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.