Porzig H, Becker C
Department of Pharmacology, University of Bern, Switzerland.
Mol Pharmacol. 1988 Aug;34(2):172-9.
We have studied allosteric effects of the Ca channel blockers d-(cis)-diltiazem, (+/-)-verapamil, and (S)- and (R)-devapamil on the specific binding of the 1,4-dihydropyridine derivative (+)-[3H]PN 200-110 to intact tissue cultured rat heart cells. In polarized cells (membrane potential, -38 +/- 4 mV) d-(cis)-diltiazem (5 microM) increased the affinity of the radiolabel 2-4-fold causing 100-187% enhancement of binding at (+)-PN 200-110 concentrations below 0.5 nM. (+/-)-Verapamil (0.1-3 microM) had a similar, although smaller, effect on (+)-PN 200-110 binding. The two enantiomers of devapamil were without effect. In depolarized cells (membrane potential, 0 mV) d-(cis)-diltiazem had a small and the phenylalkylamines a strong inhibitory effect on (+)-PN 200-110 binding, mainly due to a reduction of binding affinity. At 50% receptor occupation by the radioligand, (R)-devapamil, (+/-)-verapamil, and (S)-devapamil displaced 40, 55, and 75%, respectively, of specifically bound radiolabel. Half-maximal effects were reached with 50, 20, and 4.5 nM, respectively, of the three compounds. Compared with nominally Ca-free medium (containing 3-5 microM Ca), addition of 1.25 mM CaCl2 caused an increase in the maximal binding capacity for (+)-PN 200-110 in both polarized and depolarized cells. However, Ca had only marginal effects on the allosteric interactions between (+)-PN 200-110, d-(cis)-diltiazem, and verapamil. We conclude from our results that positive cooperative interactions between Ca channel blockers prevail under conditions in which the voltage-dependent Ca channel can fluctuate between closed, open, and inactivated states. Negative cooperativity is usually observed under conditions in which all channels are inactivated (depolarized cells, fragmented membranes). Therefore, it is impossible to predict the type and the extent of allosteric interactions in vivo from studies in cell homogenates.
我们研究了钙通道阻滞剂d-(顺式)-地尔硫䓬、(±)-维拉帕米以及(S)-和(R)-去甲维拉帕米对1,4-二氢吡啶衍生物(+)-[3H]PN 200-110与完整的组织培养大鼠心肌细胞特异性结合的变构效应。在极化细胞（膜电位为-38±4 mV）中，d-(顺式)-地尔硫䓬（5 μM）使放射性标记物的亲和力提高了2至4倍，在(+)-PN 200-110浓度低于0.5 nM时，结合增强了100%至187%。(±)-维拉帕米（0.1至3 μM）对(+)-PN 200-110结合有类似但较小的影响。去甲维拉帕米的两种对映体则无作用。在去极化细胞（膜电位为0 mV）中，d-(顺式)-地尔硫䓬对(+)-PN 200-110结合有较小的抑制作用，而苯烷基胺类有较强的抑制作用，主要是由于结合亲和力降低。当放射性配体占据50%的受体时，(R)-去甲维拉帕米、(±)-维拉帕米和(S)-去甲维拉帕米分别取代了40%、55%和75%的特异性结合放射性标记物。三种化合物分别在50 nM、20 nM和4.5 nM时达到半数最大效应。与名义上无钙的培养基（含有3至5 μM钙）相比，添加1.25 mM氯化钙使极化和去极化细胞中(+)-PN 200-110的最大结合容量增加。然而，钙对(+)-PN 200-110、d-(顺式)-地尔硫䓬和维拉帕米之间的变构相互作用仅有微小影响。我们从结果中得出结论，钙通道阻滞剂之间的正协同相互作用在电压依赖性钙通道可在关闭、开放和失活状态之间波动的条件下占主导。负协同性通常在所有通道均失活的条件下观察到（去极化细胞、破碎膜）。因此，不可能从细胞匀浆研究中预测体内变构相互作用的类型和程度。
