Identification of the Molecular Site of Ivabradine Binding to HCN4 Channels

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.伊伐布雷定（Ivabradine）是一款获批用于治疗慢性稳定性心绞痛的特异性降心率药物。其作用机制为选择性特异性阻断超极化激活环核苷酸门控阳离子通道（HCN channels）——该通道是窦房结“f”（funny）通道的分子组成成分。多项研究表明，伊伐布雷定的结合位点位于HCN通道的内前庭区域，但伊伐布雷定结合的分子细节仍未明确。为此，本研究拟通过诱变技术与计算机模拟分析（in silico analysis），探究窦房结中表达的HCN4通道（HCN4 channel）的哪些残基参与了伊伐布雷定的结合过程。本研究采用同源建模（homology modeling）技术，验证了通道孔道下方存在内腔，并鉴定出构成该内腔内壁的残基；随后将这些残基单独或联合替换为丙氨酸（alanine）或缬氨酸（valine），并将野生型（WT）与突变型通道在HEK293细胞（HEK293 cells）中进行表达。通过膜片钳（patch-clamp）技术检测突变型与野生型通道的阻断效率，结果显示残基Y506、F509与I510参与了伊伐布雷定的结合。针对每一种突变型通道的分子对接模拟（docking simulations），均可通过与伊伐布雷定结合亲和力成比例降低的角度，合理解释其阻断效率下降的现象。综上，本研究证实伊伐布雷定结合于通道孔道下方的内腔，并鉴定出朝向该内腔、可与伊伐布雷定分子相互作用并使其稳定的特异性残基。本研究还对伊伐布雷定阻断f/HCN4通道的已知特性进行了解释，包括“开放通道阻断”（open channel block）现象、阻断的电流依赖性（current-dependence），以及药物分子在通道关闭构象中“捕获”的特性。