title: Benzothiazepine binding domain of purified L-type calcium channels: direct labeling using a novel fluorescent diltiazem analogue.
abstract: 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.
authors.affiliation: Institut für Biochemische Pharmakologie, Innsbruck, Australia.
authors.forename: T
authors.initials: T
authors.lastname: Brauns
authors.forename: Z W
authors.initials: ZW
authors.lastname: Cai
authors.forename: S D
authors.initials: SD
authors.lastname: Kimball
authors.forename: K C
authors.initials: KC
authors.lastname: Kang
authors.forename: R P
authors.initials: RP
authors.lastname: Haugland
authors.forename: W
authors.initials: W
authors.lastname: Berger
authors.forename: S
authors.initials: S
authors.lastname: Berjukov
authors.forename: S
authors.initials: S
authors.lastname: Hering
authors.forename: H
authors.initials: H
authors.lastname: Glossmann
authors.forename: J
authors.initials: J
authors.lastname: Striessnig
mesh_terms: D000818:Animals; D001552:Benzazepines / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D015220:Calcium Channels / Q000145:classification / Q000378:metabolism*; D002460:Cell Line; D004110:Diltiazem / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000737:chemistry; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry; D007700:Kinetics
chemical_list: D001552:Benzazepines; D015220:Calcium Channels; D005456:Fluorescent Dyes; D004110:Diltiazem