Purified calcium channels have three allosterically coupled drug receptors.
(-)-[3H]Desmethoxyverapamil and (+)-[3H]PN 200-110 were employed to characterize phenylalkylamine-selective and 1,4-dihydropyridine-selective receptors on purified Ca2+ channels from guinea-pig skeletal muscle t-tubules. In contrast to the membrane-bound Ca2+ channel, d-cis-diltiazem (EC50 = 4.5 +/- 1.7 microM) markedly stimulated the binding of (+)-[3H]PN 200-110 to the purified ionic pore. In the presence of 100 microM d-cis-diltiazem (which binds to the benzothiazepine-selective receptors) the Bmax for (+)-[3H]PN 200-110 increased from 497 +/- 81 to 1557 +/- 43 pmol per mg protein, whereas the Kd decreased from 8.8 +/- 1.7 to 4.7 +/- 1.8 nM at 25 degrees C. P-cis-Diltiazem was inactive. (-)-Desmethoxyverapamil, which is a negative heterotropic allosteric inhibitor of (+)-[3H]IN 200-110 binding to membrane-bound channels, stimulated 1,4-dihydropyridine binding to the isolated channel. (-)-[3H]Desmethoxyverapamil binding was stimulated by antagonistic 1,4-dihydropyridines [(+)-PN 200-110 greater than (-)(R)-202-791 greater than (+)(4R)-Bay K 8644] whereas the agonistic enantiomers (+)(S)-202-791 and (-)(4S)-Bay K 8644 were inhibitory and (-)-PN 200-110 was inactive. The results indicate that three distinct drug-receptor sites exist on the purified Ca2+ channel, two of which are shown by direct labelling to be reciprocally allosterically coupled.
J
J
Striessnig
A
A
Goll
K
K
Moosburger
H
H
Glossmann
D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; D000494:Allosteric Regulation; D000588:Amines / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers; D004095:Dihydropyridines; D004110:Diltiazem / Q000494:pharmacology; D006168:Guinea Pigs; D007473:Ion Channels / Q000187:drug effects / Q000378:metabolism*; D017275:Isradipine; D009132:Muscles / Q000648:ultrastructure*; D009543:Nifedipine / Q000031:analogs & derivatives / Q000494:pharmacology; D010069:Oxadiazoles / Q000378:metabolism / Q000494:pharmacology; D011725:Pyridines / Q000378:metabolism* / Q000494:pharmacology; D011955:Receptors, Drug / Q000378:metabolism*; D014700:Verapamil / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology
D000588:Amines; D002121:Calcium Channel Blockers; D004095:Dihydropyridines; D007473:Ion Channels; D010069:Oxadiazoles; D011725:Pyridines; D011955:Receptors, Drug; D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; C038806:1,4-dihydropyridine; C043184:4-desmethoxyverapamil; D014700:Verapamil; D004110:Diltiazem; D009543:Nifedipine; D002118:Calcium; D017275:Isradipine