Identification of the molecular site of ivabradine binding to HCN4 channels.
Identification of the molecular site of ivabradine binding to HCN4 channels.
复制标题
依伐布拉丁与HCN4通道结合的分子位点的鉴定。
DOI：
10.1371/journal.pone.0053132
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
DiFrancesco D
中科院分区：
文献类型：
--
作者：
Bucchi A;Baruscotti M;Nardini M;Barbuti A;Micheloni S;Bolognesi M;DiFrancesco D
伊伐布雷定是一种特殊的心率降低剂，被批准用于治疗慢性稳定型心绞痛。它的作用方式涉及选择性和特异性阻断HCN通道，即窦房“滑稽”(f)通道的分子成分。不同的研究表明，伊瓦布雷定的结合位点位于HCN通道的内前庭，但伊瓦布雷定结合的分子细节尚不清楚。因此，我们试图通过诱变和硅分析来研究HCN4通道的残基，即在窦房结中表达的HCN亚型，与伊伐布雷定的结合有关。通过同源建模，我们验证了通道孔下方存在一个内腔，并确定了腔内的残留物；这些残基被丙氨酸（或缬氨酸）单独或联合取代，在HEK293细胞中表达了WT和突变体通道。通过膜片钳法比较突变体与WT通道的阻滞效率，发现残基Y506、F509和I510参与了伊伐布雷定的结合。对于每个突变通道，对接模拟正确地解释了依瓦布雷定结合亲和力按比例降低的块效率。总之，我们的研究表明，伊伐布雷定占据了通道孔下方的一个空腔，并确定了面对该空腔的特定残基，这些残基相互作用并稳定了伊伐布雷定分子。本研究解释了伊伐布雷定对f/HCN4通道阻滞的已知性质，如“开放通道阻滞”、阻滞的电流依赖性以及药物分子在封闭构型中的“捕获”特性。
Ivabradine is a specific heart rate-reducing agent approved as a treatment of chronic stable angina. Its mode of action involves a selective and specific block of HCN channels, the molecular components of sinoatrial "funny" (f)-channels. Different studies suggest that the binding site of ivabradine is located in the inner vestibule of HCN channels, but the molecular details of ivabradine binding are unknown. We thus sought to investigate by mutagenesis and in silico analysis which residues of the HCN4 channel, the HCN isoform expressed in the sinoatrial node, are involved in the binding of ivabradine. Using homology modeling, we verified the presence of an inner cavity below the channel pore and identified residues lining the cavity; these residues were replaced with alanine (or valine) either alone or in combination, and WT and mutant channels were expressed in HEK293 cells. Comparison of the block efficiency of mutant vs WT channels, measured by patch-clamp, revealed that residues Y506, F509 and I510 are involved in ivabradine binding. For each mutant channel, docking simulations correctly explain the reduced block efficiency in terms of proportionally reduced affinity for ivabradine binding. In summary our study shows that ivabradine occupies a cavity below the channel pore, and identifies specific residues facing this cavity that interact and stabilize the ivabradine molecule. This study provides an interpretation of known properties of f/HCN4 channel block by ivabradine such as the “open channel block”, the current-dependence of block and the property of "trapping" of drug molecules in the closed configuration.
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影响因子：
56.9
作者：
Doyle, DA;Cabral, JM;MacKinnon, R
通讯作者：
MacKinnon, R
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作者：
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通讯作者：
DiFrancesco, Dario