title: [35S]sadopine, a novel high affinity, high specific activity, L-type Ca2+ channel probe: characterization of two equipotent diastereomers with opposite allosteric properties.
abstract: 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.
authors.affiliation: Institute for Biochemical Pharmacology, Innsbruck, Austria.
authors.forename: H G
authors.initials: HG
authors.lastname: Knaus
authors.forename: J
authors.initials: J
authors.lastname: Striessnig
authors.forename: S
authors.initials: S
authors.lastname: Hering
authors.forename: S
authors.initials: S
authors.lastname: Marrer
authors.forename: E
authors.initials: E
authors.lastname: Schwenner
authors.forename: H D
authors.initials: HD
authors.lastname: Höltje
authors.forename: H
authors.initials: H
authors.lastname: Glossmann
mesh_terms: D000818:Animals; D001345:Autoradiography; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000378:metabolism*; D003198:Computer Simulation; D004095:Dihydropyridines / Q000138:chemical synthesis / Q000378:metabolism* / Q000494:pharmacology; D006168:Guinea Pigs; D066298:In Vitro Techniques; D007700:Kinetics; D008954:Models, Biological; D011817:Rabbits; D051381:Rats; D011919:Rats, Inbred Strains; D011978:Receptors, Nicotinic / Q000378:metabolism; D013237:Stereoisomerism; D013462:Sulfur Radioisotopes
chemical_list: D002121:Calcium Channel Blockers; D015220:Calcium Channels; D004095:Dihydropyridines; D011978:Receptors, Nicotinic; D013462:Sulfur Radioisotopes; C060119:sadopine