Benzothiazepinone Binding Domain of Purified L-Type Calcium Channels: Direct Labeling Using a Novel Fluorescent Diltiazem Analog

Thomas Brauns, Wolfgang Berger, Stanislav Berjukov, Steffen Hering, Hartmut Glossmann, Jörg Striessnig, Zhen Wei Cai, S. David Kimball, Hee Chol Kang, Richard P. Haugland

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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, 5max = 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.

Original languageEnglish (US)
Pages (from-to)3461-3469
Number of pages9
JournalBiochemistry
Volume34
Issue number10
DOIs
Publication statusPublished - Jan 1 1995
Externally publishedYes

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All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Brauns, T., Berger, W., Berjukov, S., Hering, S., Glossmann, H., Striessnig, J., ... Haugland, R. P. (1995). Benzothiazepinone Binding Domain of Purified L-Type Calcium Channels: Direct Labeling Using a Novel Fluorescent Diltiazem Analog. Biochemistry, 34(10), 3461-3469. https://doi.org/10.1021/bi00010a039