Introduction

Top Summary Introduction Experimental Procedures Results Discussion References

The nicotinic acetylcholine receptor (nAChR) isolated from the electric organ of the marine ray Torpedo is the best characterized ligand-gated ion channel (for reviews, see Karlin and Akabas, 1995; Hucho et al., 1996). The nAChR is composed of four homologous subunits in the stoichiometry ₂, which electron microscopic studies show are arranged pseudosymmetrically about a central axis that is the ion channel (Unwin, 1993). The M2 hydrophobic segments from each subunit line the lumen of the ion channel (Hucho et al., 1986; Imoto et al., 1988, 1991; Charnet et al., 1990; Revah et al., 1990) and undergo a conformational change when the nAChR binds agonist (White and Cohen, 1992; Akabas et al., 1994; Unwin, 1995). Within the M2 domain, a conserved leucine (Leu²⁵¹ and the homologous position in the other subunits) appears to be important for the gating of the ion channel. Cryoelectron microscopy reveals an agonist-dependent rotation of a kink in the M2 helices near the predicted position of these leucines (Unwin, 1995), referred to as position 9 with reference to the conserved Lys at the N-terminal end of each M2 segment, and site-directed mutagenesis demonstrates that this position is an important determinant of the equilibrium between closed and open channel states (Filatov and White, 1995; Labarca et al., 1995). However, it is unclear whether this ring of leucines forms the permeability barrier in the closed channel because cysteine mutants below position 9 are accessible to water-soluble sulfhydryl reagents in the absence of agonist (Akabas et al., 1994; Pascual and Karlin, 1998).

Noncompetitive antagonists (NCAs), which are compounds that block the permeability response without preventing the binding of agonist, have also been used to characterize the structure of the ion channel pore. For nAChR in the desensitized state, [³H]chlorpromazine is photoincorporated into residues at position 6 of each subunit's M2 segment as well as into position 2 in M2 and 9 in M2 and M2 (Giraudat et al., 1986, 1987, 1989; Revah et al., 1990). The labeling of these residues establishes that [³H]chlorpromazine's binding site is within the lumen of the ion channel toward the cytoplasmic end of the pore and helps to identify the pore-lining faces of the M2 segments. In contrast, in the desensitized nAChR, the NCA [³H]meproadifen mustard reacts specifically with Glu²⁶² at position 20 at the extracellular end of the pore (Pedersen et al., 1992).

Unlike chlorpromazine and meproadifen, the uncharged NCA 3-(trifluoromethyl)-3-(M-[¹²⁵I]iodophenyl)diazirine ([¹²⁵I]TID) binds with similar affinity to both the resting and desensitized states (White et al., 1991) and therefore is a useful probe of the channel in the absence as well as the presence of agonist. In the absence of agonist, [¹²⁵I]TID specifically photoincorporates into M2 residues at positions 9 and 13, whereas in the desensitized state, it labels residues deeper in the pore (positions 2 and 6) as well as the residues at 9 and 13 (White and Cohen, 1992). This study directly demonstrated a structural change in the pore between resting (closed channel) and desensitized states and suggested that the aliphatic residues at position 9 from each subunit associate in the lumen to provide the permeability barrier in the closed channel.

To better characterize the structure of the ion channel in the closed state, we identified the binding site of the NCA [³H]tetracaine, in the absence of agonist. Unlike the NCAs used in previous photoaffinity labeling studies, tetracaine binds to the nAChR with high affinity in the absence of agonist (K_eq = 0.3 µM) and with 100-fold lower affinity in the presence of agonist (Blanchard et al., 1979). Middleton et al. (1999) established that [³H]tetracaine binds with a 1:1 stochiometry to the nAChR and that ultraviolet irradiation at 302 nm resulted in specific [³H]tetracaine photoincorporation into each subunit. We report here the identification of the individual amino acids of the nAChR that photoincorporated [³H]tetracaine. These data show that the high-affinity [³H]tetracaine-binding site is located within the lumen of the ion channel. The labeled amino acids define the surface of the M2 helix extending one helical turn above and below position 9 from each subunit that lines the lumen of the channel in the absence of agonist.

	Experimental Procedures

Top Summary Introduction Experimental Procedures Results Discussion References

Materials. [³H]Tetracaine was prepared at New England Nuclear Research Products (Boston, MA) by palladium/charcoal-catalyzed reduction in 3,5-dibromotetracaine with carrier-free ³H₂ gas. [³H]Tetracaine was purified from the tritium reduction products by silica thin-layer chromatography (5:4:1 cyclohexane/chloroform/diethylamine; R_f = 0.17). The purity (>90%) and specific activity (48 Ci/mmol) were determined by silica HPLC (Hibar Si-60 10 µm, 200:1 acetonitrile/diethylamine isocratic elution). nAChR-rich membranes were isolated from electric organs of Torpedo californica (Marinus Inc., Westchester, CA) as described previously (Pedersen et al., 1992) and stored at 80°C in 38% sucrose. Endoproteinase Lys-C (EKC) was obtained from Boehringer-Mannheim (Indianapolis, IN). l-1-Tosylamido-2-phenylethylchloromethylketone-treated trypsin was obtained from Worthington Biochemical (Freehold, NJ). Staphylococcus aureus glutamyl endopeptidase (V8 protease) was purchased from ICN Biomedical Inc. (Costa Mesa, CA). Oxidized glutathione and tricine were from Sigma Chemical Co. (St. Louis, MO). o-Phthalaldehyde (OPA), 10% Genapol C-100, and trifluoroacetic acid (TFA) were obtained from Pierce Chemical Co. (Rockford, IL). 1-Azidopyrene (1-AP) was purchased from Molecular Probes (Eugene, OR). dl-perhydrohistrionicotoxin (HTX) was kindly provided by Dr. Y. Kishi (Harvard University, Cambridge, MA).

Photolabeling of nAChR-Rich Membranes with [³H]Tetracaine and Isolation of Labeled nAChR Subunits. Freshly thawed suspensions of nAChR-rich membranes (10-14 mg protein) were diluted 1:2 in Torpedo physiological saline (TPS; 250 mM NaCl, 5 mM KCl, 3 mM CaCl₂, 2 mM MgCl₂, 5 mM NaPi, pH 7.0) and pelleted (15,000g) for 30 min at 4°C. The membrane pellets were then resuspended at ~2 mg protein/ml in TPS supplemented with 50 mM oxidized glutathione (Middleton et al., 1999). [³H]Tetracaine was then added at a final concentration of 5 µM, as well as 30 µM HTX for the samples used to define nonspecific photolabeling. The suspensions (2.5-ml volumes) were incubated for 30 min at room temperature with stirring in covered glass crystallization dishes (3 cm diameter) that were flushed with Ar(g) for 15 min immediately before photolysis. The samples, contained in a water bath that served to keep the sample temperature below 25°C, were then irradiated at 302 nm for 30 min with stirring. The lamp (Spectroline model EB-280C; Spectronics, Westbury, NY) was at a distance of 12 cm and had an intensity of ~1000 µW/cm² (as measured by a Spectroline DIX 300 digital radiometer). After irradiation, samples were pelleted, resuspended to ~2 mg/ml in TPS, and photolabeled as described (Blanton and Cohen, 1994) with the fluorescent, hydrophobic probe 1-AP. The membranes were then pelleted and resuspended in electrophoresis sample loading buffer for isolation of nAChR subunits by SDS-polyacrylamide gel electrophoresis (PAGE). Labeling with 1-AP was used to facilitate the initial identification and isolation of nAChR subunits and for the subsequent isolation of their hydrophobic fragments (Blanton and Cohen, 1994).

SDS-PAGE. nAChR subunits were resolved by SDS-PAGE using the Laemmli buffer system (Laemmli, 1970) with 1.5-mm-thick, 8% acrylamide slab gels containing 0.32% bisacrylamide. After electrophoresis, the unstained gels were illuminated on a 365-nm UV light box, and the nAChR subunits were visualized by 1-AP fluorescence. The nAChR , , and  subunits were excised from the gel and eluted in 10 ml of elution buffer (0.1 M NH₄HCO₃/0.1% SDS). The band containing the nAChR  subunit was excised from the gel and placed on top of the stacking portion of a 15% mapping gel for in gel digestion with S. aureus V8 protease (Pedersen et al., 1986). The  subunit V8 protease fragments of 20 kDa (V8-20, Ser¹⁷³-Glu³³⁸) and 10 kDa (V8-10; Asn³³⁹-Gly⁴³⁷) were visualized by 1-AP fluorescence (Blanton and Cohen, 1994), excised from the gel, and eluted as above. After 4 days of elution, subunits (or subunit fragments) were concentrated with Centriprep-30 (or -10) Microconcentrators (Amicon, Beverly, MA) to approximately 400 µl, and excess SDS was removed by acetone precipitation at 20°C. The precipitates were resuspended in 15 mM Tris (pH 8.1)/0.1% SDS at 0.4 mg protein/ml. Protein concentration was measured using a bicinchoninic acid-based protein assay (Micro BCA Protein Assay, Pierce Chemical Co.), and [³H]tetracaine incorporation was determined by scintillation counting.

Purification of Proteolytic Digests of [³H]Tetracaine/1-AP -Labeled nAChR Subunits. nAChR V8-20 and  subunit at 0.4 to 0.6 mg protein/ml in 15 mM Tris (pH 8.1)/0.1% SDS were digested with 4 U/ml EKC or with 1:1 (w/w) S. aureus V8 protease at ambient temperature. The  and  subunits were digested with trypsin (25% w/w) at ambient temperature in 15 mM Tris (pH 8.1)/0.1% SDS supplemented with 0.5% Genapol C-100. The digestion times are indicated in the figure legends. The proteolytic digests were fractionated by Tricine SDS-PAGE as described (Schagger and von Jagow, 1987; Blanton and Cohen, 1994). To identify regions of the gel that contained specifically labeled fragments, an aliquot (5 µg) of each digest was run in a separate lane of the 1.5-mm-thick preparative gel, and another lane contained prestained molecular weight standards (Life Technologies, Gaithersburg, MD): insulin B (2300) and A (3400) chains, bovine trypsin inhibitor (6200), lysozyme (14,300), -lactoglobulin (18,400), carbonic anhydrase (29,000), and ovalbumin (43,000). The analytical lanes were cut into 3-mm slices and placed into scintillation tubes. After dissolution of the acrylamide in 800 µl of 30% H₂O₂ heated to 80°C for more than 1 h, the ³H in each sample was determined by scintillation counting in Dimiscint (National Diagnostics, Atlanta, GA).

Sequence Analysis. N-terminal sequence analysis was performed on an ABI model 477A (Applied Biosystems, Foster City, CA) protein sequencer with gas phase cycles. HPLC fractions that contained specifically labeled material were pooled and dried by vacuum centrifugation. The dried samples were resuspended in 25 µl of 100 mM NH₄HCO₃/0.05% SDS and then loaded onto chemically modified glass-fiber disks (Beckman, Palo Alto, CA) that were used rather than polybrene-treated filters to improve the sequencing yields of hydrophobic peptides (Pedersen et al., 1992). Before beginning the Edman degradation cycles, the sample in the sequencer cartridge was exposed to TFA vapor for 4 min to fix the sample to the filter, and then SDS was removed by a 5-min wash of the filter with ethyl acetate. Approximately 30% of each degradation cycle was injected into the on-line ABI model 120 A amino acid analyzer to determine the identity of the released phenylthiohydantoin (PTH)-amino acid derivatives, and 60% was delivered to a fraction collector for scintillation counting. During the sequencing of samples containing -M1, sequencing of contaminating peptides was blocked by treatment of the sample on the filter with OPA. OPA reacts with primary, but not secondary amines, and can be used to block Edman degradation of any peptide not containing an N-terminal proline (Brauer et al., 1984). OPA treatment was carried out as described previously (Middleton and Cohen, 1991). The Applied Biosystem model 610A Data Analysis Program Version 1.2.1 was used to analyze the sequencer chromatograms and calculate the background-subtracted amount of PTH-amino acid present in each cycle of Edman degradation. Initial peptide mass (I₀) and repetitive yield (R) were determined by nonlinear least-squares regression (Sigma Plot) of the function I = I₀ × Rⁿ, where I is the observed mass of PTH-derivative at cycle n. The background-subtracted mass of the PTH derivatives of Ser, Cys, Arg, His, and Trp were excluded from the fits because of known problems of measuring their mass yields.

	Results

Top Summary Introduction Experimental Procedures Results Discussion References

To identify the amino acids of the nAChR that specifically photoincorporated [³H]tetracaine, we labeled nAChR-rich membranes (5 mg protein, 2 mg/ml) equilibrated with 5 µM [³H]tetracaine in both the absence and presence of 30 µM HTX. The nAChR subunits were purified in good yield (typically 100-200 µg). Then , , and  subunit preparations each incorporated about 1800 to 2000 ³H cpm/µg protein, with  subunit labeling ~75% specific (i.e., inhibitable by HTX) and  and  subunit labeling ~55 and ~70% specific. Although intact  subunit was not routinely isolated, preparations of V8-20 incorporated ~2500 ³H cpm/µg with an 80% reduction for V8-20 isolated from nAChRs labeled with [³H]tetracaine in the presence of HTX. The levels of HTX-inhibitable ³H incorporation in each subunit indicated specific incorporation of [³H]tetracaine in ~0.1% of subunits, a level somewhat lower than that seen in the analytical experiments of Middleton et al. (1999).

[³H]Tetracaine Photoincorporation in  Subunit. In initial experiments, we performed a series of analytical scale digests (5-µg aliquots of labeled  subunit) with EKC that were analyzed both by the ³H distribution after Tricine SDS-PAGE and by the profile of ³H release when the total digest was sequenced. Digestion conditions that resulted in a fragment (or fragments) releasing ³H after 9 and 13 cycles of Edman degradation were identified (not shown). The digest contained a single, specifically labeled fragment of ~10 kDa, as shown in Figure 1A, the analytical lanes (5-µg aliquots) of a preparative scale digest of  subunits (~150 µg protein) isolated from nAChRs labeled in the absence or presence of HTX. The peak of ³H corresponded to a band of 1-AP fluorescence in the preparative lanes and a 5-mm strip containing the fluorescent band, as well as sections below and above the fluorescent band, were excised from each preparative lane, and the protein in them was eluted. The eluates from the fluorescent band (band C) and the band above (band D) contained the highest amounts of ³H, which when combined accounted for 38% of the eluted cpm. The material eluted from these two sections was pooled and is referred to as EKC-10.

View larger version (43K): [in this window] [in a new window]   Fig. 1.   Proteolytic mapping of [3H]tetracaine incorporation in nAChR  subunit with endoproteinase Lys-C (A-C) or S. aureus V8 protease (D). All panels display results from parallel photolabelings of nAChR-rich membranes with [3H]tetracaine in the absence () or presence () of HTX. Solutions (0.4 mg/ml) of [3H]tetracaine-labeled  subunit were digested with EKC (3U/ml) for 5 days at ambient temperature and fractionated by Tricine SDS-PAGE with 5-µg aliquots (HTX, 10,780 cpm; +HTX, 3050 cpm) electrophoresed in analytical lanes adjacent to the bulk digests. A, distribution of 3H in the analytical lanes is shown in relation to molecular weight markers (above) and the strips (A-F) excised from the preparative lanes. For the HTX sample, 543,300 cpm was loaded onto the preparative lane, with 14,640, 17,000, 66,560, 31,640, 11,520, and 7600 cpm recovered from strips A to F, respectively. The eluates from strips C and D were combined and designated as EKC-10. B, reversed phase HPLC purification of EKC-10 (5% of the HPLC fractions) were assayed for 3H. Specifically labeled material (66% of loaded cpm) eluted in a single peak (fractions 20-23). Fractions 21 and 22 were pooled for sequence analysis. C, 3H and mass release on N-terminal sequencing of EKC-10 purified by reversed phase HPLC. The only sequence detected in either sample began at Met257, the N terminus of M2 (, HTX, I0 = 10 pmol, R = 94%, 10,000 cpm loaded/1800 cpm remained on the filter; +HTX, I0 = 11 pmol, R = 94%). The sequence of the identified peptide is shown above. D, 3H release from N-terminal sequencing of V8-3. Solutions of [3H]tetracaine-labeled  subunit (0.6 mg/ml) were digested with an equal weight of S. aureus V8 protease for 14 days at ambient temperature and then separated by Tricine SDS-PAGE (not shown). A 3-kDa specifically labeled fragment (V8-3) was excised, purified by reversed phase HPLC (not shown), and sequenced (HTX, 13,700 cpm loaded/2300 cpm remained on the filter). As many as five different PTH derivatives were identified in each cycle of Edman degradation.

[³H]Tetracaine Photoincorporation in  Subunit. White and Cohen (1992) showed that digestion of [¹²⁵I]TID-labeled  subunit with trypsin generated a 7-kDa fragment beginning at the N terminus of M2 and extending through the M3 segment, as well as 3- and 10-kDa fragments that also contained M2. Therefore, we digested [³H]tetracaine-labeled  subunit (160 µg, 290,000 cpm) with trypsin [25% (w/w)] for 4 days at ambient temperature and then purified the digestion products by Tricine SDS-PAGE. Aliquots (5 µg) were electrophoresed in analytical lanes adjacent to the bulk digest. The ³H distribution in these analytical lanes (Fig. 2A) showed that specifically labeled material migrated in bands at 7 and 3 kDa. Material was eluted from gel strips (bands A-G) that were excised from the preparative lanes that spanned from below the 3-kDa marker to above the 14-kDa marker. Bands B and C, which contained 54,000 cpm (19% of loaded counts), were combined and denoted T-3, and bands E and F, which contained 52,000 cpm (18% of loaded counts), were combined and denoted T-7.

View larger version (49K): [in this window] [in a new window]   Fig. 2.   Proteolytic mapping with trypsin of [3H]tetracaine incorporation in the nAChR  subunit. All panels display results from parallel photolabelings of nAChR-rich membranes with [3H]tetracaine in the absence () or presence () of HTX. Solutions (0.7 mg/ml) of [3H]tetracaine-labeled  subunit were digested with trypsin (0.2 mg/ml) for 4 days at ambient temperature and then fractionated by Tricine SDS-PAGE with 5-µg aliquots (HTX, 9000 cpm; +HTX, 2630 cpm) electrophoresed in analytical lanes adjacent to the bulk digests. A, distribution of 3H in the analytical lanes is shown in relation to molecular weight markers (above) and the strips (A-G) excised from the preparative lanes. For the HTX sample, 213,600 cpm was loaded on the preparative lane, with 19,200, 29,200, 25,000, 20,450, 25,650, 26,350, and 12,850 cpm recovered from strips A to G, respectively. The eluates from strips B and C (~3 kDa) were combined and designated as fragment T-3, and the eluates from strips E and F (~7 kDa) were combined and designated as fragment T-7. B and D, reversed phase HPLC purification of T-7 and T-3 (5% of the HPLC fractions were assayed for 3H). B, for T-7 (HTX), 46% of the 51,000 cpm injected was recovered in a single peak associated with two absorbance peaks. Fractions 22 to 24 were combined for sequence analysis. D, for T-3 (HTX), specifically labeled material eluted in three peaks that together accounted for 70% of the injected 3H. Fractions 13 to 15 (T-3a, 30% of injected 3H) and fractions 16 to 19 (T-3b, 20% of injected 3H) were pooled for sequence analysis. C, 3H and mass release on N-terminal sequencing of T-7 purified by reversed phase HPLC. For both samples, the primary sequence began at Met249, the N terminus of M2 (, HTX, I0 = 13 pmol, R = 92%, 16,420 cpm loaded/3180 cpm remained on the filter; +HTX, I0 = 12 pmol, R = 96%), and a secondary sequence began at Lys216, the N terminus of M1 (HTX, I0 = 6 pmol, R = 92%; +HTX, I0 = 8 pmol, R = 95%). The sequences of the identified peptides are shown above. Additional PTH derivatives were present at <20% the level the primary sequence. E, sequence analysis of T-3b. After one cycle of Edman degradation, the sequencing filter was treated with OPA, which reacts selectively with the free N termini of amino acids other than proline, a secondary amine, blocking Edman degradation (Brauer et al., 1984). After OPA block, the primary sequence in both samples began at Lys216, the N terminus of M1 (HTX, I0 ~ 1 pmol, R = 95%, 10,400 cpm loaded/410 cpm remained on the filter). The sequence of the identified peptide is shown above.

[³H]Tetracaine Photoincorporation in  Subunit. The specific [³H]tetracaine photolabeling in the  subunit is contained within V8-20 (Ser¹⁶²/Ser¹⁷³ to Glu³³⁸/Asn³³⁹), a proteolytic fragment produced by an "in gel" digest of  subunit with V8 protease (Middleton et al., 1999). To determine whether that specific labeling was within M2, [³H]tetracaine-labeled V8-20 was digested with endoproteinase Lys-C, which had been used previously (Pedersen et al., 1992) to generate an ~8-kDa fragment beginning at the N terminus of M2 in studies of the site of labeling of [³H]meproadifen mustard. When the EKC digest was fractionated by Tricine SDS-PAGE, the distribution of ³H in the analytical lanes (Fig. 3A) contained a peak of specifically labeled material migrating at ~8 kDa, as well as material aggregated near the top of the gel and a band of ~14 kDa. The preparative lanes of the gels were cut into strips (referred to as bands A-G), which together spanned the gel from below the 6-kDa marker to above the 14-kDa molecular weight marker. For the digest of V8-20 labeled in the absence of HTX, 14,000 cpm of the 74,000 cpm loaded on the gel was recovered from bands D and E. This material, which was combined and denoted as EKC-8, accounted for 40% of the ³H cpm eluted. When EKC-8 was further purified by HPLC (Fig. 3B), 50% of the loaded cpm was recovered in a single peak (fractions 25-27) associated with single peaks in fluorescence and absorbance. For the sample labeled in the presence of HTX, the ³H in that peak was reduced by >90%, whereas the peaks in fluorescence and absorbance were similar in size to those seen for the specifically labeled sample (not shown).

View larger version (45K): [in this window] [in a new window]   Fig. 3.   Proteolytic mapping of [3H]tetracaine incorporation in the nAChR  subunit with endoproteinase Lys-C (A-C) or S. aureus V8 protease (D). All panels display results from parallel photolabelings of nAChR-rich membranes with [3H]tetracaine in the absence () or presence () of HTX. Solutions (0.6 mg/ml) of V8-20 isolated from [3H]tetracaine-labeled  subunit were digested with EKC (3 U/ml) for 6 days at ambient temperature and fractionated by Tricine SDS-PAGE with 5-µg aliquots (HTX, 16,060 cpm; +HTX, 3315 cpm) electrophoresed in analytical lanes adjacent to the bulk digests. A, the distribution of 3H in the analytical lanes is shown in relation to molecular weight markers (above) and the strips (A-G) excised from the preparative lanes. For the HTX sample, 73,900 cpm was loaded on the preparative lane, with 1550, 2750, 2050, 7150, 7550, 2000, and 1850 cpm recovered from strips A to G, respectively. The eluates from strips D and E were combined and designated as EKC-8. B, reversed phase HPLC purification of EKC-8 (5% of the HPLC fractions were assayed for 3H). For the HTX sample, 50% of the 14,000 cpm injected was recovered in a single peak associated with a single peak in absorbance and fluorescence (not shown). Fractions 25 to 27 were combined for sequence analysis. C, 3H and mass released from N-terminal sequencing of EKC-8 purified by reversed phase HPLC. The only sequence detected in either sample began at Met243, the N terminus of M2 (, HTX, I0 = 11 pmol, R = 86%, 6740 cpm loaded/680 cpm remained on the filter; +HTX, I0 = 27 pmol, R = 86%). The sequence of the identified peptide is shown above. This sequence was present at a 10-fold higher level than any secondary sequence. D, 3H release on N-terminal sequencing of an S. aureus V8 protease digest of V8-20. Solutions of [3H]tetracaine-labeled V8-20 (0.3 mg/ml) were further digested with equal weight of V8 protease for 28 days at ambient temperature and then separated by reversed phase HPLC (not shown). The 3H peak (43% of recovered 3H), which eluted at 88% organic, was sequenced (HTX, 14,500 cpm loaded/3640 cpm remained on the filter). The presence of multiple PTH derivatives in each cycle of Edman degradation precluded the identification of peptides in this sample.

[³H]Tetracaine Photoincorporation in  Subunit. Attempts to isolate a labeled fragment of  subunit were unsuccessful. Digestion of intact  subunit with either endoproteinase Lys-C or trypsin resulted in specifically labeled fragment or fragments of <3 kDa, which when repurified by HPLC eluted in common with many peptides. After digestion of  subunit (170 µg) with trypsin, 31% of the specifically labeled material migrated in a band below 3 kDa (T-3), a result consistent with the presence of trypsin sites at the N and C termini of M2. When further purified by reversed phase HPLC, 34% of the specifically labeled cpm were recovered in a peak centered at 48% organic, consistent with a previously characterized 3-kDa peptide containing M2 (White and Cohen, 1992). Sequence analysis of HPLC-purified T-3 revealed the presence of a complex mixture of unidentifiable peptides. There was a peak of ³H release in cycle 9 (40 cpm), which, if it originated from M2, would correspond to Leu²⁶⁰, the amino acid homologous to the most highly labeled amino acids in the other subunits.

	Discussion

Top Summary Introduction Experimental Procedures Results Discussion References

In this report, we mapped the binding site for [³H]tetracaine in the closed channel state of the Torpedo nAChR by identifying the amino acids specifically photolabeled by this drug. N-terminal sequencing demonstrated that the specific photoincorporation within the , , and  subunits was contained within proteolytic fragments that included the M2 segments. Sites of specific ³H release in these fragments corresponded to labeling within the M2 segments of Ile²⁴⁷, Leu²⁵¹, Val²⁵⁵, Leu²⁵⁷, Leu²⁶⁵, Ala²⁶⁸, and Val²⁶⁹. Trypsin digestion of labeled  subunit produced a labeled proteolytic fragment whose size and hydrophobicity were consistent with the known properties of isolated M2 segments, and sequence analysis of this material showed release of ³H nine cycles after the N terminus that, if originating from M2, corresponds to Leu260.

Each of the labeled residues lies on a common helical face of the M2 segments (Fig. 4A). Because [³H]tetracaine binds to the nAChR with a 1:1 stoichiometry (Middleton et al., 1999), the photolabeling data provide clear evidence that this binding site is located within the lumen of the closed ion channel and identify the amino acids within M2 that are in close proximity to bound tetracaine. Although [¹²⁵I]TID labels many of the same residues (White and Cohen, 1992), the additional residues labeled by [³H]tetracaine (Ile²⁴⁷, Ala²⁶⁸) extend the definition of the surface of the M2 helix that is oriented toward the lumen of the closed ion channel (Fig. 4B; [³H]tetracaine:  arc = 100 degrees,  arc = 80 degrees; [¹²⁵I]TID: , ,  arcs = 40 degrees).

View larger version (58K): [in this window] [in a new window]   Fig. 4.   The pattern of [3H]tetracaine photoincorporation defines the surface of the M2 segments lining the closed channel and suggests a model for tetracaine binding within the nAChR ion channel. A, space-filling, -helical models of each subunit's M2 segment and a model at the same scale of tetracaine in an extended conformation were made using the molecular modeling software Insight (Biosym, Inc.). The M2 residues labeled by [3H]tetracaine, which are shaded, lie on a single helical face. The presumed photoreactive aromatic atoms of tetracaine are positioned at the same level as the labeled residues of the M2 helices. In this orientation, tetracaine's dimethylaminoethyl group would be in proximity to the hydrophilic side chains at position M2-6, whereas its N-butyl group is aligned with the hydrophobic residues toward the C terminus of the M2-segment. B, when depicted on a helical wheel, the  carbons of the labeled residues in the  subunit (positions 5, 9, and 13) span 80 degrees of the helix cylinder, and those of the  subunit (positions 9, 12, and 13) span 100 degrees of the helical face. Thus, identification of amino acids labeled by [3H]tetracaine extends the definition of the surface of the M2 helices lining the lumen of the closed channel beyond those side labeled by [125I]TID (residues 9 and 13; White and Cohen, 1992).

The photochemistry of [³H]tetracaine is unknown. However, the photolysis products of para-aminobenzoic acid (Chignell et al., 1980; Gasparro, 1985) include reactive radicals at the aromatic nitrogen and on the adjacent carbon on the benzene ring. This suggests that reactive radicals should be produced at tetracaine's aromatic atoms as well. In the model in Figure 4A, tetracaine has been aligned with its benzene ring positioned at the level of M2 position 9 (i.e., at the level of the amino acids that are the most efficiently labeled in each M2 segment). In this orientation, tetracaine's benzene ring, as well as its aryl nitrogen and carboxyl, could interact with the labeled residues at positions 5, 9, 12, and 13 and account for all the specific photolabeling. With the benzene ring at this position, chemical intuition suggests that it would be energetically favorable for tetracaine to be oriented as shown with its N-butyl substituent interacting with the hydrophobic residues at positions 16 and 17 (leucines, valines, and phenylalanines) and the dimethylamino group (probably protonated) interacting with hydrophilic side chains (serines and threonines) at position 6.

An orientation of tetracaine with its N-butyl group interacting with the hydrophobic residues at positions M2-16 and M2-17 is consistent with structure-activity studies of tetracaine analogs. Procaine, a tetracaine analog that lacks the butyl group on the aryl nitrogen, is bound by the Torpedo nAChR in the absence of agonist with 1000-fold lower affinity than tetracaine (Middleton et al., 1999). Although the hydrophobic side chains at M2-16 and M2-17 would form an energetically favorable environment for the butyl group of tetracaine, no such stabilization would be expected for the unsubstituted aryl nitrogen of procaine. In addition, if the desensitized state of the nAChR has a more open pore structure at the level of positions 2, 6, and 9, as is required to accommodate drugs such as trimethylphenylphosphonium and chlorpromazine and (Hucho et al., 1986; Revah et al., 1990; White and Cohen, 1992), then the dimethylamino of tetracaine may contribute less to the energetics of binding in the desensitized state than does a diethyl group. This interpretation is consistent with the analysis of tetracaine analogs by Middleton et al. (1999). This proposed orientation of bound tetracaine is also consistent with the observation that tetracaine is 10 times more potent as an antagonist of Torpedo than of mouse muscle nAChR (Eterovic et al., 1993). The Torpedo and mouse muscle nAChRs have nearly identical M2 segments, with substitutions at three positions in  subunit (S2G, S6F, A10T) and one in  subunit (S6N). The differences include an increase in hydrophobicity at position 6 in the mouse  subunit. If our model of tetracaine's interaction with the Torpedo receptor is correct, then the mouse muscle nAChR may interact less strongly with the charged dimethylamino group of tetracaine.

The positioning of tetracaine's dimethylamino group near position M2-6 is in apparent contrast to the proposed permeability barrier. Previous mutagenesis (Revah et al., 1991; Filatov and White, 1995; Labarca et al., 1995), electron microscopy (Unwin, 1993, 1995), and photolabeling (White and Cohen, 1992; Blanton et al., 1998) studies have suggested that the hydrophobic side chains at M2-9 could form the permeability barrier in the closed channel. However, the kinetics of [³H]tetracaine binding to the Torpedo nAChR in the absence of agonist are characterized by very low association (k₊ = 4 × 10⁴ M¹ min¹) and dissociation (k = 3 × 10⁴ s¹) rate constants at 4°C (Cohen et al., 1986; Strnad, 1988). This indicates that tetracaine's access to its high-affinity binding site in the closed channel, as well as its kinetics of dissociation, is greatly hindered and depend on slow changes in the structure of the pore region that are distinct from the movements required for channel opening. In support of this interpretation, TID, which has a much greater association rate constant at 4°C (k₊ ~ 2 × 10⁶ M¹ min¹; Wu et al., 1994), is not in contact with (i.e., does not label) residues below M2-9 in the absence of agonist.

An alternative model for the permeability barrier, based on the access from the extracellular side of water-soluble sulfhydryl reagents in the absence of agonist to positions as low as M2-2, places the permeability barrier below M2-2 (Akabas et al., 1994; Pascual and Karlin, 1998). However, for 2-aminoethyl methanethiosulfonate, the rate of reaction with a cysteine at position 2 in M2 was 3 × 10⁴ M¹ min¹ (Pascual and Karlin, 1998), which is close to the observed association rate constant for [³H]tetracaine binding to Torpedo nAChR, whereas the rates of reaction at positions 6, 9, and 10 were 1 to 3 × 10³ M¹ min¹. Although it is clear that factors other than the kinetics of access do contribute to the rate constant for cysteine alkylation, the observed accessibility and reactivity of cysteines within the M2 channel domain in the absence of agonist are not incompatible with a structural barrier made up by the aliphatic residues at M2-9 that contributes to the hindered binding of tetracaine and to the permeability barrier to inorganic cations in the closed channel. There is no reason to expect that the kinetics of tetracaine association and dissociation from the closed channel would be determined by a structural constraint below the level of position 2 in the M2 domain.

Tetracaine is an unusual NCA in that it binds with 100-fold higher affinity to the resting state than to the desensitized state, and this preferential binding in the absence of agonist emphasizes the restricted dimensions of drugs that can be accommodated within the structure of the closed channel. In the absence of agonist, a benzene ring (~6.5 Å) as well as the butylamino group (~4 Å wide in extended configuration) can be accommodated within the ion channel in a hydrophobic environment that does not exist in the desensitized state of the nAChR. The hindered binding kinetics indicates that the structure of the pore must relax somewhat to accommodate tetracaine, but the change in structure is far smaller than that required for transitions to either the open channel or desensitized state. Although the width of tetracaine may be larger than the restriction in the closed channel in the absence of drug, it is not nearly as large as the desensitizing NCAs such as chlorpromazine that bind at the level of M2-2 and M2-6 in the desensitized state (Revah et al., 1990). The hydrophobic ring made up by the leucines at M2-9 must be more splayed apart in the desensitized than in the resting state (White and Cohen, 1992), and there is no longer a domain within the pore with a structure favorable for the high-affinity binding of a molecule with tetracaine's dimensions.