5-((3-azidophenethyl)methylamino)-2-(3-4-5-trimethoxyphenyl)-2-isopropylvaleronitrile and azidopine

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Affinity Labels; Animals; Azides; Calcium Channels; Cloning, Molecular; Dihydropyridines; DNA; Glycoproteins; Muscles; Phosphorylation; Protein Conformation; Protein Kinases; Receptors, Nicotinic; Verapamil

This study identifies calcium-antagonist-receptor-carrying polypeptides of calcium channels in guinea-pig hippocampus membranes. The arylazide ligands (-)-[3H]azidopine and [N-methyl-3H]LU49888 [(-)-5-[(3-azidophenethyl) [N-methyl-3H]methylamino]-2-(3,4,5-trimethoxyphenyl-2- isopropylvaleronitrile] were used to selectively label 1,4-dihydropyridine and phenylalkylamine receptors respectively. In the absence of u.v. light, both ligands reversibly bound to a single class of high-affinity receptors with a calcium-channel-typical pharmacological profile. [N-methyl-3H]LU49888 bound to the extent of 849 +/- 188 fmol/mg of protein (mean +/- S.D., n = 3) with a dissociation constant (Kd) of 1.4 +/- 0.3 nM. Under identical assay conditions (-)-[3H]azidopine labelled to the extent of 562 +/- 132 fmol/mg of protein with a Kd of 0.096 +/- 0.024 nM. After u.v. irradiation of the [N-methyl-3H]LU49888- and (-)-[3H]azidopine-labelled membranes, both photo-affinity probes were found to be incorporated specifically into a 190-195 kDa band as shown by SDS/polyacrylamide-gel electrophoresis (SDS/PAGE). Photoincorporation occurred with a protection profile identical with that produced by reversible binding-inhibition. [N-methyl-3H]LU49888, but not (-)-[3H]-azidopine, specifically labelled an additional 265 kDa band. Both photolabelled bands had an identical electrophoretic mobility on SDS/PAGE, irrespective of pretreatment either with 10 mM-N-ethylmaleimide or 10 mM-dithiothreitol. The electrophoretic properties of the 195 kDa polypeptide and the lability of receptor-incorporated (-)-[3H]azidopine to nucleophilic agents resemble those of the previously described drug-receptor-carrying alpha 1 subunit of the purified skeletal-muscle calcium channel. The data suggest that this polypeptide carries both the high-affinity 1,4-dihydropyridine as well as the phenylalkylamine receptor of neuronal calcium channels in guinea-pig hippocampus and is a component of the L-type calcium channel.

Topics: Affinity Labels; Animals; Azides; Calcium Channel Blockers; Calcium Channels; Cell Membrane; Dihydropyridines; Guinea Pigs; Hippocampus; In Vitro Techniques; Kinetics; Muscles; Peptides; Radioimmunoassay; Receptors, Nicotinic; Tritium; Verapamil

The dihydropyridine receptor purified from rabbit skeletal muscle yields in the presence of dithiothreitol and sodium dodecyl sulfate on polyacrylamide gels bands of apparent molecular mass 165 +/- 5, 130 +/- 5, 55 +/- 3, 32 +/- 2 and 28 +/- 1 kDa (chi +/- SEM, n = 12). Under nonreducing conditions, the 130 kDa and 28-kDa peptides migrate as a single peptide of 165 kDa. These peptides were separated on a HPLC size-exclusion column. The specific absorption coefficients of the isolated peptides were determined. From these a stoichiometry of 1:1.7 +/- 0.2:1.4 +/- 0.3 (chi +/- SEM of 12 experiments with three different preparations) was calculated for the 165-kDa, 55-kDa and 32-kDa peptides. The relative amount of the 130/28-kDa peptide varied with different preparations. Tryptic, chymotryptic and V-8 protease peptides of the isolated proteins suggested that the 130/28-kDa peptide was not related to the 165-kDa peptide. The dihydropyridine photoaffinity analog (+/-)-azidopine was specifically incorporated only into the 165-kDa peptide with an efficiency of about 2.4%. The azido analog of desmethoxyverapamil, LU 49888, was specifically incorporated into the same peptide with an efficiency of 1.5%. These results suggest that only the 165-kDa peptide contains the regulatory sites detected so far in the voltage-operated L-type calcium channel. They suggest further that the 130/28-kDa peptide, which migrates as a 165-kDa peptide under nonreducing conditions, does not contain high-affinity binding sites for the calcium channel blockers.

Topics: Affinity Labels; Animals; Azides; Calcium; Calcium Channels; Dihydropyridines; Ion Channels; Isradipine; Kinetics; Molecular Weight; Muscles; Oxadiazoles; Pyridines; Rabbits; Receptors, Nicotinic; Verapamil