INVIVO LABELING OF L-TYPE CA(2+) CHANNELS BY FLUORESCENT DIHYDROPYRIDINES - EVIDENCE FOR A FUNCTIONAL, EXTRACELLULAR HEPARIN-BINDING SITE

We have synthesized and characterized fluorescently labeled dihydropyridines (DHPs) as probes for L-type Ca2+ channels. Racemic as well as (+)- and (-)-1,4-dihydro-2,6-dimethyl-4-(2-trifluoromethylphenyl)-3,5-pyridinecarboxylic acid 2-(aminoethyl)ethyl ester hydrochlorides were coupled to boron dipyrromethane (Bodipy) derivatives. (4,4-Difluoro-5,7-dimethyl-4-bora-3a,4a-diaza)-3-(s-indacene)propionic acid (DMBodipy)-DHP and (4,4-difluoro-7-styryl-4-bora-3a,4a-diaza)-3-(s-indacene)propionic propionic acid (STBodipy)-DHP have K(d) values in the nanomolar range for membrane-bound or partially purified skeletal muscle and for neuronal L-type Ca2+ channels. (-)- and (+)-STBodipy-DHPs block Ca-45(2+) uptake through L-type Ca2+ channels into GH3 cells with IC50 Values of 14.8 and 562 nM, respectively. The measurement of bound fluorescence after removal of free DMBodipy-DHP with charcoal shows that the probes can substitute for radioactive ligands to study the properties (equilibrium binding, kinetics, allosteric regulation) of partially purified L-type Ca2+ channels from skeletal muscle. L-type Ca2+ channels on GH3 cells were stereoselectively visualized by using the optical enantiomers of STBodipy-DHP. Heparin inhibited GH3 cell labeling by (-)-STBodipy-DHP with an IC50 value of 9.7-mu-g/ml and blocked L-type Ca2+-channel-mediated Ca-45(2+) uptake with an IC50 value of 32-mu-g/ml. These findings argue for an extracellular orientation of the heparin-binding domain of the Ca2+ channel that is coupled to the DHP receptor.