Molecular determinants of dofetilide block of HERG K+ channels
Molecular determinants of dofetilide block of HERG K+ channels
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DOI：
10.1161/01.res.82.3.386
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发表时间：
1998-02-23
影响因子：
20.1
通讯作者：
Brown, AM
中科院分区：
文献类型：
--
作者：
Ficker, E;Jarolimek, W;Brown, AM
人类 ether-a-go-go 相关基因 (HERG) 编码的 K+ 通道具有与心脏延迟整流 K+ 电流 (I-Kr) 的快速成分几乎相同的生物物理特性。 HERG/I-Kr 通道是心律失常药物治疗的主要靶点，可被 III 类抗心律失常甲磺酰苯胺药物（例如多非利特、E4031 和 MK-499）在亚微摩尔浓度下选择性阻断。相比之下，密切相关的牛 ether-a-go-go 通道 (BEAG) 对多非利特的敏感性要低 100 倍。为了鉴定多非利特阻断的分子决定因素，我们首先设计了 HERG 和 BEAG 之间的嵌合体，然后使用定点诱变来定位负责阻断的单个氨基酸残基。使用在非洲爪蟾卵母细胞中异源表达的构建体，我们发现将 BEAG 的 S5-S6 连接子移植到 HERG 中可以消除多非利特的高亲和力阻断。 S5-S6 连接区的点突变 HERG S620T 废除了高亲和力阻断并干扰了 C 型失活。因此，我们的结果表明多非利特结合的重要决定因素位于 HERG 的孔区域。由于高亲和力药物结合的丧失始终与 C 型失活的丧失相关，因此观察到的药物结合变化可能是由于通道蛋白结构的间接变构修饰，而不是多非利特与相应突变位点链的直接相互作用。然而，嵌合方法无法将 HERG 通道的 S5-S6 连接区之外的域识别为参与药物结合的假定候选域。此外，回复突变BEAG T432S增加了BEAG K+通道对多非利特的亲和力，而C型失活无法恢复。因此，HERG 620位的丝氨酸可能直接参与多非利特结合；然而，完整的 C 型失活过程似乎对于高亲和力药物结合至关重要。
The human ether-a-go-go-related gene (HERG) encodes a K+ channel with biophysical properties nearly identical to the rapid component of the cardiac delayed rectifier K+ current (I-Kr). HERG/I-Kr channels are a prime target for the pharmacological management of arrhythmias and are selectively blocked by class III antiarrhythmic methanesulfonanilide drugs, such as dofetilide, E4031, and MK-499, at submicromolar concentrations. By contrast, the closely related bovine ether-a-go-go channel (BEAG) is 100-fold less sensitive to dofetilide. To identify the molecular determinants for dofetilide block, we first engineered chimeras between HERG and BEAG and then used site-directed mutagenesis to localize single amino acid residues responsible for block. Using constructs heterologously expressed in Xenopus oocytes, we found that transplantation of the S5-S6 linker from BEAG into HERG removed high-affinity block by dofetilide. A point mutation in the S5-S6 Linker region, HERG S620T, abolished high-affinity block and interfered with C-type inactivation. Thus, our results indicate that important determinants of dofetilide binding are localized to the pore region of HERG. Since the loss of high-affinity drug binding was always correlated with a loss of C-type inactivation, it is possible that the changes observed in drug binding are due to indirect allosteric modifications in the structure of the channel protein and not to the direct interaction of dofetilide with the respective mutated site chains. However, the chimeric approach was not able to identify domains outside the S5-S6 linker region of the HERG channel as putative candidates involved in drug binding. Moreover, the reverse mutation BEAG T432S increased the affinity of BEAG K+ channels for dofetilide, whereas C-type inactivation could not be recovered. Thus, the serine in position HERG 620 may participate directly in dofetilide binding; however, an intact C-type inactivation process seems to be crucial for high-affinity drug binding.