Photoaffinity-labelling of the calcium-channel-associated 1,4-dihydropyridine and phenylalkylamine receptor in guinea-pig hippocampus. A 195 kDa polypeptide carries both drug receptors and has similarities to the alpha 1 subunit of the purified skeletal-muscle calcium channel.
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.
Striessnig
J
J
Institut für Biochemische Pharmakologie, Universität Innsbruck, Austria.
Knaus
H G
HG
Glossmann
H
H
D000345:Affinity Labels; D000818:Animals; D001386:Azides; D002121:Calcium Channel Blockers / Q000378:metabolism*; D015220:Calcium Channels; D002462:Cell Membrane / Q000378:metabolism; D004095:Dihydropyridines; D006168:Guinea Pigs; D006624:Hippocampus / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D009132:Muscles / Q000378:metabolism; D010455:Peptides / Q000378:metabolism*; D011863:Radioimmunoassay; D011978:Receptors, Nicotinic / Q000378:metabolism*; D014316:Tritium; D014700:Verapamil / Q000031:analogs & derivatives
D000345:Affinity Labels; D001386:Azides; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D004095:Dihydropyridines; D010455:Peptides; D011978:Receptors, Nicotinic; D014316:Tritium; C051537:LU 49888; C041130:azidopine; D014700:Verapamil