Schilling W P, Zaher M C, Rampe D
Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas 77030.
Mol Pharmacol. 1990 Jan;37(1):80-9.
The effects of the inorganic Ca2+ channel blockers Cd2+ and La3+ on dihydropyridine (DHP) binding in highly enriched cardiac sarcolemma preparations has been examined. Cd2+ produced an apparent competitive inhibition of DHP binding with a Ki of 60 microM. DHP binding in the presence of La3+ produced nonlinear Scatchard plots when performed in intact membrane vesicle preparations. Evaluation of DHP binding in saponin-permeabilized vesicles or in the presence of the ionophore A23187 yielded linear Scatchard profiles in the presence of La3+. Under these conditions, La3+ produced a mixed-type inhibition, with effects on both Kd and Bmax. These results suggest that La3+ must have access to the interior of sealed vesicles for expression of full inhibitory activity and that La3+ may produce inhibition of DHP binding by interaction with only one surface of the membrane. In order to evaluate the sidedness of the La3+ interaction, membrane preparations consisting of 74% right side out and 26% leaky vesicles were isolated. In the absence of saponin, La3+ decreased maximum DHP binding in this preparation approximately 25%, with no significant change in Kd. When binding was performed in saponin-permeabilized preparations, however, La3+ produced dramatic decreases in both DHP binding affinity and capacity. These results are consistent with the hypothesis that La3+ produces inhibition of DHP binding by interaction with sites accessible only from the cytoplasmic membrane surface. To obtain additional support for this hypothesis. DHP binding was examined in rat ventricular myocytes grown in culture. La3+ and Cd2+, at concentrations in the extracellular buffer that substantially inhibited K+ depolarization-induced 45Ca2+ influx, had little or no effect on DHP binding.
研究了无机钙离子通道阻滞剂Cd2+和La3+对高度富集的心肌肌膜制剂中二氢吡啶（DHP）结合的影响。Cd2+对DHP结合产生明显的竞争性抑制，其抑制常数Ki为60 microM。在完整膜囊泡制剂中进行实验时，La3+存在下的DHP结合产生非线性Scatchard图。在皂素通透的囊泡中或存在离子载体A23187的情况下评估DHP结合，在La3+存在时产生线性Scatchard图。在这些条件下，La3+产生混合型抑制，对解离常数（Kd）和最大结合量（Bmax）均有影响。这些结果表明，La3+必须能够进入密封囊泡内部才能表现出完全的抑制活性，并且La3+可能仅通过与膜的一个表面相互作用来抑制DHP结合。为了评估La3+相互作用的方向性，分离了由74%外翻和26%渗漏囊泡组成的膜制剂。在没有皂素的情况下，La3+使该制剂中的最大DHP结合减少约25%，Kd无显著变化。然而，当在皂素通透的制剂中进行结合实验时，La3+使DHP结合亲和力和容量均显著降低。这些结果与以下假设一致：La3+通过与仅从细胞质膜表面可及的位点相互作用来抑制DHP结合。为了获得对该假设的更多支持，在培养的大鼠心室肌细胞中检测了DHP结合。在细胞外缓冲液中浓度能显著抑制钾离子去极化诱导的45Ca2+内流的La3+和Cd2+，对DHP结合几乎没有影响。
