[35S]sadopine, a novel high affinity, high specific activity, L-type Ca2+ channel probe: characterization of two equipotent diastereomers with opposite allosteric properties

HG Knaus, J Striessnig, S Hering, S Marrer, E Schwenner, HD Holtje and H Glossmann

Institute for Biochemical Pharmacology, Innsbruck, Austria.

A novel 35S-labeled dihydropyridine (DHP), 1,4-dihydro-2,6-dimethyl-4- (2-trifluoromethylphenyl)-pyridine-3,5-dic arboxyl-3- [2-(N-tert- butoxycarbonyl-L-[35S]methionyl)-aminoethyl]-ester-5-ethyl ester, ([35S]sadopine) (800-1400 Ci/mmol), the respective (+)- and (-)- diastereomers, and unlabeled (+/- )-, (-)-, and (+)-sadopine were synthesized. [35S]Sadopine is an excellent high affinity, high specific activity radioligand to label selectively the DHP receptor of L-type Ca2+ channels in tissue sections as well as in membrane fragments. Both diastereomers bind to the DHP receptors in a saturable and reversible manner, with equal, subnanomolar, dissociation constants. Despite their similar affinities, (+)- and (-)-sadopine differ with respect to their kinetic properties [the association and dissociation rate constants are 10-fold higher for (+)-[35S]sadopine at 22 degrees] and their allosteric modulation of the phenylaklylamine or benzothiazepine binding domain. (+)-Sadopine is a negative but (-)-sadopine a positive allosteric modulator of (-)-[N-methyl-3H]LU49888 or (+)-cis-[3H] diltiazem binding at 30 degrees. Both diastereomers act as L-type Ca2+ channel blockers in cardiac and smooth muscle cells. Computer-based analysis of the electrostatic potentials of the two diastereomers and calculation of the interaction energies with a hypothetical DHP receptor model predicted not only the similar affinities of (+)- and (- )-sadopine but also their Ca2+ channel-blocking effects. The temperature-dependent allosteric differences between the diastereomers suggest that two distinct conformational states of the DHP receptor are stabilized in vitro, both corresponding to a nonconducting state of the channel. Our data indicate that access to the DHP receptor site, but not binding affinity, is a function of the opposite stereochemistry of the sadopine diastereomers. Therefore, labeled and unlabeled (+)- and (- )-sadopine will be useful probes to further characterize the molecular basis of DHP-Ca2+ channel interaction and the pharmacological and physiological significance of the different allosteric conformations of the channel induced by Ca2+ channel-active drugs.

Volume 41, Issue 2, pp. 298-307, 02/01/1992
Copyright © 1992 by American Society for Pharmacology and Experimental Therapeutics