INTRODUCTION

Top Abstract Introduction Procedures Results Discussion References

Ca²⁺ antagonists bind with high affinity to L-type Ca²⁺ channels and block the entry of extracellular Ca²⁺. Three specific classes of Ca²⁺ antagonists have been identified and include 1,4-dihydropyridines (DHP),¹ phenylalkylamines (PAA), and benzothiazepines (BTZ), which are represented by the parent compounds, nifedipine, verapamil, and diltiazem, respectively. These drugs bind to different sites on the ₁ subunit of Ca²⁺ channels (1), and logically explain the well known allosteric interactions with one another (2). Using photoaffinity labeling and antibody mapping techniques, all three drugs have been shown to bind to different regions in more than one motif. Several other Ca²⁺ antagonists have different chemical structures and somewhat different pharmacological actions than DHP, PAA, and BTZ (3-5). Semotiadil (SD-3211) is a novel Ca²⁺ antagonist with a unique 1,4-benzothiazine ring structure (3) (Fig. 1).

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Comparison of chemical structures of semotiadil, diltiazem, and [3H]D51-4700.

The benzothiadine ring is homologous to the benzothiazepine ring of diltiazem whereas the ring components of the two drugs might contribute different properties in the action on Ca²⁺ channels. Studies on structure-function relationships of diltiazem (reviewed in Ref. 6) suggest that the acetoxy and 2-(dimethyamino)ethyl groups play important roles in the calcium antagonistic activity. It is likely that the benzothiazepine ring of diltiazem is a structure on which various side groups can be inserted, which may change the position of the ring in binding and subsequent inhibition of the Ca ²⁺ channel. For example, the hydrophobic 4-methoxyphenyl group as well as the acetoxy and 2-(dimethyamino)ethyl groups, probably confer specific activities of diltiazem and other BTZs. In contrast, the calcium antagonist activity of semotiadil depends, in part, on the long side chain of Ar-O-CH₂CH₂CH₂-N(Me)-CH₂CH₂-O-Ar at the C-3 position of the 1,4-benzothiazine. This idea is supported by the comparison of the three-dimensional structures between semotiadil and diltiazem based on their conformational analyses by x-ray crystallography and spectroscopy in solution (7, 8). There is no apparent similarity in the orientation of the side chains as well as in the common methoxyphenyl group between two drugs, when the phenyl ring of the benzothiazepine and 1,4-benzothiazine are overlaid by the computer. The hypothesis that the long side chain at the C-3 position of the 1,4-benzothiazine ring is a part of the pharmacophore for calcium antagonist activity (13) is supported by the fact that a similar structural component: Ar-C(R₁R₂)-CH₂CH₂CH₂-N(Me)-CH₂CH₂-Ar exists in verapamil and other PAAs. It is apparent that the 1,4-benzothiazine ring of semotiadil plays an additional role that contributes to the enhanced potency.

For example, in considering the pharmacological characteristics as a Ca²⁺ antagonist, semotiadil is longer-lasting than diltiazem and nifedipine and shows a higher selectivity for blood vessels compared with cardiac tissues than diltiazem but lower selectivity than nifedipine (9, 10). In addition, semotiadil increases the dissociation rate of [³H](+)PN200-110, [³H]diltiazem and [³H]verapamil binding sites (11-13). These results suggest that semotiadil has a strong allosteric interaction with the three classes of Ca²⁺ antagonists, as exemplified by differential displacement of [³H]PN200-110, [³H]diltiazem, and [³H]verapamil from their specific sites on the Ca²⁺ channels (11-13).

Localization of the semotiadil binding site would provide information about a putative new class of Ca²⁺ antagonists but more importantly might uncover overlapping binding region(s), if any, with conventional Ca²⁺ antagonists. The binding sites for DHP, PAA, and BTZ have been localized by photoaffinity labeling of Ca²⁺ channels followed by defined proteolysis and antibody mapping using sequence-directed antibodies (14-18). By comparing the results of the latter, with those derived from mutagenesis experiments (19-25), one can demonstrate that sequence stretches photolabeled by DHP, PAA, and BTZ indeed contain amino acid residues that directly participate in binding. However, some recent mutagenesis experiments (26-28) have revealed sites that are not labeled by photoligands. As an initial work to identify the binding site for semotiadil, we employed techniques of photoaffinity labeling of Ca²⁺ channels isolated from rabbit skeletal muscles with [³H]D51-4700, an azidophenyl derivative of [³H]semotiadil (29), and the localizing of the site(s) of photolabeling and comparing with those for DHP, PAA, and BTZ.

	EXPERIMENTAL PROCEDURES

Top Abstract Introduction Procedures Results Discussion References

Materials-- [³H]D51-4700 (77.6 Ci/mmol) was synthesized as described (29). Semotiadil was obtained from Daiichi Pharmaceutical Co., Ltd. Enzymes and chemicals were obtained from the following sources: N-p-toluenesulfonyl-L-phenylalanine chloromethyl ketone-treated trypsin (TPCK-trypsin from bovine pancreas) from Worthington; endoprotease Glu-C from Boehringer Mannheim; Achromobacter lyticus protease I (Lys-C) and digitonin from Wako Pure Chemicals (Osaka, Japan); N-hydroxysuccinimidyl m-maleimidobenzoate and bovine thyroglobulin from Sigma; bovine serum albumin from Nacalai Tesque (Kyoto, Japan); protein A-Sepharose CL-4B and WGA-Sepharose 4B from Pharmacia Biotech Inc.; prestained low molecular weight standard from Life Technologies, Inc.; unstained high molecular weight standard and Dowex 1-X8 from Bio-Rad; scintillation mixture ACS II from Amersham; dimethyl pimelidate from Pierce.

Peptide Synthesis and Antibody Production-- Polyclonal antibodies were raised in rabbits against synthetic peptides corresponding to particular regions of the skeletal muscle ₁ subunit sequence (30): 1320-1332 (anti-(1320-1332)), 1338-1351 (anti-(1338-1351)), 1382-1400 plus the N-terminal Gly-Cys (anti-(1382-1400)), 1401-1414 plus C-terminal Cys-Gly [anti-(1401-1414)]. The peptide was conjugated to bovine serum albumin or bovine thyroglobulin via a cysteine residue using N-hydroxysuccinimidyl m-maleimidobenzoate. Japanese white rabbits were immunized with the conjugate emulsified in Freund's complete adjuvant. After 3 weeks, the immunizaton was repeated 5 times at 2-wk intervals with the conjugate in Freund's incomplete adjuvant.

Membrane Preparation-- Triad membranes were isolated from rabbit skeletal muscle as described by Mitchell et al. (31).

Photoaffinity Labeling and Purification of Rabbit Skeletal Ca²⁺ Channels-- Rabbit triad membranes (300 pmol of [³H](+)-PN200-110 binding sites, 20 mg of proteins) were incubated with 100 nM [³H]D51-4700 in 10 ml of binding buffer (25 mM Tris-HCl (pH 7.2), 0.1 mM phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin A, 1 µg/ml leupeptin, 10 µg/ml soybean trypsin inhibitor) in the presence and absence of 10 µM semotiadil at 30 °C for 60 min. The incubation mixture was transferred into a glass Petri dish on ice, and irradiated for 20 min with a 100 watt black light/blue lamp (Ultra-Violet Products, Inc., San Gabriel, CA) at distance of 10 cm. After photolysis, the [³H]D51-4700-labeled Ca²⁺ channels were solubilized in 1% (w/v) digitonin and purified by affinity chromatography on WGA-Sepharose 4B according to the described method (30). The sample was dialyzed against 1 mM Tris-HCl (pH 7.3) and lyophilized.

Reductive Carboxymethylation and Gel Permeation High Pressure Liquid Chromatography-- The photolabeled and lyophilized protein was resuspended in 0.1 M Tris-HCl (pH 8.0), 1% (v/v) 2-mercaptoethanol, 1.5% (w/v) SDS (final volume of 0.3 ml). After incubation at room temperature for 30 min, iodoacetic acid was added to a final concentration of 84 mM. After incubation for 1 h, the photolabeled ₁ subunits were further purified by gel permeation liquid chromatography as described (14). Fractions corresponding to the ₁ subunit were pooled, lyophilized, and stored at 30 °C until use.

Proteolytic and CNBr Cleavage of [³H]D51-4700-labeled ₁ Subunits-- The photolabeled ₁ subunit was dissolved in deionized water (0.5 ml) and dialyzed against 6 M urea as described (14), followed by dialysis against 0.01% Triton X-100 for 6 h. The sample was digested with Lys-C (50 µg/ml) in 50 mM Tris-HCl (pH 9.0) containing 0.05% (w/v) SDS and 0.01% (v/v) Triton X-100 (final volume of 100 µl) at 37 °C for 6 h. For trypsin digestion, the sample was incubated with TPCK-trypsin (100 µg/ml) at 37 °C for 12 h in 50 mM Tris-HCl (pH 8.0) containing 0.01% (v/v) Triton X-100 and 2 mM CaCl₂. The reaction was stopped by heating at 90 °C for 3 min. Prior to Lys-C/Glu-C digestion and CNBr cleavage, Lys-C digests were dialyzed against H₂O for 6 h using a microdialyzer apparatus with a 1 kDa cut-off dialysis tube (Spectra/Por 6, Spectrum). For Lys-C/Glu-C digestion, the dialyzed sample was incubated with Glu-C (0.5 mg/ml) in 50 mM sodium phosphate buffer (pH 7.8) containing 0.05% (w/v) SDS for 12 h at 37 °C. For CNBr cleavage, the dialyzed sample was lyophilized and then incubated with CNBr (5 mg/ml) in 70% (v/v) formic acid for 12 h at 37 °C. After incubation, the mixture was lyophilized.

Immunoprecipitation-- Antibodies were bound to protein A-Sepharose CL-4B gel by incubating 1 volume of antiserum with 1 volume of the swollen gel in the buffer A (10 mM Tris-HCl (pH 7.2), 150 mM NaCl, 0.1% (v/v) Triton X-100 and 1 mg/ml bovine serum albumin) for 2 h at 4 °C. The gel was washed with the ice-cold buffer A before addition of digested or nondigested [³H]D51-4700-labeled ₁ subunits. After incubation for 2 h at room temperature, the gel was washed with buffer A. Immunoprecipitated radioactivity was directly determined by liquid scintillation counting of the protein A-Sepharose CL-4B gel containing 100 mM sodium citrate (pH 3.0). Immunoprecipitated labeled fragments were extracted from the gel with a sampling buffer for SDS-PAGE (50 mM Tris-HCl (pH 6.8), 4% (w/v) SDS, 2% (v/v) 2-mercaptoethanol and 12% (v/v) glycerol) for 3 min at 90 °C and analyzed by SDS-PAGE. To determine the immunoprecipitated fragments size, the antibody-protein A Sepharose complex was cross-linked with dimethyl pimelidate as described by Schneider et al. (32).

SDS-PAGE-- Intact ₁ subunits were analyzed on SDS-PAGE using an 8% polyacrylamide gel according to Laemmli (33) and a sampling buffer (10 mM Tris-HCl (pH 7.6), 1% (w/v) SDS, 20 mM dithiothreitol, 4 mM ethylenediaminetetraacetic acid and 2% (w/v) sucrose). For separation of proteolytic and CNBr-cleaved fragments, the gel system described by Schägger and von Jagow (34) (4% stacking gel, 10% spacer gel, and 16.5% separating gel, 3 or 6% cross-linking) was used.

Radioluminography and Gel Slicing-- Instead of fluorography, a higher sensitive visualization method ("radioluminography") of the tritiated proteins and peptides was used. In brief, the gel after electrophoresis was electrophoretically transferred onto a polyvinylidene difluoride membrane in a transfer buffer (25 mM Tris, 193 mM glycine, 10% methanol) by using a semidry blotting assembly. The blotted membrane was stained with Coomassie Brilliant Blue R250, followed by drying completely in air. The membrane was then placed in contact with an imaging plate, BAS-TR2040S (Fuji Photo Film Co.) in a cassette at room temperature for 2 days. The imaging plate was scanned and analyzed by a Bio-Imaging Analyzer BAS 1000 model (Fuji Photo Film Co.). Scanning conditions were at a sensitivity 10,000, latitude 4, gradation 1024, and resolution 100. Printouts were performed by a high quality pictorial copy apparatus. Alternatively, individual gel lanes were manually cut into 3-mm slices and radioactivity was determined in ACSII with 3% (v/v) H₂O₂.

	RESULTS

Top Abstract Introduction Procedures Results Discussion References

Specific Photoincorporation of the 170-kDa ₁ Subunit of Rabbit Skeletal Muscle Tubules-- The synthesis and pharmacological characterization of the photoaffinity ligand [³H]D51-4700 have been reported (29). [³H]D51-4700 photolabeled several polypeptides as shown in Fig. 2 (lane 1). However, only the ₁ subunit bound label (170 kDa) of the Ca²⁺ channel was selectively inhibited in the presence of excess of semotiadil (lane 2). The selective labeling was also confirmed when the photolabeled triad preparation was solubilized by digitonin and purified by a WGA-Sepharose column (30). In the purified sample, a single band of 170 kDa was photolabeled (lane 3), whereas the labeled band was not observed when photolabeling was done in the presence of excess semotiadil (lane 4).

View larger version (40K): [in this window] [in a new window]   Fig. 2.   Photolabeling of Ca2+ channel preparations with [3H]D51-4700. Triad membranes from rabbit skeletal muscles (2 mg/ml) were photolabeled with 100 nM [3H]D51-4700 in the absence (lane 1) and presence (lane 2) of 10 µM semotiadil. Aliquots (20 µl) of the photolabeled mixture were spun down, and the pellet was solubilized by the sampling buffer for SDS-PAGE and analyzed on a 8% polyacrylamide gel followed by radioluminography. The photolabeled samples were also solubilized with 1% (w/v) digitonin and partially purified by WGA-Sepharose 4B. The purified samples (2.5 µg) that were photolabeled in the absence (lane 3) and presence (lane 4) of 10 µM semotiadil were similarly analyzed on the SDS-PAGE followed by radioluminography. The migration of the 1 subunit and of molecular mass markers (shown in kDa) is indicated.

[³H]D51-4700 Labeling Occurs Only within Repeat IV-- To determine the localization of photolabeled site within the ₁ subunit, we first subjected the photolabeled ₁ subunit to protease digestion with an endoprotease Lys-C and probed the Lys-C fragment by immunoprecipitation with a series of sequence-directed antibodies (see "Experimental Procedures") against different regions of ₁. The Lys-C digestion of ₁ is shown in Fig. 3, resulting in a labeled fragment of 8.3 ± 0.7 kDa (n = 5) (Fig. 3A, lane 1). The fragment contained 92 ± 4% (n = 5) of the ₁-associated radioactivity as determined by gel slicing (not shown). Immunoprecipitation with sequence-directed antibodies revealed that only two antibodies directed against epitopes located near segment S6 in repeat IV (anti-(1338-1351) and anti-(1382-1400), see Fig. 7) immunoprecipitated the photolabeled fragments, whereas anti-(1320-1332) and anti-(1401-1414) did not immunoprecipitate at all (Fig. 3B). Other antibodies against repeat I, repeat III, and repeat IV efficiently immunoprecipitated the nondigested labeled ₁ but did not immunoprecipitate Lys-C fragments (not shown). About 56-68 and 57-75% of the ₁-associated labeling were associated with a fragment recognized by anti-(1338-1351) and anti-(1382-1400), respectively (Fig. 3B). After the immunoprecipitated radioactivities were normalized with respect to the radioactivities immunoprecipitated in nondigested samples (100%), the values calculated were 125-147 and 124-163%, respectively (Fig. 3B). The reason why the calculated values were over 100% will be discussed later (see "Discussion"). The radioactivity applied was recognized quantitatively by anti-(1338-1351) and anti-(1382-1400) suggesting that both of the antibodies were immunoprecipitating the same 8.3-kDa band. This was confirmed by SDS-PAGE analysis of the antibody bound radioactivity (Fig. 3A, lane 2 and 3). Since the extracellular ₁ (1338-1351) or intracellular ₁ (1382-1400) epitope is located within a single Lys-C fragment that contains IVS6 and intracellular residues, or IVS6 and extracellular residues, respectively, the 8.3-kDa fragment represents the correct digested product at Lys¹³³⁶ and Lys¹⁴⁰³ (calculated mass 7.9 kDa, see Fig. 7).

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Lys-C digestion of photolabeled 1 subunits. A, [3H]D51-4700-labeled 1 was digested with Lys-C (50 µg/ml, 37 °C, 6 h) and 30-µl aliquots were separated on a Schägger and von Jagow (34) gel (lane 1). A 30-µl aliquot was also subjected to immunoprecipitation with anti-(1338-1351) (lane 2) and anti-(1382-1400) (lane 3). The photolabeled band was visualized by radioluminography. The arrow indicates the 8.3-kDa fragment. The migration of prestained molecular mass markers (given in kDa) is indicated. In the separate experiments, 92 ± 4% (approximately 850 dpm) of the radioactivity applied on the gel was detected in the band centered on 8.3 kDa by liquid scintillation counting of 3-mm gel slices (not shown). Five runs were carried out. B, immunoprecipitation of [3H]D51-4700-labeled peptide fragment from Lys-C digests. Photolabeled 1 subunits were digested in the absence (control) or presence of Lys-C. Both samples were probed in parallel with the antibodies against the indicated 1 peptides. The immunoprecipitated percentages (immunoprecipitated dpm per applied dpm) are shown as filled bars and the immunoprecipitated dpm were normalized with respect to the dpm immunoprecipitated in nondigested samples (100%) are shown as open bars. Means ± S.D. are given for n = 3.

[³H]D51-4700 Labeling Is Located in Tryptic Fragments Containing the S6 Segment in Repeat IV-- Since the Lys-C fragment contains cleavable sites by trypsin, the photolabeled ₁ subunits were digested with TPCK-trypsin to refine the photolabeled sites. SDS-PAGE revealed two smaller labeled fragments with apparent molecular masses of 8.3 ± 0.8 (n = 3) and 6.6 ± 0.7 kDa (n = 3). A radioluminogram of a gel where two peaks are clearly separated is shown in Fig. 4A. 84 ± 8% of the ₁-associated radioactivity was recovered in these peaks and no other smaller fragments were observed as determined by gel slicing (not shown). Location of the photolabeled tryptic fragments was assessed by immunoprecipitation using anti-(1338-1351) and anti-(1382-1400). About 53-61% of the ₁-associated labeling were associated with fragments recognized by anti-(1338-1351) (Fig. 4B). The immunoprecipitated peptides were 8.3 and 6.6 kDa, determined by SDS-PAGE analysis (Fig. 4A, lane 2). The 6.6-kDa peptide was immunoprecipitated to a greater extent than the 8.3-kDa peptide, which is in accordance with the fact that the 6.6-kDa band was the major labeled peptide (Fig. 4A). Therefore, both peptides must contain the full epitope sequence of anti-(1338-1351) and the 6.6-kDa peptide must be the smallest labeled peptide obtained by trypsin digestion.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Trypsin digestion of photolabeled 1 subunits. A, [3H]D51-4700-labeled 1 was digested with TPCK-trypsin (100 µg/ml, 37 °C, 12 h) and 30 µl aliquots were separated on a Schägger and von Jagow gel (lane 1). A 30-µl aliquot was also subjected to immunoprecipitation with anti-(1338-1351) (lane 2). The photolabeled bands were visualized by radioluminography. The arrows indicate 8.3- and 6.6-kDa fragments. The migration of prestained molecular mass markers (given in kDa) is indicated. In the separate experiments, 84 ± 8% (approximately 780 dpm) of the radioactivity applied on the gel was detected in the bands for 8.3- and 6.6-kDa by liquid scintillation counting of 3-mm gel slices. (Resolution was not enough in the sliced gels.) Three runs were carried out. B, immunoprecipitation of [3H]D51-4700-labeled peptide fragments from TPCK-trypsin digests. The immunoprecipitated percentages (immunoprecipitated dpm per applied dpm) by anti-(1338-1351) and anti-(1382-1400) are shown. Means ± S.D. are given for n = 3.

Isolation and Characterization of Smaller Photolabeled Fragments by Glu-C Digestion-- Since the Lys-C fragment also contains potential cleavable sites by Glu-C, the photolabeled Lys-C fragment was subsequently digested with endoprotease Glu-C to further restrict the photolabeled sites. As shown in Fig. 5A, a radioluminogram of a gel revealed two smaller labeled fragments with apparent molecular masses of 7.8 ± 0.9 (n = 3) and 6.1 ± 0.7 kDa (n = 3). The ₁-associated radioactivity was recovered in 86 ± 7% in these peaks and no other smaller fragments were observed as determined by gel slicing (not shown).

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Further proteolysis with Glu-C after Lys-C digestion of photolabeled 1 subunits. A, [3H]D51-4700-labeled 1 was first digested with Lys-C (50 µg/ml, 37 °C, 6 h), followed by Glu-C digestion (0.5 mg/ml, 37 °C, 12 h). After digestions, 30 µl aliquots were separated on a Schägger and von Jagow gel (lane 1). A 30-µl aliquot was also subjected to immunoprecipitation with anti-(1382-1400) (lane 2). The photolabeled bands were visualized by radioluminography. The arrows indicate 7.8- and 6.1-kDa fragments. The migration of prestained molecular mass markers (given in kDa) is indicated. In the separate experiments, 86 ± 7% (approximately 830 dpm) of the radioactivity applied on the gel was detected in the bands for 7.8- and 6.1-kDa by liquid scintillation counting of 3-mm gel slices. (Resolution was not enough in the sliced gels.) Three runs were carried out. B, immunoprecipitation of [3H]D51-4700-labeled peptide fragments from successive digestion with Lys-C and Glu-C. The immunoprecipitated percentages (immunoprecipitated dpm per applied dpm) by anti-(1338-1351) and anti-(1382-1400) are shown. Means ± S.D. are given for n = 3.

Isolation and Characterization of Smaller Photolabeled Fragments by CNBr Cleavage-- Since the Lys-C fragment contains two methionine residues, the photolabeled Lys-C fragment was subsequently treated with CNBr to further restrict the photolabeled sites. As shown in Fig. 6A, a radioluminogram of a gel revealed three smaller labeled fragments with apparent molecular masses of 5.7 ± 0.6 (n = 3), 3.4 ± 0.4 (n = 3), and 1.8 ± 0.3 kDa (n = 3). During the incubation with CNBr in 70% formic acid, almost 70% of the photolabeled radioactivity was liberated and migrated to the dye front position on SDS-PAGE (Fig. 6B). However, the liberated radioactivity was not blotted on the polyvinylidene difluoride membrane sheet, and therefore it did not interfere with the analysis of newly generated labeled fragments in the radioluminogram (Fig. 6A). In the immunoprecipitation experiments, anti-(1338-1351) showed apparent binding activity (10 ± 3%, n = 3) to the total radioactivity applied after CNBr cleavage, whereas anti-(1382-1400) did not immunoprecipitate at all. As the radioactivity associated with peptide fragments was only 30% of the radioactivity in the applied sample, the immunoprecipitated value of 10% can be corrected to 33%. This value is further corrected to 73% after normalization with respect to immunoprecipitation avidity of anti-(1338-1351) in uncleaved ₁ subunits (45%).

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Further CNBr cleavage after Lys-C digestion of photolabeled 1 subunits. A, [3H]D51-4700-labeled 1 was first digested with Lys-C (50 µg/ml, 37 °C, 6 h), followed by CNBr treatment in 70% formic acid (5 mg/ml, 37 °C, 12 h). After cleavage, the reaction mixture was lyophilized and resuspended with buffer A (10 mM Tris-HCl (pH 7.2), 150 mM NaCl and 0.1% (v/v) Triton X-100). A 30-µl aliquot was separated on a Schägger and von Jagow gel (lane 1). The arrows indicate 5.7-, 3.4-, and 1.8-kDa fragments. Another 30-µl aliquot was also subjected to immunoprecipitation with anti-(1338-1351) (lane 2). The photolabeled bands were visualized by radioluminography. The arrows indicate 5.7- and 3.4-kDa fragments. The migration of prestained molecular mass markers (given in kDa) is indicated. B, [3H]D51-4700-labeled 1 was digested with Lys-C followed by CNBr cleavage and analyzed on the Schägger and von Jagow gel. Labeled peptide fragments were detected by liquid scintillation counting of 3-mm gel slices. About 70% of the radioactivity was migrated at the dye front due to its liberation from the labeled sites during CNBr treatments. Three runs were carried out.

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Location of labeled fragments in the IVS6 region. A schematic alignment of the transmembrane segment S6 (shadowed) in repeat IV and antibodies (a-d) used and shown in the previous figures is indicated at the top. Antibodies shown here are: a, anti-(1320-1332); b, anti-(1338-1351); c, anti-(1382-1400); d, anti-(1401-1414). In the second line, particular amino acid residues that are potential cleavage sites by the following protease digestions and CNBr treatment are shown as single letters. In the third to sixth lines, the smallest labeled fragment(s) observed by each protease digestion (Lys-C or trypsin) or its combination with subsequent proteolysis (Lys-C/Glu-C) or CNBr treatment (Lys-C/CNBr) are indicated with its size (kDa) and the N and C terminus amino acid residues. Consequently, the smallest labeled fragments can be deduced as Tyr1350-Met1366 (Y1350-M1366) and Leu1367-Met1381 (L1367-M1381).

	DISCUSSION

Top Abstract Introduction Procedures Results Discussion References

Semotiadil Receptor Site of the ₁ Subunit-- [³H]D51-4700, a photoaffinity probe of semotiadil, selectively labeled the ₁ subunit of Ca²⁺ channels in skeletal triad membranes. In the absence of unlabeled semotiadil, the probe labeled several polypeptides including the ₁ subunit. This may explain the observation that reversible binding of [³H]D51-4700 to triad membrane preparations is rather difficult to show due to the high level of nonspecific binding (not shown). However, the photoincorporation to the ₁ subunit occurred in a specific manner since the Ca²⁺ channels purified by WGA column showed a single photolabeled band of 170 kDa, and the label was totally blocked by excess unlabeled semotiadil. The specifically photolabeled site was localized within the ₁ subunit by an antibody mapping method employed previously for the DHP-, PAA-, and BTZ-binding domains (14-18). As shown in the results of Lys-C digestion, we observed that the normalized values of the immunoprecipitated percentage of the protease-digested fragment gave more than 100% with respect to those of the nondigested samples. Similar results were reported in the literature (16) where the labeled site was localized to a single peptide fragment. This is probably due to the fact that higher reactivity of the anti-peptide antibody occurs to the peptide fragment rather than to the nondigested polypeptide ₁.

Implication of the Semotiadil Binding Site Compared with Other Ca²⁺ Antagonists-- The labeled fragments by [³H]D51-4700 are identified as Tyr¹³⁵⁰-Met¹³⁶⁶ and Leu¹³⁶⁷-Met¹³⁸¹ in IVS6 after CNBr cleavage. They are included in the Glu-C fragment of Tyr¹³⁵⁰-Trp¹³⁹¹, which was identified as the labeled peptide by [³H]LU49888 (16), a photoaffinity probe of PAA, but the intracellular region of Asp¹³⁸²-Trp¹³⁹¹ is not included in the [³H]D51-4700 labeled fragments. Since smaller [³H]LU49888 fragments than those generated by Glu-C digestion have not been mapped, we cannot exclude the possibility that [³H]LU49888 did not label the intracellular region of Asp¹³⁸²-Trp¹³⁹¹. However, semotiadil does not compete with the binding of PAA but rather allosterically inhibits binding or vice versa (11, 12). This suggests that the binding site for semotiadil is similar but not identical to that for PAA. The present results are consistent with this interpretation.