Fiset C, Feng Z P, Wang L, Sheldon R S, Duff H J
Cardiovascular Research Group, University of Calgary, Alberta, Canada.
J Mol Cell Cardiol. 1996 May;28(5):1085-96. doi: 10.1006/jmcc.1996.0100.
Dofetilide is a Class III antiarrhythmic agent known to selectively block the rapid component of the delayed rectifier K+ current (IKr). [3H]Dofetilide binds to a low and a high affinity sites on guinea-pig myocytes. The purposes of this study were: (1) to find biological models which express solely the high or the low [3H]dofetilide binding sites; (2) to characterize the single binding site models; and (3) to establish which of the high or the low affinity binding sites is associated with IKr. We compared and characterized the [3H]dofetilide binding on guinea-pig myocytes, neonatal mouse ventricular homogenate and untransfected CHO cells. These tissue preparations were selected since the neonatal mouse tissue expresses IKr while this current is absent from CHO cells. We compared the IC50 concentrations of dofetilide and two other known IKr blockers E-4031 and sotalol, on [3H]dofetilide binding to these three preparations. Using steady-state and kinetic binding techniques, we characterized the interaction of E-4031 and sotalol with the high and the low [3H]dofetilide binding sites. We found that neonatal mouse ventricle manifest solely the high affinity site (Kd 20 +/- 4 nmol/l, Bmax 18 +/- 4 fmol/mg) while CHO cells manifest solely the low affinity binding site (Kd 1.6 +/- 0.1 mumol/l, Bmax 5.8 +/- 0.8 pmol/mg). We demonstrated that the high and low affinity binding sites present on guinea-pig myocytes show characteristics similar to the single high affinity site expressed on neonatal mouse homogenate and to the single low affinity site expressed on CHO cells, respectively. Class III antiarrhythmic drugs inhibited binding to the high affinity site at concentrations similar to those required to inhibit 50% of IKr current in electrophysiologic studies. In contrast, dofetilide and E-4031 inhibited [3H]dofetilide binding to the low affinity site only at supra-pharmacologic concentrations. We next demonstrated that Class III drugs interact in a competitive manner with the high affinity site on neonatal mouse tissue while they interact with a site allosterically coupled to the low binding site on CHO cells. These data suggest that dofetilide interacts with the high and low affinity sites in a fundamentally different manner. We defined biological models which express solely the high or low [3H]dofetilide binding sites. Only the high affinity site is related to IKr.
多非利特是一种Ⅲ类抗心律失常药物，已知其可选择性阻断延迟整流钾电流（IKr）的快速成分。[3H]多非利特可与豚鼠心肌细胞上的一个低亲和力位点和一个高亲和力位点结合。本研究的目的是：（1）寻找仅表达高或低[3H]多非利特结合位点的生物学模型；（2）对单结合位点模型进行表征；（3）确定高或低亲和力结合位点中哪一个与IKr相关。我们比较并表征了[3H]多非利特在豚鼠心肌细胞、新生小鼠心室匀浆和未转染的CHO细胞上的结合情况。选择这些组织标本是因为新生小鼠组织表达IKr，而CHO细胞中不存在这种电流。我们比较了多非利特以及另外两种已知的IKr阻滞剂E-4031和索他洛尔在[3H]多非利特与这三种标本结合时的IC50浓度。使用稳态和动力学结合技术，我们表征了E-4031和索他洛尔与高和低[3H]多非利特结合位点的相互作用。我们发现新生小鼠心室仅表现出高亲和力位点（Kd 20±4 nmol/L，Bmax 18±4 fmol/mg），而CHO细胞仅表现出低亲和力结合位点（Kd 1.6±0.1 μmol/L，Bmax 5.8±0.8 pmol/mg）。我们证明，豚鼠心肌细胞上存在的高亲和力和低亲和力结合位点分别表现出与新生小鼠匀浆上表达的单一高亲和力位点以及CHO细胞上表达的单一低亲和力位点相似的特征。Ⅲ类抗心律失常药物在与电生理研究中抑制50% IKr电流所需浓度相似时，可抑制与高亲和力位点的结合。相比之下，多非利特和E-4031仅在超药理浓度下才抑制[3H]多非利特与低亲和力位点的结合。接下来我们证明，Ⅲ类药物与新生小鼠组织上的高亲和力位点以竞争性方式相互作用，而它们与CHO细胞上与低结合位点变构偶联的一个位点相互作用。这些数据表明，多非利特与高亲和力和低亲和力位点的相互作用方式存在根本差异。我们定义了仅表达高或低[3H]多非利特结合位点的生物学模型。只有高亲和力位点与IKr相关。
