From the Faculty of Pharmaceutical Sciences, Kumamoto University,
5-1 Ohe-Honmachi, Kumamoto 862, Japan, Laboratory of
Signal Transduction, National Institute of Environmental Health
Services, Research Triangle Park, North Carolina 27709, § New Product Research Laboratories II, Daiichi
Pharmaceutical Co. Ltd., 1-16-13, Kita-Kasai, Edogawa-Ku, Tokyo 134, Japan, and ¶ Institute for Molecular Pharmacology and Biophysics,
University of Cincinnati College of Medicine,
Cincinnati, Ohio 45267-0828
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ABSTRACT |
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To identify the binding domain of a new
Ca2+ antagonist semotiadil on L-type Ca2+
channels from skeletal muscle, photolabeling was carried out by using
an azidophenyl derivative of [3H]semotiadil.
Photoincorporation was observed in several polypeptides of membrane
triad preparations; the only specific photoincorporation was in the
1 subunit of the Ca2+ channel. After
solubilization and purification, the photolabeled
1
subunit was subjected to proteolytic and CNBr cleavage followed by
antibody mapping. Specific labeling was associated solely with the
region of transmembrane segment S6 in repeat IV. Quantitative immunoprecipitation was found in the tryptic and the Lys-C/Glu-C fragments of 6.6 and 6.1 kDa, respectively. Further CNBr cleavage of
the Lys-C digests produced two smaller fragments of 3.4 and 1.8 kDa
that were included in the tryptic and Lys-C/Glu-C fragments. The
smallest labeled fragments were:
Tyr1350-Met1366 and
Leu1367-Met1381 containing IVS6, a possible
pore-forming region. The data suggest that semotiadil binds to a region
that is overlapped with but not identical to those for
phenylalkylamines, dihydropyridines and benzothiazepines. The present
study also provides evidence that region IV represents an important
component of a binding pocket for Ca2+ antagonists.
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INTRODUCTION |
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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).
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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.
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EXPERIMENTAL PROCEDURES |
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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.
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RESULTS |
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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).
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[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).
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[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.
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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).
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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%).
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DISCUSSION |
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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.
The two labeled fragments by [3H]D51-4700 are not only overlapped with the [3H]LU49888 labeled site but also are part of the labeled regions by DHP (14, 15) and BTZ (17, 18). The association of the newly identified semotiadil site with those of the three typical Ca2+ antagonists (DHP, BTZ, and PAA) within the pore-forming regions of the channel allows allosteric interactions among semotiadil and these drugs. Although a few reports are available concerning the pharmacological interaction of semotiadil and other Ca2+ antagonists (11-13), the observed negative allosteric effect of semotiadil on the binding of DHP, PAA, and BTZ to canine skeletal muscle membranes (12) suggests that the binding sites for all these drugs are in close apposition in the Ca2+ channel but not identical. This is clearly consistent with the present photoaffinity labeling results. In contrast to the photolabeled sites for DHP (14, 15) and BTZ (18), the identified fragments for photolabeling with [3H]D51-4700 do not contain any peptides in repeat III. It is tempting to conclude that the semotiadil binding site is different from those for DHP and BTZ. However, there are complexities between the results obtained by photoaffinity labeling and those obtained by molecular biological techniques. In BTZ, for example, IIIS6 as well as IVS6 were identified as the photolabeled fragments (18), whereas only the IVS6 was shown to be sufficient for BTZ sensitivity for L-type Ca2+ channels (23). With regard to PAA, only the IVS6 with the adjacent extracellular and intracellular stretches were identified by the photolabeling technique, whereas not only IVS6 (24, 25) but also IIIS6 appear to be determinants of high affinity binding for ( ![]() |
ACKNOWLEDGEMENTS |
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We thank Dr. Yoshifumi Watanabe for the synthesis of D51-4700 and Dr. Kazunobu Harano for helpful discussions.
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FOOTNOTES |
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* This work was supported in part by the Monbusho International Scientific Research Program 08044306, Grants 07229102, 08219133 for the research priority areas "Natural Supramolecules: Chemistry and Function" and 07457543, 08557138 for the general subject from Ministry of Education, Science, and Culture of Japan (to H. N.), and Grant PO1 HL22619 from the National Institutes of Health (to A. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
81-96-371-4357; Fax: 81-96-372-7182; E-mail:
jin{at}gpo.kumamoto-u.ac.jp.
1 The abbreviations used are: DHP, 1,4-dihydropyridine; BTZ, benzothiazepine; dpm, disintegrations per minute; [3H]D51-4700, (+)-(R)3,4-dihydro-2-[5-methoxyl-2-[3-[N-[3H]methyl-N-[2-(3-azidophenoxy)ethyl]amino]propoxyl]phenyl]-4-methyl-3-oxo-2H-1,4-benzothiazine; Glu-C, endoprotease Glu-C; Lys-C, endoprotease Lys-C; PAA, phenylalkylamine; PAGE, polyacrylamide gel electrophoresis; TPCK, N-p-toluenesulfonyl-L-phenylalanine chloromethylketone; WGA, wheat germ agglutinin.
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REFERENCES |
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