A unique fluorescent phenylalkylamine probe for L-type Ca2+ channels. Coupling of phenylalkylamine receptors to Ca2+ and dihydropyridine binding sites.

Knaus, Hans−Günther; Moshammer, Thomas; Kang, Hee Young; Haugland, Richard P.; Glossmann, Hartmut

doi:10.1016/s0021-9258(18)45860-7

Cited by 41 publications

(12 citation statements)

References 53 publications

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“…Figure 3A shows the fluorescent properties of DMBODIPY-BAZ. As expected for the DMBODIPY fluorophore (Knaus et al, 1992a;Knaus et al, 1992b), the maxima of the excitation and emission spectra were at 511 and 515 nm, respectively. DMBODIPY-BAZ concentrations were routinely determined after direct excitation at 488 nm, measuring emission at 517 nm (see Materials and Methods section).…”

Section: Resultssupporting

confidence: 79%

“…Radioligand binding assays to particulate or partially purified channels were carried out using previously published procedures . Binding of fluorescent DMBODIPY-BAZ was determined using a charcoal assay (Knaus et al, 1992b). DMBODIPY-BAZ and channel protein were incubated at 25 °C in incubation buffer (50 mM Tris/HCl, pH 7.4, 0.1% (w/v) digitonin, 0.25 mg/ mL BSA) in a final volume of 1 mL in the absence and presence of unlabeled drugs.…”

Section: Dmbodipy-bazmentioning

confidence: 99%

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

Brauns¹,

Zw²,

Sd³

et al. 1995

Biochemistry

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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.

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Section: Resultssupporting

confidence: 79%

Section: Dmbodipy-bazmentioning

confidence: 99%

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

Brauns¹,

Zw²,

Sd³

et al. 1995

Biochemistry

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“…Fluorescence Binding Studies (Charcoal Assay). Binding studies with DMBODIPY-DHP were carried out at 25 °C in binding buffer [50 mM Tris-HCl, pH 7.6, 0.1% (w/v) digitonin, 0.25 mg/mL bovine serum albumin (essentially fatty acid free), 0.1 mM CaCh] in a final volume of 1 mL as previously described (Knaus et al, 1992a). Fluorescent and nonfluorescent drugs were diluted in dimethyl sulfoxide (DMSO) and directly added to the incubation mixture.…”

Section: Methodsmentioning

confidence: 99%

Complex Molecular Mechanism for Dihydropyridine Binding to L-Type Ca2+-Channels As Revealed by Fluorescence Resonance Energy Transfer

Berger¹,

Prinz²,

Striessnig³

et al. 1994

Biochemistry

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We analyzed binding-induced changes in the fluorescence properties of the 1,4-dihydropyridine (DHP), DMBODIPY-DHP [(-)-1,4-dihydro-2,6-dimethyl-4-(2-trifluromethylphenyl)- 3,5-pyridinedicarboxylic acid 2-[4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-3- (s-indacene)propionylamino]ethylethyl ester)], to study the molecular mechanisms underlying the interaction of DHPs with the alpha 1-subunit of skeletal muscle L-type Ca2+ channels. The quantum yield of the fluorophore DMBODIPY was similar in solvents of different polarity. In contrast, the quantum yield of DMBODIPY-DHP was low in buffer but increased with solvent polarity and upon specific binding. This indicates the existence of binding-induced changes of intramolecular quenching of the fluorophore by the DHP moiety. Specific ligand binding also induced fluorescence resonance energy transfer (FRET) between one or more tryptophanes of the channel protein and the DMBODIPY-DHP fluorophore. The specific FRET signal was successfully used to directly measure DHP binding at high time resolution. It revealed complex association and dissociation kinetics of DMBODIPY-DHP although no site heterogeneity was detected in equilibrium experiments. We therefore fitted our data to a binding scheme considering one or more intermediate conformational states for the formation of the ligand-receptor complex. Such a step-wise binding mechanism explains previously observed differences in the binding site densities and the kinetic constants determined for different DHPs using conventional binding (for example filtration) assays.

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“…When added at different times during the association time course, the amplitude of the DHP-induced FRET increase was proportional to the concentration of bound DMBODIPY-BAZ (n ) 2). Moreover, the effect was absent when, instead of the BTZ site, the PAA binding domain of the channel was labeled with the fluorescent verapamil analogue DMBODIPY-PAA (Knaus et al, 1992b) (n ) 2, data not shown).…”

Section: Dhps Modulate Dmbodipy-baz Fret Fluorescencementioning

confidence: 99%

L-Type Calcium Channels: Binding Domains for Dihydropyridines and Benzothiazepines Are Located in Close Proximity to Each Other

et al. 1997

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We investigated the binding of a fluorescent diltiazem analogue (3R,4S)-cis-1-[2-[[3-[[3-[4,4-difluoro-3a,4-dihydro-5,7-dimethyl-4-bo ra-3a,4a-diaza-s-indacen-3-yl]propionyl]amino]propyl]amin o]ethy]-1,3,4,5-tetrahydro-3-hydroxy-4-(4-methoxyphenyl)-6-(triflu oromethyl)-2H-1-benzazepin-2-one (DMBODIPY-BAZ) to L-type Ca2+ channels in the presence of different 1,4-dihydropyridines (DHPs) by using fluorescence resonance energy transfer (FRET) [Brauns, T., Cai, Z.-W., Kimball, S. D., Kang, H.-C., Haugland, R. P., Berger, W., Berjukov, S., Hering, S., Glossmann, H., & Striessnig, J. (1995) Biochemistry 34, 3461]. When channels are occupied with DMBODIPY-BAZ, a rapid fluorescence change occurred upon addition of different DHPs. The direction of this intensity modulation was found to be only dependent on the chemical composition of the dihydropyridine employed. DHPs containing a nitro group decreased, whereas others (e.g., isradipine) enhanced the fluorescence signal. In addition, all DHPs markedly decreased the association rate constant for DMBODIPY-BAZ without affecting equilibrium binding. Both observations together are best explained by a steric model where the DHP binding site is located in close proximity to the accession pathway of DMBODIPY-BAZ.

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A unique fluorescent phenylalkylamine probe for L-type Ca2+ channels. Coupling of phenylalkylamine receptors to Ca2+ and dihydropyridine binding sites.

Cited by 41 publications

References 53 publications

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

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

Complex Molecular Mechanism for Dihydropyridine Binding to L-Type Ca2+-Channels As Revealed by Fluorescence Resonance Energy Transfer

L-Type Calcium Channels: Binding Domains for Dihydropyridines and Benzothiazepines Are Located in Close Proximity to Each Other

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