High-throughput analysis of drug binding interactions for the human cardiac channel, Kv1.5
High-throughput analysis of drug binding interactions for the human cardiac channel, Kv1.5
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DOI：
10.1016/j.bcp.2008.09.035
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发表时间：
2009-01-15
影响因子：
5.8
通讯作者：
Spencer, Robert H.
中科院分区：
文献类型：
--
作者：
Karczewski, Jerzy;Kiss, Laszlo;Spencer, Robert H.
电压门控钾通道Kv1.5是人心房肌细胞膜电位复极的关键调节因子之一，被认为是治疗心房颤动的潜在药物靶点。在这项研究中，我们试图确定DPO-1作用分子机制，DPO-1是一种二苯基氧化膦衍生物，最近显示可终止实验性房性心律失常而不影响心室不应期。此外，我们对另外两种小分子阻断剂提供了类似的分析，这两种小分子阻断剂代表不同的结构类别：环己酮（PAC）和降三萜类化合物（correhenoids）。为了快速鉴定Kv1.5通道内对阻断这些分子活性至关重要的残基，采用两种功能性高通量离子通道测定法以及定点诱变。我们的研究表明，对DPO-1的阻断活性至关重要的残基包括位于孔外口的T480和沿着S6螺旋的两个残基：V505和I508。PAC的重叠位点被鉴定，包括残基T480和V505。与DPO-1相反，I508 A突变导致PAC对Kv1.5的阻断仅适度减少（9倍）。Correxide是所检测的最大分子，沿着孔的整个长度（从T480到V516）沿着进行广泛的相互作用。总之，我们已经鉴定了参与形成Kv1.5阻断剂的高亲和力结合位点的多个残基。高通量离子通道技术的类似方法与定点突变相结合，可以并行、快速和准确地分析离子通道与多种化合物的相互作用，并有助于设计更有效和更有选择性的离子通道阻滞剂。(C)2008年爱思唯尔公司All rights reserved.
The voltage-gated potassium channel Kv1.5 is one of the key regulators of membrane potential repolarization in human atrial myocytes and is considered a potential drug target to treat atrial fibrillation. in this study we sought to determine molecular mechanism of action of DPO-1, a diphenylphosphine oxide derivative recently shown to terminate experimental atrial arrhythmia without affecting ventricular refractory period. in addition, we provided similar analysis for additional two small molecule blockers, representing different structural classes: cyclohexanones (PAC) and nor-triterpenoids (correolide). To rapidly identify the residues within the Kv1.5 channel critical for blocking activity of these molecules, two functional high-throughput ion channel assays were employed together with site-directed mutagenesis. Our study revealed that the residues critical for blocking activity of for DPO-1 include T480, localized at the outer mouth of the pore, and two residues along S6 helix: V505 and I508. The overlapping site was identified for PAC and included residues T480 and V505. In contrast to DPO-1, the I508A mutation resulted in only a modest reduction in the block of Kv1.5 by PAC (9-fold). Correolide, the largest molecule examined, made widespread interactions along the entire length of the pore (from T480 to V516). In summary, we have identified multiple residues involved in forming high affinity binding site for Kv1.5 blockers. Similar approaches of high-throughput ion channel technologies, combined with site-directed mutagenesis, may allow for parallel, rapid and accurate analysis of ion channel interactions with multiple compounds and could facilitate the design of more potent and selective ion channel blockers. (C) 2008 Elsevier Inc. All rights reserved.