Photoaffinity labelling of the cardiac calcium channel. (-)-[3H]azidopine labels a 165 kDa polypeptide, and evidence against a [3H]-1,4-dihydropyridine-isothiocyanate being a calcium-channel-specific affinity ligand.
The arylazide 1,4-dihydropyridine (-)-[3H]azidopine binds to a saturable population of sites in guinea-pig heart membranes with a dissociation constant (KD) of 30 +/- 7 pM and a density (Bmax.) of 670 +/- 97 fmol/mg of protein. This high-affinity binding site is assumed to reside on voltage-operated calcium channels because reversible binding is blocked stereoselectively by 1,4-dihydropyridine channel blockers and by the enantiomers of Bay K 8644. A low-affinity (KD 25 +/- 7 nM) high-capacity (Bmax. 21.6 +/- 9 pmol/mg of protein) site does not bind (-)- or (+)-Bay K 8644, but is blocked by high concentrations (greater than 500 nM) of dihydro-2,6-dimethyl-4-(2-isothiocyanatophenyl)-3,5-pyridinedicarboxy lic acid dimethyl ester (1,4-DHP-isothiocyanate) or, e.g., (+/-)-nicardipine. (-)-[3H]Azidopine was photoincorporated covalently into bands of 165 +/- 8, 39 +/- 2 and 35 +/- 3 kDa, as determined by SDS/polyacrylamide-gel electrophoresis. Labelling of the 165 kDa band is protected stereoselectively by 1,4-dihydropyridine enantiomers at low (nM) concentrations and by (-)- and (+)-Bay K 8644, whereas the lower-Mr bands are not. Thus, only the 165 kDa band is the calcium-channel-linked 1,4-dihydropyridine receptor. Photolabelling of the 39 or 35 kDa bands was only blocked by 10 microM-1,4-DHP-isothiocyanate or 50 microM-(+/-)-nicardipine but not by 10 microM-(-)-Bay K 8644. [3H]-1,4-DHP-isothiocyanate binds to guinea-pig heart membranes with a KD of 0.35 nM and dissociates with a k-1 of 0.2 min-1 at 30 degrees C. [3H]-1,4 DHP-isothiocyanate irreversibly labels bands of 39 and 35 kDa which are protected by greater than 10 microM-(+/-)-nicardipine or unlabelled ligand but not by 10 microM-(-)-Bay K 8644. Thus, [3H]-1,4-DHP-isothiocyanate is not an affinity probe for the calcium channel.
D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D001386:Azides / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D004095:Dihydropyridines; D004591:Electrophoresis, Polyacrylamide Gel; D006168:Guinea Pigs; D006321:Heart / Q000187:drug effects; D006592:Hexobendine / Q000494:pharmacology; D007473:Ion Channels / Q000187:drug effects / Q000378:metabolism*; D017879:Isothiocyanates; D009206:Myocardium / Q000378:metabolism*; D009529:Nicardipine / Q000494:pharmacology; D009543:Nifedipine / Q000031:analogs & derivatives* / Q000378:metabolism; D010455:Peptides / Q000378:metabolism*; D011725:Pyridines / Q000378:metabolism*; D051381:Rats; D013045:Species Specificity; D013861:Thiocyanates / Q000378:metabolism*
D000345:Affinity Labels; D001386:Azides; D004095:Dihydropyridines; D007473:Ion Channels; D017879:Isothiocyanates; D010455:Peptides; D011725:Pyridines; D013861:Thiocyanates; C041130:azidopine; C045531:2,6-dimethyl-3,5-dicarbomethoxy-4-(2-isothiocyano)phenyl-1,4-dihydropyridine; D006592:Hexobendine; D009529:Nicardipine; D009543:Nifedipine; D002118:Calcium