scholarly journals 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

1987 ◽  
Vol 243 (1) ◽  
pp. 127-135 ◽  
Author(s):  
D R Ferry ◽  
A Goll ◽  
H Glossmann
Keyword(s):  
Guinea Pig ◽  
Calcium Channel ◽  
Polyacrylamide Gel Electrophoresis ◽  
Dimethyl Ester ◽  
High Capacity ◽  
Bay K 8644 ◽  
Channel Blockers ◽  
Guinea Pig Heart ◽  
Affinity Ligand ◽  
Heart Membranes

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.

Ca2+ channels in chick neural retina cells characterized by 1,4-dihydropyridine antagonists and activators

1989 ◽  
Vol 67 (5) ◽  
pp. 506-514 ◽  
Author(s):  
X. Y. Wei ◽  
A. Rutledge ◽  
Q. Zhong ◽  
J. Ferrante ◽  
D. J. Triggle
Keyword(s):  
Calcium Channel ◽  
Intact Cell ◽  
Neural Retina ◽  
Bay K 8644 ◽  
Single Type ◽  
Channel Blockers ◽  
Binding Affinities ◽  
High Affinity ◽  
Voltage Dependent ◽  
High Affinity Binding Site

The voltage-sensitive calcium channel in cultured chick neural retina cells was characterized by the actions of the enantiomers of Bay K 8644 and 202-791 and other 1,4-dihydropyridines. These cells showed time- and voltage-dependent Ca2+ uptake that was stimulated by K+ depolarization and blocked by the inorganic calcium channel blockers Cd2+ and Co2+. A small fraction only (15% maximum) of the uptake was inactivated by predepolarization of the cells with 80 mM K+. Ca2+ uptake was sensitive to the 1,4-dihydropyridine calcium channel antagonists and activators. (S)-Bay K 8644 and (S)-202-791 stimulated the Ca2+ uptake, and (R)-Bay K 8644 and (R)-202-791 as well as nitrendipine and PN 200-110 inhibited Ca2+ uptake stimulated by K+ depolarization or channel activators. The K+ depolarization-stimulated uptake was inhibited by 90%, but the activator-stimulated uptake was completely blocked by the 1,4-dihydropyridine antagonists. The potencies of these agents as inhibitors of Ca2+ uptake were significantly lower than the binding affinities in membrane preparations from the same cells or their binding and pharmacologic affinities in vascular smooth muscle. K+ depolarization or (S)-Bay K 8644 induced 45Ca2+ uptake was not observed in a glial cell culture. [3H]Nitrendipine and [3H]PN 200-110 bound to membrane preparations of the cells consistent with the presence of a single type of high affinity binding site. [3H]PN 200-110 bound with higher affinity (KD = 7.09 ± 0.90 × 10−11 M) than did [3H]nitrendipine (KD = 4.10 ± 0.92 × 10−10 M), but the Bmax values were similar for the two ligands (98.9 ± 4.1 and 99.4 ± 6.58 fmol/mg protein, respectively). The discrepancy between binding and pharmacologic activities of the antagonist ligands does not appear to be due to the presence of 1,4-dihydropyridine-insensitive Ca2+ channels, but may relate to the inability of these agents to access a high affinity inactivated state in the intact cell or to the presence of discrete categories of binding sites. This marked discrepancy between affinities does not exist for the activator ligands studied. This study confirms the presence of voltage-dependent 1,4-dihydropyridine sensitive Ca2+ channels in chick neural retina cells.Key words: Ca2+ channels, 1,4-dihydropyridines, chick neural retina, retinal neurons.

The effects of the calcium channel agonist, Bay K-8644, on guinea-pig ileum and rat uterine horn

1987 ◽  
Vol 39 (11) ◽  
pp. 954-957 ◽  
Author(s):  
Diana Conte-Camerino ◽  
Marcello D. Lograno ◽  
Annamaria Luca ◽  
Michele Persichella ◽  
Flavia Franconi
Keyword(s):  
Guinea Pig ◽  
Calcium Channel ◽  
Uterine Horn ◽  
Bay K 8644 ◽  
Guinea Pig Ileum ◽  
Channel Agonist

scholarly journals A novel 1,4-dihydropyridine-binding site on mitochondrial membranes from guinea-pig heart, liver and kidney

1988 ◽  
Vol 253 (1) ◽  
pp. 49-58 ◽  
Author(s):  
G Zernig ◽  
H Glossmann
Keyword(s):  
Guinea Pig ◽  
Binding Site ◽  
Binding Sites ◽  
Ph Dependence ◽  
High Capacity ◽  
Mitochondrial Membranes ◽  
Guinea Pig Heart ◽  
Heat Stable ◽  
Liver And Kidney ◽  
Dihydropyridine Binding Site

The 1,4-dihydropyridine (+/-)-[3H]nitrendipine reversibly binds to mitochondrial preparations from guinea-pig heart with a dissociation constant (Kd) of 593 +/- 77 nM and a maximum density of binding sites (Bmax.) of 1.75 +/- 0.27 nmol/mg of protein. This low-affinity high-capacity 1,4-dihydropyridine-binding site does not discriminate between the enantiomers of nitrendipine and is also found in mitochondrial membranes from guinea-pig liver (Kd 586 +/- 91 nM; Bmax. 0.36 +/- 0.04 nmol/mg of protein) and kidney (Kd 657 +/- 149 nM; Bmax. 0.56 +/- 0.12 nmol/mg of protein). Phenylalkylamines (e.g. verapamil) inhibit (+/-)-[3H]nitrendipine binding with micromolar inhibition constants, but the benzothiazepine D-cis-diltiazem, a potent Ca2+-channel blocker, is without effect. The binding is heat-stable, shows a V-shaped pH-dependence with a minimum around pH 7.0, and is strongly dependent on ionic strength in the incubation medium. The cations La3+ greater than Cd2+ much greater than Co2+ greater than Ca2+ much greater than Ba2+ greater than Mg2+ greater than Li+ greater than Na+ and the anions NO3- greater than C1- greater than or equal to F- stimulate the binding, whereas PO4(3-) greater than SO4(2-) slightly inhibit it. The low-affinity (+/-)-[3H]nitrendipine-binding site located on the mitochondrial inner membrane is biochemically and pharmacologically different from the 1,4-dihydropyridine-receptor domain of the L-type Ca2+ channel. Furthermore, it is not identical with any of the low-affinity 1,4-dihydropyridine-binding sites described so far.

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