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J. Biol. Chem., Vol. 267, Issue 4, 2179-2189, 02, 1992

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

HG Knaus, T Moshammer, HC Kang, RP Haugland and H Glossmann
Institute for Biochemical Pharmacology, Innsbruck, Austria.

The first fluorescently labeled phenylalkylamine, DMBODIPY-PAA (5-(3-[3- (4,4-difluoro-5,7-dimethyl-3a, 4a-diaza-4-bora-indacen-3- yl)propionamido] phenethyl-N-methylamino)-2-isopropyl-2-(3,4,5- trimethoxyphenyl)-valer onitrile) has been introduced for L-type Ca2+ channel research. DMBODIPY-PAA binds reversibly to L-type Ca2+ channels purified from rabbit skeletal muscle microsomes by wheat germ agglutinin-Sepharose chromatography. In this preparation DMBODIPY-PAA labels 412 pmol of phenylalkylamine receptors/mg of protein with a Kd of 6.82 nM and a favorable signal-to-noise ratio. Therefore DMBODIPY- PAA has a higher affinity for purified Ca2+ channels than the commonly employed radioligands and consequently has assisted in channel purification after prelabeling by simply monitoring receptor-bound fluorescence. (+)-PN200-110 (which is stimulatory for (-)- [3H]desmethoxyverapamil binding to purified Ca2+ channels) inhibits DMBODIPY-PAA labeling. Since these drug interactions are reciprocal, the phenylalkylamine and dihydropyridine binding sites of the alpha 1- subunit are tightly coupled. Kinetic and equilibrium binding studies with (-)-[3H]desmethoxyverapamil and DMBODIPY-PAA show that phenylalkylamine binding to L-type Ca2+ channels is dependent on Ca2+. Chelation of divalent metal ions converts phenylalkylamine receptors into a very low affinity state. This conversion is temperature- and time-dependent and completely reversible (K0.5 for free Ca2+ = 58 nM). This study demonstrates the utility of fluorescent ligands for binding studies with L-type Ca2+ channels and provides evidence for coupling between Ca2+ binding sites and phenylalkylamine receptors.
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