Garcia M L, King V F, Siegl P K, Reuben J P, Kaczorowski G J
J Biol Chem. 1986 Jun 25;261(18):8146-57.
Stereospecific saturable and reversible binding of d-cis-diltiazem has been demonstrated in cardiac sarcolemmal membrane vesicles. Analysis of binding by either equilibrium or kinetic techniques indicates the presence of a single class of benzothiazepine receptors which bind diltiazem with a KD of 80 nM at 25 degrees C. Benzothiazepine receptors copurify with other sarcolemmal marker activities and exist in a complex with distinct receptors for dihydropyridine and aralkylamine Ca2+ entry blockers in a 1:1:1 stoichiometry. Ligand binding to one receptor of this complex influences binding reactions at the other two sites in a manner that depends on ambient temperature. Binding of either dihydropyridine agonists or antagonists causes partial inhibition of diltiazem binding at 25 degrees C (Bmax effect), while most dihydropyridine antagonists stimulate and agonists inhibit diltiazem binding at 37 degrees C (both are KD effects). This temperature-dependent change in receptor coupling was confirmed by Scatchard analyses and study of diltiazem dissociation kinetics. Verapamil, interacting at the aralkylamine receptor, inhibits diltiazem binding equivalently at 25 and 37 degrees C (KD effects). In addition, both classes of dihydropyridines inhibit verapamil binding in a temperature-independent fashion, as does diltiazem (all are KD effects). Allosteric coupling between benzothiazepine and dihydropyridine receptors is manifested in cardiac muscle since the negative inotropic potency of diltiazem is increased by nitrendipine and decreased by 4-(O-trifluromethy(phenyl)-2,6-dimethyl-5-nitro-1,4-dihydropyridin e-3- carboxylic acid, methyl ester. These results suggest a model in which the Ca2+ entry blocker receptor complex undergoes a change between 25 and 37 degrees C so that at the latter temperature all sites are directly coupled. Allosteric coupling may have important consequences in vivo since it can be detected in functional assays of Ca2+ channel activity.
在心肌肌膜囊泡中已证实d - 顺式地尔硫䓬存在立体特异性、可饱和且可逆的结合。通过平衡或动力学技术对结合进行分析表明存在一类单一的苯并硫氮䓬受体，其在25℃时与地尔硫䓬结合的解离常数（KD）为80 nM。苯并硫氮䓬受体与其他肌膜标记活性共同纯化，并以1:1:1的化学计量比与二氢吡啶和芳烷基胺钙通道阻滞剂的不同受体形成复合物存在。配体与该复合物中一个受体的结合以依赖于环境温度的方式影响另外两个位点的结合反应。在25℃时，二氢吡啶激动剂或拮抗剂的结合会导致地尔硫䓬结合的部分抑制（最大效应），而在37℃时，大多数二氢吡啶拮抗剂刺激且激动剂抑制地尔硫䓬结合（均为解离常数效应）。通过Scatchard分析和地尔硫䓬解离动力学研究证实了受体偶联的这种温度依赖性变化。维拉帕米作用于芳烷基胺受体，在25℃和37℃时对地尔硫䓬结合的抑制作用相同（解离常数效应）。此外，两类二氢吡啶均以与温度无关的方式抑制维拉帕米结合，地尔硫䓬也是如此（均为解离常数效应）。苯并硫氮䓬和二氢吡啶受体之间的变构偶联在心肌中表现出来，因为尼群地平会增加地尔硫䓬的负性肌力作用，而4 - (O - 三氟甲基苯基)-2,6 - 二甲基 - 5 - 硝基 - 1,4 - 二氢吡啶 - 3 - 羧酸甲酯会降低其负性肌力作用。这些结果提示了一个模型，其中钙通道阻滞剂受体复合物在25℃至37℃之间发生变化，使得在后者温度下所有位点直接偶联。变构偶联在体内可能具有重要意义，因为它可以在钙通道活性的功能测定中被检测到。
