BENZOTHIAZEPINONE BINDING DOMAIN OF PURIFIED L-TYPE CALCIUM CHANNELS - DIRECT LABELING USING A NOVEL FLUORESCENT DILTIAZEM ANALOG

We have synthesized a series of N-propylamino-substituted benzazepinon es (NPSBs) as specific probes for the benzothiazepinone (BTZ) binding domain of muscle L-type calcium channels (LTCCs). NPSBs were identifie d which possess high affinity for the channel after purification. We s ynthesized a fluorescent NPSB, DMBODIPY-BAZ, as the first benz(othi)az epinone derivative known to reversibly label partially purified LTCCs. DMBODIPY-BAZ binds to the partially purified channel with high affini ty (K-d = 25 nM, B-max = 580 pmol/mg of protein). Fluorescence resonan ce energy transfer (FRET) occurred between tryptophan residues of the channel protein and the DMBODIPY fluorophore upon specific drug bindin g. FRET was exploited to allow highly time-resolved detection of speci fic drug binding kinetics. We found that the dissociation half-life (t in) of DMBODIPY-BAZ decreased with the concentration of an unlabeled c ompetitor, which indicates ligand-induced accelerated dissociation. In contrast, t(1/2) was concentration-dependently increased by the dihyd ropyridine (DHP) (+)-isradipine. These kinetic properties of DMBODIPY- BAZ indicate that a high-affinity BTZ binding domain also exists on pu rified LTCCs. NPSBs represent novel tools to provide further insight i nto the molecular pharmacology of the BTZ binding domain on LTCCs.