COMPLEX MOLECULAR MECHANISM FOR DIHYDROPYRIDINE BINDING TO L-TYPE CA2+-CHANNELS AS REVEALED BY FLUORESCENCE RESONANCE ENERGY-TRANSFER

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-trifluoromethylphenyl)-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.