Benzothiazepinone Binding Domain of Purified L-Type Calcium Channels: Direct Labeling Using a Novel Fluorescent Diltiazem Analog
| Autor: | Cai Zw, Jörg Striessnig, Wolfgang Berger, Kang Kc, Stanislav Berjukov, Brauns T, S D Kimball, Hartmut Glossmann, Steffen Hering, Richard P. Haugland |
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| Rok vydání: | 1995 |
| Předmět: |
Binding Sites
Fluorophore Voltage-dependent calcium channel Chemistry Dihydropyridine Benzazepines Biochemistry Fluorescence Receptor–ligand kinetics Cell Line Diltiazem Kinetics chemistry.chemical_compound Förster resonance energy transfer medicine Biophysics Animals L-type calcium channel Calcium Channels Fluorescent Dyes medicine.drug Binding domain |
| Zdroj: | Biochemistry. 34:3461-3469 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi00010a039 |
| Popis: | We have synthesized a series of N-propylamino-substituted benzazepinones (NPSBs) as specific probes for the benzothiazepinone (BTZ) binding domain of muscle L-type calcium channels (LTCCs). NPSBs were identified which possess high affinity for the channel after purification. We synthesized a fluorescent NPSB, DMBODIPY-BAZ, as the first benz(othi)azepinone derivative known to reversibly label partially purified LTCCs. DMBODIPY-BAZ binds to the partially purified channel with high affinity (Kd = 25 nM, Bmax = 580 pmol/mg of protein). Fluorescence resonance energy transfer (FRET) occurred between tryptophan residues of the channel protein and the DMBODIPY fluorophore upon specific drug binding. FRET was exploited to allow highly time-resolved detection of specific drug binding kinetics. We found that the dissociation half-life (t1/2) of DMBODIPY-BAZ decreased with the concentration of an unlabeled competitor, which indicates ligand-induced accelerated dissociation. In contrast, t1/2 was concentration-dependently increased by the dihydropyridine (DHP) (+)-isradipine. These kinetic properties of DMBODIPY-BAZ indicate that a high-affinity BTZ binding domain also exists on purified LTCCs. NPSBs represent novel tools to provide further insight into the molecular pharmacology of the BTZ binding domain on LTCCs. |
| Databáze: | OpenAIRE |
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