Identification of benz(othi)azepine-binding regions within L-type calcium channel alpha1 subunits.

To identify the binding domain for diltiazem-like Ca2+ antagonists on L-type Ca2+ channel α1 subunits we synthesized the benzazepine [3H]benziazem as a novel photoaffinity probe. [3H]Benziazem reversibly labeled the benzothiazepine (BTZ)-binding domain of partially purified skeletal muscle Ca2+ channels with high affinity (Kd = 12 nM) and photoincorporated into its binding domain with high yield (>66%). Antibody mapping of proteolytic labeled fragments revealed specific labeling of regions associated with transmembrane segments S6 in repeats III and IV. More than 50% of the labeling was found in the tryptic fragment alanine 1023-lysine 1077 containing IIIS6 together with extracellular and intracellular amino acid residues. The remaining labeling was identified in a second site comprising segment S6 in repeat IV and adjacent residues. Unlike for dihydropyridines, no labeling was observed in the connecting IIIS5-IIIS6 linker. The [3H]benziazem photolabeled regions must be in close contact to the drug molecule when bound to the channel. We propose that the determinants for high affinity BTZ binding are located within or in close proximity to segments IIIS6 and/or IVS6. Therefore the binding domain for BTZs, like for the other main classes of Ca2+ antagonists, must be located in close proximity to pore-forming regions of the channel.

To identify the binding domain for diltiazem-like Ca 2؉ antagonists on L-type Ca 2؉ channel ␣1 subunits we synthesized the benzazepine [ 3 H]benziazem as a novel photoaffinity probe. [ 3 H]Benziazem reversibly labeled the benzothiazepine (BTZ)-binding domain of partially purified skeletal muscle Ca 2؉ channels with high affinity (K d ‫؍‬ 12 nM) and photoincorporated into its binding domain with high yield (>66%). Antibody mapping of proteolytic labeled fragments revealed specific labeling of regions associated with transmembrane segments S6 in repeats III and IV. More than 50% of the labeling was found in the tryptic fragment alanine 1023-lysine 1077 containing IIIS6 together with extracellular and intracellular amino acid residues. The remaining labeling was identified in a second site comprising segment S6 in repeat IV and adjacent residues. Unlike for dihydropyridines, no labeling was observed in the connecting IIIS5-IIIS6 linker. The [ 3 H]benziazem photolabeled regions must be in close contact to the drug molecule when bound to the channel. We propose that the determinants for high affinity BTZ binding are located within or in close proximity to segments IIIS6 and/or IVS6. Therefore the binding domain for BTZs, like for the other main classes of Ca 2؉ antagonists, must be located in close proximity to pore-forming regions of the channel.
Voltage-dependent L-type Ca 2ϩ channels are blocked by different groups of chemically unrelated compounds, termed Ca 2ϩ antagonists. These drugs cause vasodilation and cardiac depression by blocking L-type Ca 2ϩ channels in smooth and cardiac and are used for the therapy of cardiovascular disorders.
One of the clinically most widely used Ca 2ϩ antagonist is the benzothiazepine (ϩ)-cis-diltiazem. Like other Ca 2ϩ antagonists (e.g. dihydropyridines and phenylalkylamines (1)) it interacts with a high affinity binding domain on the pore-forming ␣1 subunit of L-type Ca 2ϩ channels (2) in heart muscle, brain, and skeletal muscle. The benzothiazepine (BTZ) 1 selective domain is coupled to the dihydropyridine (DHP)-and phenylalkylamine (PAA)-binding domains via non-competitive mechanisms (1).
To gain deeper insight into the mechanisms of L-type channel modulation by Ca 2ϩ antagonists the molecular motifs involved in drug binding must be identified. DHP and PAA interaction sites within the primary structure of L-type ␣1 subunits were successfully localized by antibody mapping of proteolytic fragments derived from their photoaffinity labeled domains (3)(4)(5). Within these regions individual amino acid residues participating in drug interaction were then identified using site-directed mutagenesis (6 -9).
As compared to DHPs and PAAs, photoaffinity ligands developed for the BTZ-binding domain display low binding affinity and incorporation yields (2,10,11). Therefore a complete localization of the BTZ-binding domain using photoaffinity labeling and antibody mapping was impossible (10).
We report here the synthesis of a novel photoreactive diltiazem derivative (benziazem) that reversibly labels the BTZbinding domain of partially purified L-type Ca 2ϩ channels with high affinity. Benziazem specifically photoincorporates into the BTZ-binding domain of skeletal muscle ␣1 subunits with high labeling efficiency. Antibody mapping of photolabeled peptides revealed that the critical determinants of benziazem binding are exclusively localized within regions of ␣1 that also participate in the formation of the DHP-and PAA-binding domain. Our results suggest that all major classes of Ca 2ϩ antagonists bind within the pore-forming regions of repeats III and IV of L-type Ca 2ϩ channel ␣1 subunits.

EXPERIMENTAL PROCEDURES
Materials-Reagents were obtained from the following sources: L-1tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (TPCKtrypsin, from bovine pancreas), Protein A-Sepharose, and bovine serum albumin (essentially globulin free) from Sigma; endoproteinase Lys-C (Lys-C) from Boehringer Mannheim; prestained molecular weight marker proteins from Bio-Rad (high range) and Life Technologies, Inc. (low range); Amplify from Amersham; digitonin from Biosynth AG (Basel, Switzerland). (Fig. 1) was synthesized by coupling N-hydroxysuccinimidyl-benzoyl dihydrocinnammic acid to the free amino group of a benzazepine precursor ((3R,4R)-cis-1,3,4,5 -tetrahydro-6-trifluoromethyl-3-hydroxy-  13) were added and the mixture incubated on ice for 10 min. The solvent was evaporated under a gentle stream of nitrogen. To remove the Boc protecting group, 0.1 ml of methylene chloride followed by 0.1 ml of trifluoroacetic acid were added and the mixture incubated for 60 min on ice. Methylene chloride/ trifluoroacetic acid were removed under vacuum, the reaction products resuspended in 50 l of ethanol and then separated on a Silica Gel 60 thin layer plate developed in benzene/methanol/diethylamine (74/25/1). [ 3 H]Benziazem migrated with an R F value of 0.57 Ϯ 0.06 (n ϭ 3). After extraction of the radioactive peak into ethanol more than 50% of the applied radioactivity was recovered as [ 3 H]benziazem. Radioactive benziazem was stored in the dark at Ϫ25°C.
Radioligand Binding Studies-Binding experiments with [ 3 H]benziazem were performed as described (15)  After incubation, the assay mixture was cooled on melting ice for 5 min. One mg of bovine serum albumin and ␥-globulin were added followed by 4 ml of a solution containing 10% (w/v) polyethylene glycol 6000, 10 mM MgCl 2 . After precipitation of the labeled complexes for 3 min on ice, free ligand was removed by filtration over GF/C Whatman filters. Filters were washed 5 times with ice-cold buffer (20 mM Tris-HCl, pH 7.4) and then counted for radioactivity.
Preparation of Photolabeled ␣ 1 Subunit-Ca 2ϩ channels were solubilized in digitonin and partially purified by affinity chromatography on wheat germ agglutinin-Sepharose 4B, as described (16). One to three milligrams of affinity purified protein was incubated with 3.5-4.5 nM [ 3 H]benziazem in a final volume of 20 -40 ml for 90 min at 25°C in the absence or presence of 10 M unlabeled competitor (Bz-BAZ) to determine nonspecific binding and photolabeling. Reversible binding prior to photolabeling was determined by filtration (15) of 200-l aliquots of the incubation mixture. Samples were transferred to siliconized glass Petri dishes and irradiated for 90 min at 4°C with ultraviolet light (Philips 38W/TL black light lamp) at a distance of 25 mm. Photolabeled samples were dialyzed against deionized water at room temperature for 12 h and lyophilized.
Reductive Carboxamidomethylation of [ 3 H]Benziazem-labeled ␣ 1 Subunits-Dialyzed and lyophilized samples were reduced for 15 min at 57°C with 1% (v/v) 2-mercaptoethanol in the presence of 1.6% (w/v) SDS and 400 mM Tris-HCl (pH 8.8) in a final volume of 0.425 ml. Freshly prepared iodoacetamide was then added to a final concentration of 100 mM. After incubation for 30 min at 22°C the sample was separated by HPLC size exclusion chromatography on a TSK4000SW column eluted with 0.04 M sodium phosphate, 0.1% (w/v) SDS (pH 7.2) at a flow rate of 1 ml/min. Fractions containing the radiolabeled ␣ 1 subunit were pooled and subjected to proteolytic cleavage and immunoprecipitation.
Immunoprecipitation-Sequence directed antibodies were bound to Protein A-Sepharose 4B by incubating 1-3 volumes of serum with 1 volume of swollen gel in radioimmunoassay buffer containing 1% (v/v) Triton X-100, 0.5 mg/ml bovine serum albumin, 150 mM NaCl, and 20 mM Tris-HCl (pH 7.4) for 30 min at 22°C. The Sepharose was washed with ice-cold radioimmunoassay buffer before digested or nondigested samples were added. After incubation for 2 h at room temperature immunoprecipitated radioactivity was either determined directly by liquid scintillation counting of the washed Protein A-Sepharose pellet or analyzed by SDS-PAGE.
Statistics-Data are given as means Ϯ S.D. of the indicated number of experiments. Analysis of equilibrium and kinetic binding experiments was as described previously (12).

[ 3 H]Benziazem, A High Affinity Photoligand for the BTZbinding Domain of Skeletal Muscle L-type Ca 2ϩ
Channels-A series of benzazepinones (BAZs) have recently been developed as potent diltiazem-related Ca 2ϩ antagonists (12). Extensive analysis of their structure-activity relationship identified the methoxy group (hydrogen acceptor) on the 4-aryl ring and the basic amine in ethyl linkage at N-1 as the critical pharmacophores for high affinity interaction with the channels BTZbinding domain (19). We have recently shown (12) that introduction of bulky side chains at some distance from the basic amine yields potent Ca 2ϩ antagonists that bind with high affinity to partially purified L-type Ca 2ϩ channels. To obtain a photoaffinity ligand we introduced a tritiated photoreactive benzophenone moiety (13,20) into the benzazepine structure at the appropriate position (Fig. 1). As shown in Fig. 2, the resulting compound, [ 3 H]benziazem (35 Ci/mmol), bound reversibly and with high affinity (K d ϭ 11.9 Ϯ 1.85 nM, B max ϭ 361 Ϯ 92 pmol/mg protein) to partially purified skeletal muscle Ca 2ϩ channels. Binding was completely blocked by (ϩ)-cis-diltiazem (IC 50 ϭ 2.6 Ϯ 1.7 M, n ϭ 3) and the more potent diltiazem derivative Bz-BAZ (IC 50 ϭ 21.0 Ϯ 4.5 nM, n ϭ 3) (12,21). At 22°C the DHP (ϩ)-isradipine (1 M) dramatically slowed (10 -18-fold, n ϭ 2) [ 3 H]benziazem dissociation kinetics. This noncompetitive interaction with DHPs is typical for drugs that bind selectively to the BTZ-binding domain of the channel (12,22,23). Irradiation of [ 3 H]benziazem-channel complexes resulted in efficient photoaffinity labeling under our experimental conditions. More than 66% (n ϭ 8, Fig. 2B) of the reversibly bound ligand was irreversibly incorporated into the ␣1 subunit after irradiation with ultraviolet light as determined by immunoprecipitation of labeled ␣1 subunits (Fig. 2B). Labeling was absent when 10 M unlabeled competitor (Bz-BAZ) were included in the binding reaction (Fig. 2, B and C). SDS-PAGE also revealed that photolabeling occurred into the ␣1 subunit without labeling of nonspecific bands (Fig. 2C). Taken together, [ 3 H]benziazem represents a novel high affinity probe for the BTZ-binding domain of L-type Ca 2ϩ channels.

FIG. 2. Reversible and irreversible labeling properties of [ 3 H]benziazem.
A, saturation analysis: increasing concentrations of [ 3 H]benziazem were incubated with 5 g/ml partially purified Ca 2ϩ channel protein for 90 min at 22°C in the absence (control) and presence (nonspecific binding) of 10 M Bz-BAZ. Bound ligand was determined by a filtration assay. The following binding parameters were derived by fitting the data to a monophasic saturation isotherm: B max ϭ 2.27 nM (454 pmol/mg of protein), K d ϭ 10 nM. One of two similar experiments is shown. B, specific ␣1 photolabeling with [ 3 H]benziazem: partially purified Ca 2ϩ channels were photolabeled with [ 3 H]benziazem (3 nM; 0.026 mg/ml protein) in the absence (T, total) or presence (NS, nonspecific) of 10 M Bz-BAZ. Reversible binding prior to photolysis was determined by filtration. After photolysis, samples were diluted 10-fold with radioimmunoassay buffer containing 1% Triton X-100 to dissociate reversibly bound ligand (29). Diluted samples were then immunoprecipitated with a saturating concentration of anti-␣1 219 -309 . More than 90% of reversible binding activity was irreversibly incorporated into ␣1. One of eight similar experiments is shown. C, aliquots (1 ml) of the photolabeled mixture were collected on 40 l of heparin-Sepharose CL-6B (30) and analyzed on a 8% polyacrylamide gel followed by fluorography (48-h exposure time). The migration of the ␣1 subunit (arrow) and of prestained molecular mass markers (given in kDa) is indicated.
containing S6 Segments in Repeats III and IV-As the Lys-C fragments also contain trypsin cleavage sites the photolabeled ␣1 subunits were digested with TPCK-trypsin (10 g/ml) to further restrict the sites of photolabeling. SDS-PAGE revealed two small photolabeled tryptic fragments with apparent molecular masses of 8.8 Ϯ 0.9 and 7.1 Ϯ 0.5 kDa (n ϭ 4). A fluorogram of a gel where these peaks were clearly separated is shown in Fig. 4A. The ␣1-associated [ 3 H]benziazem photolabeling was quantitatively recovered (n ϭ 3) in these peaks. In experiments with at least three different ␣1 preparations, tryptic digests revealed no smaller photolabeled polypeptides (Fig.  4, A and C).
The larger peptide must be derived from the 22-35-kDa Lys-C fragments as it was selectively immunoprecipitated by anti-␣1 1382-1400 (Fig. 4, A and C). The other repeat IV antibodies (anti-␣1 1339 -1354 and anti-␣1 1290 -1356 ) also immunoprecipitated radioactivity from the same tryptic digests to a similar extent (Fig. 4D). The labeled peptide must therefore contain IVS6 together with adjacent extracellular and cytoplasmic amino acid residues resulting most likely from cleavage at lysine 1336 and lysines in positions 1403 or 1414 (calculated molecular masses 7.9 -9.2 kDa).

Benz(othi)azepine Labeling Occurs Near the DHP and PAA
Interaction Sites of the ␣1 Subunit-We synthesized [ 3 H]benziazem as a novel photoaffinity probe to selectively label the BTZ-binding domain with high yield. Specifically photolabeled proteolytic fragments derived from this domain contained transmembrane segments S6 of repeats III and IV together with extracellular and intracellular sequence stretches. Residues within these regions must therefore be in close contact with the bound drug molecule. A preliminary study (10) of photolabeling by azidobutyryl clentiazem, a photoaffinity probe with much lower affinity and labeling efficiency, also implicated the S5-S6 region of repeat IV in benzothiazepine binding, but the labeled peptides could not be specifically identified by SDS-PAGE and characterized because of the low level of specific labeling. Nevertheless, this earlier work adds further support for our conclusion that a component of the benz(othi)azepine receptor site is located in transmembrane segment S6 of domain IV.
Previous functional studies revealed that a membrane-permeable diltiazem analogue can access its site only via an extracellular permeation pathway (24,25). We therefore propose that the high affinity BTZ-binding domain is formed by residues within the photolabeled regions that can be accessed from the extracellular surface of the ␣1 subunit.
Investigation of the molecular organization of the local anaesthetic binding domain of voltage-gated Na ϩ -channels has shown that residues not directly involved in high affinity drug binding in segment IVS6 control drug access to the adjacent binding site (26). This indicates that drug access involves steric interactions with amino acids near the drug-binding domains. It is therefore possible that [ 3 H]benziazem not only photoaffinity labels high affinity determinants for BTZ interaction but also adjacent residues modifying drug access to the binding domain.
As only one high affinity BTZ-binding domain exists on Ltype Ca 2ϩ channel ␣1 subunits, the photolabeled regions must be located in close proximity to each other in the folded struc-  ture of ␣1. This closely resembles the topology of the DHPbinding domain, where high affinity determinants for drug interaction are also provided by repeats III and IV including the respective S6 segments (3,4,7,9). Accordingly, the [ 3 H]benziazem labeled fragments were also found to be photolabeled by different DHPs (3). In contrast, PAAs only seem to bind to residues within IVS6 (6,8). Considering that S6 segments are believed to contribute to the lining of the ion conducting pathway (6,26), our data indicate that the binding domains for the three major classes of Ca 2ϩ antagonists are located in close proximity to each other within pore-forming regions of L-type channel ␣1 subunits.
Antibody mapping of DHP photolabeled ␣1 subunit also revealed labeling within the connecting linker between segments S5 and S6 in repeat III resulting in a labeled 3-4-kDa tryptic peptide. Experiments with chimeric ␣1 subunits recently confirmed the importance of this region to confer DHP sensitivity (7). We found no evidence for [ 3 H]benziazem labeling of this fragment that should be recognized by anti-␣1 1011-1026 . Similar to the bulky photoreactive side chains of DHP photoligands (27), the relatively flexible (see legend to Fig. 1) photoreactive benzophenone group may reach even more peripheral residues of the BTZ-binding domain. Based on the absence of photolabeling in the IIIS5-IIIS6 linker in repeat III it therefore appears unlikely that this region contains major determinants for high affinity BTZ interaction.
Implications for Non-competitive Binding Interactions among Ca 2ϩ Antagonist Drugs-Although identical tryptic fragments are photolabeled by DHPs and [ 3 H]benziazem, the respective binding domains are not identical. In radioligand binding studies non-competitive interactions have been described between DHP and BTZ binding that require DHP and BTZ binding to the channel at the same time resulting in the formation of a ternary complex (1). Allosteric interactions have originally been proposed to account for these effects (1). However, the close association of all three binding domains within pore-forming regions of the channel may also allow steric interactions between these drugs. We have previously obtained evidence for an isosteric interaction between DHPs and BTZs in studies with the fluorescent Ca 2ϩ antagonist DMBODIPY-BAZ (12,28). The effects of the DHP (ϩ)-isradipine on DMBO-DIPY-BAZ binding and fluorescence suggested that these drugs are located in close proximity to each other when simultaneously bound to the ␣1 subunit in a ternary complex. Our biochemical findings further support such a model.
Taken together we demonstrate that pore-forming regions of L-type Ca 2ϩ channel ␣1 subunits in repeats III and IV provide a "hot spot" for the binding and action of chemically unrelated Ca 2ϩ antagonists. Refining the structural organization of this region may not only help to provide insight into the molecular mechanism of Ca 2ϩ antagonist action but may also aid in the development of organic channel blockers against other types of voltage-gated Ca 2ϩ channels.