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 (2-trifluoromethylphenyl)- 3,5-pyridinedicarboxylic acid a,4a-diaza-3-(s-indacene)propionylamino] ethylethyl ester)], to study the molecular mechanisms underlying the i nteraction of DHPs with the alpha(1)-subunit of skeletal muscle L-type Ca2+ channels. The quantum yield of the fluorophore DMBODIPY was simi lar 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-ind uced changes of intramolecular quenching of the fluorophore by the DHP moiety. Specific ligand binding also induced fluorescence resonance e nergy 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 resolu tion. It revealed complex association and dissociation kinetics of DMB ODIPY-DHP although no site heterogeneity was detected in equilibrium e xperiments. We therefore fitted our data to a binding scheme consideri ng one or more intermediate conformational states for the formation of the ligand-receptor complex. Such a step-wise binding mechanism expla ins previously observed differences in the binding site densities and the kinetic constants determined for different DHPs using conventional binding (for example filtration) assays.