tris-HCl, 0.1% SDS, pH 6.8) containing 250 µg of S. aureus V8 protease (500 µg V8 protease for gel pieces containing the -subunit). Electrophoresis was carried out overnight at 25 mA constant current. In the course of this work it was determined that for membranes labeled with 1-AP and [³H]DAF, the fluorescent- and ³H-labeled-AChR subunits comigrated, as did their large proteolytic fragments.

Purification of Proteolytic Digests of [³H]DAF/1-AP-labeled AChR Subunits to Isolate Fragments Containing the M2 Segment-- For trypsin digestion, acetone-precipitated subunits ( and ) were resuspended in a small volume (~50 µl) of buffer (100 mM NH₄HCO₃, 0.1% SDS, pH 7.8). The SDS concentration was then reduced by diluting with buffer without SDS, and Genapol C-100 was added, resulting in final concentrations of 0.02% SDS, 0.5% Genapol C-100, and 1-2 mg/ml protein. Trypsin was added to a 1:5 (w/w) enzyme to substrate ratio and incubated at room temperature for 4 days. For endoproteinase Lys-C (EKC) digestion, subunits () were resuspended in 15 mM Tris-HCl, 0.1% SDS, pH 8.1, at 1-2 mg/ml protein. Approximately 1.5 units of EKC was added and incubated at room temperature for 6 days. Both trypsin and EKC digests were separated on Tricine/SDS-polyacrylamide gels prepared as described (17, 28). Aliquots of each of the digests (~5%) were routinely resolved on analytical scale Tricine/SDS-polyacrylamide gels (1.0-mm thick) along with prestained molecular weight standards (Life Technologies, Inc.) as follows: ovalbumin (43,000), carbonic anhydrase (29,000), -lactoglobulin (18, 400), lysozyme (14, 300), bovine trypsin inhibitor (6, 200), the A chain of insulin (3, 400), and the B chain of insulin (2, 300). Analytical gels were soaked in 25% methanol, 10% acetic acid for 30 min, and then prepared for fluorography.

Generation and Isolation of Fragments of AChR Subunits Containing [³H]DAF/1-AP-labeled M4 Segments-- Fragments beginning near the N terminus of the M4 segment of each AChR subunit were isolated as described (17) for [¹²⁵I]TID/1-AP-labeled subunits. Briefly, in gel digestion of each isolated subunit with S. aureus V8 protease was used to generate 10-14-kDa subunit fragments as follows: V8-10 (Asn-339 to Gly-437); V8-12 (Met-384/Ser-417 to Ala-469); V8-14 (Leu-373/Ile-413 to Pro-489; V8-11 (Lys-436 to Ala-501). Trypsin digests of these fragments were fractionated by Tricine/SDS-PAGE yielding fluorescent and ³H containing bands of 3-4 kDa for -subunit (T-4K), 5 kDa for - and -subunits (T-5K and T-5K), and 6 kDa for -subunit (T-6K). Material eluted from these bands was further purified by reversed-phase HPLC. With the exception of the -subunit digest, each digest yielded a single major peak of ³H which coeluted with 1-AP fluorescence, and these peaks eluted at the same concentrations of organic solvent as had been seen for the [¹²⁵I]TID/1-AP-labeled M4 segments. Material in these fractions were pooled, dried, and resuspended for protein microsequence analysis. The tryptic digest of T-6K yielded a broader distribution of ³H without significant fluorescence, and material was pooled from the concentrations of organic eluent that had been found to contain [¹²⁵I]TID/1-AP-labeled M4.

Sequence Analysis-- N-terminal sequence analysis was performed on an Applied Biosystems model 477A protein sequencer using gas phase cycles. Pooled HPLC samples were dried by vacuum centrifugation, resuspended in a small volume of 0.05% SDS (~20 µl), and immobilized on chemically modified glass fiber disks (Beckman Instruments). Approximately 30% of the released PTH-derivatives were separated by an on-line Model 120A PTH-derivative analyzer, and approximately 60% was collected for determination of released ³H by scintillation counting of each sample for three 5-min intervals. Initial yield (I₀) and repetitive yield (R) were calculated by nonlinear least squares regression of the observed release (M) for each cycle (n): M = I₀Rⁿ (PTH-derivatives of Ser, Thr, Cys, and His were omitted from the fit).

Radioligand Binding Assays-- The equilibrium binding of [³H]PCP (6 nM), [³H]tetracaine (2 nM), and [³H]histrionicotoxin (10 nM) to Torpedo membranes was assayed by centrifugation. 500-µl aliquots of membrane suspensions (0.5 mg of protein/ml in TPS, ~0.6 µM AChR) were equilibrated with the radioligand and the nonradioactive cholinergic ligands for 2-3 h in 10 × 75-mm Pyrex disposable culture tubes (Corning) and then transferred to 1.5-ml plastic microcentrifuge tubes and pelleted for 45 min at 15,000 rpm in a Sorvall SA-600 rotor. After removal of the supernatants, the membrane pellets were solubilized in 100 µl of 10% SDS, and the pellet ³H was determined by liquid scintillation counting.

	RESULTS

Top Abstract Introduction Procedures Results Discussion References

In initial experiments, nonradioactive DAF (Fig. 1) was tested as an inhibitor of the equilibrium binding of radiolabeled, positively charged AChR NCAs. [³H]Tetracaine binds with high affinity (K_eq = 0.3 µM) in the absence of agonist to one site per AChR monomer, whereas it binds ~100-fold more weakly to desensitized AChRs (29). The sites of specific photoincorporation of [³H]tetracaine are restricted to amino acids within each M2 segment (30). In the absence of agonist, DAF produced a dose-dependent inhibition of [³H]tetracaine binding (IC₅₀ = 6 µM), with high concentrations inhibiting ~60% of specific binding² (Fig. 1A). For desensitized AChRs, [³H]phencyclidine binds with high affinity (K_eq = 1 µM) to a single site per AChR (31), and DAF also acted as an allosteric inhibitor of [³H]phencyclidine binding (Fig. 1B, IC₅₀ = 10 µM, 60% maximal inhibition). DAF also acted as an allosteric inhibitor of [³H]histrionicotoxin binding, with IC₅₀ = 2 µM and maximal inhibition of 50% in the presence of agonist (data not shown).

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Effects of diazofluorene on the binding of [3H]tetracaine and [3H]PCP in the absence and in the presence of carbamylcholine, respectively. AChR-rich membranes (0.5 mg/ml, 0.60 µM ACh binding sites) containing 2 nM [3H]tetracaine (A) or 6 nM [3H]PCP and 200 µM carbamylcholine (B) were equilibrated for 2-3 h with increasing concentrations of diazofluorene (DAF). Bound tritiated ligand () was determined by centrifugation ("Experimental Procedures"). In each panel the dashed line indicates nonspecific bound tritiated ligand in the presence of 200 µM tetracaine (A, ) or meproadifen (B, ) at 0 and 500 µM DAF.

Initial photolabeling experiments were designed to characterize the general pattern of [³H]DAF photoincorporation into Torpedo AChR-rich membranes as well as to test the sensitivity of the photoincorporation to cholinergic ligands. Membranes (2 mg/ml) were equilibrated with 5 µM [³H]DAF in the absence and in the presence of 100 µM carbamylcholine. After irradiation, membrane suspensions were pelleted and resuspended in electrophoresis sample buffer, and the pattern of incorporation was monitored by SDS-PAGE followed by fluorography. As is evident in the fluorograph of an 8% polyacrylamide gel (Fig. 2, lanes 3 and 4), there was appreciable incorporation of [³H]DAF into each of the AChR subunits. Neither the overall labeling pattern nor the relative incorporation into individual AChR subunits was affected by the inclusion of 100 µM carbamylcholine (Fig. 2, lane 4). Based on liquid scintillation counting of excised gel bands, ~1% of subunits incorporated ³H, with approximately equal incorporation in each subunit (///: 1/(0.8 ± 0.2)/(0.93 ± 0.3)/(1.2 ± 0.3)). The presence of carbamylcholine resulted in a <10% change of subunit labeling. Incorporation of [³H]DAF into the AChR subunits accounted for approximately 60% of the total labeling in polypeptides present in Torpedo AChR-rich membranes.

View larger version (99K): [in this window] [in a new window]   Fig. 2.   Photoincorporation of [3H]DAF into AChR-rich membranes in the absence and presence of carbamylcholine. AChR-rich membranes were equilibrated with [3H]DAF (5 µM) in the absence (lanes 1 and 3) and in the presence (lanes 2 and 4) of 100 µM carbamylcholine and irradiated at 365 nm for 5 min. Polypeptides were resolved by SDS-PAGE, visualized by Coomassie Blue stain (lanes 1 and 2), and processed for fluorography (4-week exposure; lanes 3 and 4). Labeled lipid and free photolysis products were electrophoresed from the gel with the tracking dye. The AChR subunits and the Na+/K+ ATPase -subunit (NK) are indicated. In addition, bands of 89 (89K), 37 (37K), 34 (34K), and 32 (32K) kDas are also indicated. These bands have been identified by N-terminal sequence analysis of purified proteolytic fragments to be the chloride channel CLC-0, calectrin (annexin V), the mitochondrial voltage-dependent anion channel, and the mitochondrial ATP/ADP translocase, respectively. The AChR-associated 43-kDa protein is not indicated but can be seen migrating with a slightly slower mobility than the AChR -subunit (lanes 1 and 2). Also not indicated is a 105-kDa band that can be seen migrating with slightly slower mobility than the -subunit of the Na+/K+ ATPase.

Two NCAs, tetracaine and proadifen, were tested at concentrations of 3, 30, and 250 µM for their effects on [³H]DAF photoincorporation into AChR-rich membranes in the absence and in the presence of 100 µM carbamylcholine, respectively. The effects of these ligands on [³H]DAF (5 µM) photoincorporation was examined by fluorography and by liquid scintillation counting of excised gel pieces. The pattern of incorporation was qualitatively very similar in each of the different conditions, with a small dose-dependent decrease (~15-25%) in the labeling of the subunits evident for both tetracaine and proadifen that was not seen for other labeled non-AChR polypeptides (data not shown). The relative distribution of ³H incorporation within the -subunit was examined by determining ³H incorporation within the four large, non-overlapping -subunit fragments that can be generated by digestion with S. aureus V8 protease (14, 26). Inspection of the fluorograph of the dried mapping gel indicated that all the visible labeling was contained within a 20-kDa fragment (V8-20, Ser-173-Glu-338) containing hydrophobic segments M1-M3 and a 10-kDa fragment (V8-10, Asn-339-Gly-437) containing the M4 segment. Based on liquid scintillation counting of the excised gel pieces, 75% of ³H cpm was incorporated in V8-10 and 25% was in V8-20, with the relative incorporation of [³H]DAF into V8-10 similar for labelings carried out in the absence (77%) and in the presence (74%) of 100 µM carbamylcholine.

Sites of [³H]DAF Incorporation in M4 Segments of Each AChR Subunit-- The M4 segments were isolated from tryptic digests of large V8-protease fragments of each of the receptor subunits as described under "Experimental Procedures." For -subunit, tryptic digestion of V8-10 produced a fluorescent and radioactive band of 3-4 kDa (T-4K), which was further purified by reversed-phase HPLC. N-terminal sequence analysis (Fig. 3A) revealed the presence of a single sequence beginning at Tyr-401 (490 pmol) that was present in a 10-20-fold greater abundance than any secondary sequence. The largest release of ³H occurred in cycle 12, with additional release in cycles 8, 15, and 18. The same pattern of ³H release was seen for the M4 segment isolated from membranes labeled in the presence of 100 µM carbamylcholine (data not shown). ³H release in cycle 12 indicated that Cys-412 was the primary site of incorporation of [³H]DAF in M4, as it was for [¹²⁵I]TID (17), with lower level reaction with His-408, Met-415, and Cys-418.

View larger version (27K): [in this window] [in a new window]   Fig. 3.   Radioactivity and mass release upon sequential Edman degradation of [3H]DAF/1-AP-labeled fragments containing the M4 segment. A, -subunit tryptic peptide T-4K isolated by HPLC from membranes labeled with 5 µM [3H]DAF (18,800 cpm loaded on the filter and 3,770 cpm remaining after 29 cycles). The only sequence detected began at Tyr-401 (I0, 490 pmol; R, 90%). B, -subunit tryptic peptide T-5K isolated by HPLC (3,480 cpm loaded on the filter and 591 cpm remaining after 29 cycles). The primary sequence began at Asn-427 before M4 (I0, 85 pmol; R, 95%), with a secondary sequence beginning at Tyr-401 before -M4 (I0, 24 pmol; R, 91% (see text3)). C, -subunit tryptic fragment T-5K isolated by HPLC (3,680 cpm loaded on the filter and 669 cpm remaining after 27 cycles). The only sequence detected began at Val-446 (I0, 110 pmol; R, 91%). D, -subunit tryptic peptide T-6K isolated by HPLC (1,660 cpm loaded on the filter and 290 cpm remaining after 24 cycles). The only sequence detected began at Leu-456 (I0, 49 pmol; R, 95%). For each sample 60% of each cycle of Edman degradation was analyzed for released 3H () and 30% for PTH-derivatives (), with the dashed lines corresponding to the exponential decay fit of the amount of detected PTH-derivatives for the peptides containing M4. The amino acid sequence of the sequenced peptide containing the M4 region is shown above each panel, with the solid line indicating the limits of the M4 regions.

[³H]DAF Labeling in the Ion Channel-- To determine the sites, the agonist sensitivity, and the specificity of [³H]DAF photoincorporation in the M2 regions of AChR subunits, T. californica AChR-rich membranes (~1.4 µM AChR) were photolabeled with 10 µM [³H]DAF under four different conditions as follows: 1) in the absence of agonist; 2) in the absence of agonist and in the presence of 100 µM tetracaine; 3) in the presence of 100 µM carbamylcholine; 4) in the presence of 100 µM carbamylcholine and in the presence of 100 µM phencyclidine (PCP).

Identification of the Sites of [³H]DAF Incorporation in -M2-- The -subunits isolated from AChRs labeled with [³H]DAF under the four different conditions (~300 µg/condition) were digested with 20% (w/w) trypsin for 4 days. The digests were resolved by Tricine/SDS-PAGE, and a 5.1-kDa fragment (T-5.1K; Fig. 4), known to contain the M2-M3 region (13), was isolated from the gel as described under "Experimental Procedures." The material eluted from the T-5.1K band was further purified by reversed-phase HPLC (Fig. 5), and for each labeling condition, the majority of ³H counts eluted in a peak centered at 74% solvent B. HPLC fractions 29-31 were pooled and sequenced (Fig. 6). For each of the labeling conditions, a single sequence was evident beginning at Met-257 at the N terminus of -M2. In addition, each of the samples sequenced with similar efficiencies and mass levels (see legend to Fig. 6). For T-5.1K labeled in the absence of agonist, ³H release occurred primarily in cycle 13 (Fig. 6A ()), a result that indicates that the labeled amino acid is Val-269 (26 cpm/pmol) in -M2. For the ³H release profile of T-5.1K labeled in the absence of agonist but in the presence of 100 µM tetracaine (, Fig. 6A), there was an approximately 90% reduction in [³H]DAF incorporation into Val-269 (3 cpm/pmol). The presence of agonist alone (Fig. 6B, ) caused a similar reduction in ³H incorporation into Val-269 (2 cpm/pmol), and there was also a small but significant release of ³H in cycles 6 and 9 that corresponds to [³H]DAF incorporation into Ser-262 (1.9 cpm/pmol) and Leu-265 (1.3 cpm/pmol) with similar efficiency as in Val-269. Finally, in the presence of both agonist and 100 µM PCP, there was no detectable ³H release in cycles 6 and 9, whereas release in cycle 13 was unaffected (Fig. 6B, ).

View larger version (53K): [in this window] [in a new window]   Fig. 4.   Trypsin digestion of -subunit from AChR photolabeled with [3H]DAF in the presence and absence of carbamylcholine (Carb) and noncompetitive antagonists. -Subunits, isolated from AChR-rich membranes labeled with [3H]DAF under the four different conditions indicated (top), were digested with 20% (w/w) trypsin for 4 days. Aliquots of the digests (~5%) were fractionated by Tricine/SDS-PAGE and then subjected to fluorography for 8 weeks. The migration of prestained molecular weight standards are indicated on the right, and the relative molecular masses of the principal [3H]DAF-labeled digestion products are shown on the left. The T-5.1K band contains a fragment beginning at Met-257 before M2 and extending through M3. The T-3.1K fragment contains the M2 region alone, N terminus: Met-257.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Reversed-phase HPLC purification of [3H]DAF-labeled tryptic fragment T-5.1K. T-5.1K isolated from tryptic digests of AChR -subunits (Fig. 4) was further purified by reversed-phase HPLC as described under "Experimental Procedures." The elution of [3H]DAF-labeled peptides was determined by scintillation counting of aliquots (25 µl) of the collected fractions (, ). Elution profiles are shown for T-5.1K material labeled in the absence of agonist but in the absence () or presence () of 100 µM tetracaine. Based upon the recovery of radioactivity, for each of the samples >90% of the material was recovered from the HPLC column.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Radioactivity and mass release upon N-terminal sequence analysis of -T-5.1K. -T-5.1K was isolated by Tricine/SDS-PAGE followed by reversed-phase HPLC (Fig. 5) and then subjected to automated Edman degradation, with 60% of each cycle analyzed for released 3H (, ) and 30% for PTH-derivatives (, with the dashed lines corresponding to the exponential decay fit of the amount of detected PTH-derivatives). The amino acid sequence of the sequenced peptide containing the M2 region is shown above each panel. A, 3H release from T-5.1K labeled in the absence of agonist (, /), or in the absence of agonist but in the presence of 100 µM tetracaine (, /+). /: I0 = 47 pmol; R = 93%; 15,000 cpm loaded/5,000 cpm remaining on filter. /+: I0 = 38 pmol; R = 94%; 9,000 cpm loaded/3,200 cpm remaining. B, 3H release from T-5.1K labeled in the presence of agonist (, +/) or in the presence of agonist and in the presence of 100 µM PCP (, +/+). +/: I0 = 51 pmol; R = 93%; 13,700 cpm loaded/3,900 cpm remaining. +/+: I0 = 57 pmol; R = 93%; 13,000 cpm loaded/3,500 cpm remaining.

Identification of the Sites of [³H]DAF Incorporation in -M2-- In a manner similar to that for -subunit, the sites of [³H]DAF photoincorporation in the M2 region of -subunit were determined by digesting the subunit (~250 µg/labeling condition) with 20% (w/w) trypsin for 4 days. The digests from each of the four labeling conditions were then fractionated by Tricine/SDS-PAGE and a 7.2-kDa band (T-7.2K), known to contain the M2-M3 region (13), was isolated (see under "Experimental Procedures"). When the gels were illuminated at 365 nm on a UV-light box, the T-7.2K fragment, which migrates as a sharp band of ³H in the fluorograph of an analytical Tricine/SDS-PAGE gel, migrated in an area of weak fluorescence between two strongly fluorescent bands of 10 and 5.5 kDas (data not shown). The material eluted from the T-7.2K fragment was further purified by reversed-phase HPLC (Fig. 7), and for each labeling condition the majority of ³H counts eluted in a peak centered at 84% solvent B. HPLC fractions 29-33 were pooled and sequenced (Fig. 8). In each of the four labeling conditions, sequence analysis revealed the presence of a single peptide beginning at Met-249 at the N terminus of M2 that was sequenced at similar efficiency and mass level for each sample (see legend to Fig. 8). For the sample labeled in the absence of any ligands other than [³H]DAF itself, the major release of ³H release was in cycle 13, with lower release in cycle 9 (, Fig. 8A). This pattern of release corresponds to [³H]DAF incorporation into Leu-257 (0.7 cpm/pmol) and Val-261 (5.3 cpm/pmol) within -M2. The presence of tetracaine (, Fig. 8A) resulted in an approximately 95% reduction in the incorporation into Val-261 (0.2 cpm/pmol). The addition of agonist (, Fig. 8B) resulted in a substantial reduction in the amount of [³H]DAF incorporated into Val-261, although the extent of inhibition was difficult to quantify because that reduced release in cycle 13 was associated with a dramatic increase in ³H release in cycles 9 and 10 that corresponds to increased incorporation of [³H]DAF into Leu-257 (2 cpm/pmol) and Ala-258 (~3 cpm/pmol). Interestingly, the presence of excess PCP (, Fig. 8B) resulted in an 80% reduction in the amount of incorporation into Leu-257 (0.4 cpm/pmol) but only a 50% reduction in the labeling of Ala-258 (1.5 cpm/pmol).

View larger version (20K): [in this window] [in a new window]   Fig. 7.   Reversed-phase HPLC purification of [3H]DAF-labeled tryptic fragment T-7.2K. A 7.2-kDa molecular mass fragment isolated by Tricine/SDS-PAGE from tryptic digests of AChR -subunit labeled under four different conditions material was then further purified by reversed-phase HPLC (see "Experimental Procedures"). The elution of [3H]DAF-labeled peptides was determined by scintillation counting of aliquots (25 µl) of the collected fractions (, ). Elution profiles are shown for T-7.2K material labeled in the absence of agonist but in the absence () or presence () of 100 µM tetracaine. Based upon the recovery of radioactivity, for each of the samples >90% of the material was recovered from the HPLC column.

View larger version (19K): [in this window] [in a new window]   Fig. 8.   Radioactivity and mass release upon N-terminal sequence analysis of -T-7.2K. T-7.2K was isolated by Tricine/SDS-PAGE followed by reversed-phase HPLC (Fig. 7) and then subjected to automated Edman degradation with 60% of each cycle analyzed for released 3H (, ) and 30% for PTH-derivatives (, with the dashed lines corresponding to the exponential decay fit of the amount of detected PTH-derivatives for the peptides containing M2). The amino acid sequence of the sequenced peptide containing the M2 region is shown above each panel. A, 3H release from T-7.2K labeled in the absence of agonist but in the absence (, /) or presence (, /+) of 100 µM tetracaine. /: I0 = 63 pmol; R = 88%; 9,200 cpm loaded/2,000 remaining on filter; /+: I0 = 61 pmol; R = 92%; 7,200 cpm loaded/1,400 cpm remaining). B, 3H release from T-7.2K labeled in the presence of agonist but in the absence (, +/) or presence of 100 µM PCP (, +/+). +/: I0 = 70 pmol; R = 90%; 10,800 cpm loaded/2,400 cpm remaining. (+/+: I0 = 70 pmol; R = 90%; 9,600 cpm loaded/2,900 cpm remaining.)

Identification of the Sites of [³H]DAF Incorporation in -M2-- The -subunits isolated from AChRs labeled under each of the four different conditions (~500 µg of -subunit/labeling condition) were each digested with 1.5 units of endoproteinase Lys-C for 6 days. The digests were then resolved by Tricine/SDS-PAGE, and an approximately 10-kDa band (K-10K), previously shown to contain the M2-M3 region (11), was excised (see "Experimental Procedures"). Inspection of the fluorogram of the gel of the analytical digests (Fig. 9) revealed that the K-10K fragment migrated with lower mobility than the major radiolabeled band of 7.8 kDa that was also intensely fluorescent (not shown). When the material eluted from the K-10K band was further purified by reversed-phase HPLC, for each labeling condition the majority of ³H counts were in a peak centered at 78% solvent B. HPLC fractions 31-34 were pooled and sequenced (Fig. 10). For each sample the primary sequence began at Met-243 before -M2 (~25 pmol), and a secondary sequence beginning at Tyr-401 before M4 was present at ~8 pmol.⁴ For all four of the samples, which correspond to each of the different labeling conditions, low level ³H release was seen in cycles 12, 15, and 18. This pattern of release corresponds to that observed for nonspecific [³H]DAF incorporation into -M4 region, i.e. labeling of Cys-412, Met-415, and Cys-418 (Fig. 3A).⁴ For both of the -K-10K samples labeled in the absence of agonist (with and without tetracaine), no other sites of ³H release were evident (Fig. 10), a result that indicates that [³H]DAF does not incorporate into -M2. Had [³H]DAF incorporated into Val-255 in -M2 at the same efficiency as it did into Val-269, release of 180 cpm would be observed in cycle 13. For the K-10K sample isolated from AChRs labeled in the presence of agonist, there was also ³H release in cycles 6 and 9 at levels similar to cycle 12 (data not shown) that was eliminated (cycle 6) by the presence of PCP. Thus, in the presence of agonist there was low level labeling (~1 cpm/pmol) of Ser-248 and Leu-251 as was seen for the equivalent amino acids in -M2 (Fig. 6B).

View larger version (55K): [in this window] [in a new window]   Fig. 9.   Endoproteinase Lys-C digestion of -subunit from AChRs photolabeled with [3H]DAF in the presence and absence of carbamylcholine and noncompetitive antagonists. -Subunit, which was isolated from AChR-rich membranes labeled with [3H]DAF under the four different conditions indicated (top), was digested with 1.5 units of endoproteinase Lys-C for 6 days. Aliquots of the digests (~5%) were fractionated by Tricine/SDS-PAGE and then subjected to fluorography for 6 weeks. The migration of prestained molecular weight standards are indicated on the right and the relative molecular masses of the principal [3H]DAF-labeled digestion products are shown on the left. The K-7.8K fragment contained the M4 region, amino termini: Ser-388, Tyr-401. The K-10K fragment contained a peptide beginning at Met-243 before M2 and a peptide beginning at Tyr-401 before M4 (see Fig. 10).

View larger version (23K): [in this window] [in a new window]   Fig. 10.   Radioactivity and mass release upon N-terminal sequence analysis of -K-10K. -K-10K was isolated by Tricine/SDS-PAGE (Fig. 9) followed by reversed-phase HPLC (see "Experimental Procedures") and then subjected to automated Edman degradation with 60% of each cycle analyzed for released 3H (, ) and 30% for PTH-derivatives (, with the dashed lines corresponding to the exponential decay fit of the amount of detected PTH-derivatives for the peptides containing M2). The amino acid sequences of the two primary peptides that were detected are both shown along the top axis. The sequence of the primary peptide began at Met-243 and contained the M2 region, whereas the secondary sequence began at Tyr-401 and contained M4. The profile of 3H release is shown for K-10K labeled in the absence of agonist (, /) or in the absence of agonist and in the presence of 100 µM tetracaine (, /+). (/ (Met-243): I0 = 25 pmol; R = 91%; (Tyr-401): I0 = 7.8 pmol; R = 93%; 14,600 cpm loaded/3,500 cpm remaining on filter after 20 cycles. /+ (Met-243): I0 = 25 pmol; R = 91%; (Tyr-401): I0 = 8.7 pmol; R = 88%; 15,700 cpm loaded/3,400 cpm remaining on filter after 20 cycles.)

	DISCUSSION

Top Abstract Introduction Procedures Results Discussion References

The results presented here demonstrate that the uncharged hydrophobic compound 2-[³H]diazofluorene ([³H]DAF) photoincorporates in a specific and agonist-sensitive fashion into residues in the channel lining M2 region of the nicotinic AChR. In addition, [³H]DAF photoincorporates in a agonist-insensitive manner into the M4 segment of each receptor subunit. Therefore, there exist two components to the labeling of the AChR by [³H]DAF as follows: a nonspecific component consistent with incorporation into residues situated at the lipid-protein interface of the AChR, and a specific component, inhibitable by noncompetitive antagonists, sensitive to state-dependent transitions, and localized to residues within the ion channel. It is significant that even in the absence of agonist the specific photoincorporation of [³H]DAF within - or -M2 can be clearly revealed only during N-terminal sequence analyses of isolated peptides, since that component of the photolabeling is 20% of the total labeling at the level of isolated subunits or even of proteolytic fragments after fractionation by SDS-PAGE and reversed-phase HPLC (Figs. 5 and 7). Unlike [¹²⁵I]TID at micromolar concentrations for which the reaction with residues within M2 segments comprises 70% of the total labeling at the subunit level, for [³H]DAF at micromolar concentrations labeling of -M2 does not predominate over labeling of residues in the M4 segments. For example, for M2 and M4 segments isolated from the same labeling experiment, the inhibitable ³H incorporation in Val-269 (7-8 cpm/pmol) is ~twice the level of nonspecific labeling in Cys-412 (3-4 cpm/pmol), the most highly labeled residue in M4.

Characterization of [³H]DAF Labeling at the Lipid-Protein Interface-- The majority of AChR labeling by [³H]DAF appears similar to the component of [¹²⁵I]TID labeling which is inhibitable neither by agonist nor by an excess of non-radioactive TID and which is consistent with photoincorporation into lipid-exposed regions of the AChR (14-17). [³H]DAF incorporation into each AChR subunit M4 segment was determined under labeling conditions done in the absence and in the presence of 100 µM carbamylcholine. In either condition [³H]DAF reacted in -M4 with His-408, Cys-412, Met-415, and Cys-418 (Fig. 3A); in -M4 with Tyr-441, Phe-443, Phe-444, and Cys-447 (Fig. 3B); in -M4 with Cys-451 and Trp-453 (Fig. 3C). The low level of ³H release within -M4 allows only a tentative assignment for Met-458 (Fig. 3D). Although the isolation of M4 fragments by alternative digestion strategies would provide unambiguous proof that the observed ³H release originates from the observed M4 peptides, this is the expected result based upon previous studies with [¹²⁵I]TID that establish that the labeled and unlabeled peptides coelute from the reversed-phase column (16, 17). It is also important to point out that due to the lags resulting from the ~90% repetitive yields inherent in the Edman degradation reaction, cycles that follow a labeled amino acid also contain prior PTH-derivatives and associated ³H. It is therefore difficult to detect the presence of labeled amino acids which immediately follow another labeled amino acid, particularly if the efficiency of incorporation into those residues is significantly lower. Without direct characterization of the ³H-labeled amino acids released in each cycle, one cannot determine whether some of the ³H release in cycles immediately following these labeled residues is due to amino acids which have also reacted with [³H]DAF.

View larger version (24K): [in this window] [in a new window]   Fig. 11.   Helical representations of the -, -, -, and -subunit M4 regions. Helical representations of the -, -, -, and -subunit M4 regions. [3H]DAF-labeled amino acid residues are indicated along the left side of the helix along with the location of the labeled amino acid in the subunit primary sequence. Asterisks denote residues that were previously shown to be labeled by [125I]TID (16, 17). Helices are oriented with N termini at the bottom to reflect the extracellular location of the carboxyl termini (46).