Conformational dynamics of the S4 helix voltage sensor of the potassium channel hERG

项目摘要 hERG（人eag相关基因; KCNH 2）编码一种电压激活钾通道，在 大脑和心脏hERG是一种延迟整流（IKr）电流，可驱动细胞的复极，具有长期作用 潜力hERG的遗传突变会导致心律失常，心律失常可导致猝死， hERG的失调也与精神分裂症有关。药物的脱靶效应抑制 这是获得性LQT综合征（一种常见的临床问题）的主要机制。尽管 hERG在无数生理功能中重要性，其电压感应能力如何在结构上 在功能上与离子门控的机制相耦合还没有被很好地理解。这主要是由于 目前研究S4螺旋响应电压的运动方法的局限性; hERG，其感测电压并将电压信号转换到通道的其余部分。克服这些 限制需要新技术来揭示S4和hERG门控的机制。我们假设 hERG的S4螺旋经历大的动态运动并间接调节通道关闭。到 为了验证我们的假设，我们直接将小的，结构上和功能上不受干扰的， 具有琥珀密码子抑制的hERG的S4螺旋中的荧光非典型氨基酸L-ANAP。我们 实施L-ANAP作为强大荧光报告分子，以严格探测 hERG通道与过渡金属Förster共振能量转移（tmFRET）。我们将测量 使用同步电压和光谱分析的hERG通道中S4螺旋的动态运动 记录（tmFRET）以测量响应于电压变化的S4螺旋移动的距离。第二、 我们将研究的作用，S4螺旋在hERG失活（关闭）门控通过测量的运动， 与野生型通道相比，具有tmFRET的S4螺旋在快速失活hERG通道中， 经过化学处理的慢失活通道。由于hERG在心脏兴奋性中的重要性， 心律失常，确定hERG门控机制直接影响健康和疾病。

Project Summary hERG (human eag-related gene; KCNH2) encodes a voltage activated potassium channel expressed in the brain and heart. hERG is the delayed-rectifier (IKr) current that drives repolarization in the cells with long action potentials. Inherited mutations in hERG cause cardiac arrhythmias which can lead to sudden death and dysregulation of hERG is also associated with schizophrenia. Off target effects of pharmaceuticals inhibit hERG and this is the primary mechanism for acquired LQT syndrome, a common clinical problem. Despite the importance of hERG in a myriad of physiological function, how its voltage sensing ability is structurally and functionally coupled to the mechanisms of ion gating is not well understood. This is predominately due to limitations in current methods to study the movements of the S4 helix in response to voltage; the key domain in hERG that senses voltage and transduces the voltage signal to the rest of the channel. To overcome these limitations new techniques are required to uncover the mechanism of S4 and hERG gating. We hypothesize that the S4 helix of hERG undergoes large dynamic movements and indirectly regulates channel closing. To test our hypothesis, we have directly incorporated the small, structurally and functionally non-perturbing, fluorescent non-canonical amino acid L-ANAP in the S4 helix of hERG with amber codon suppression. We implement L-ANAP as powerful fluorescent reporter to rigorously probe short-range conformational changes in hERG channels with transition metal Förster resonance energy transfer (tmFRET). We will measure the dynamic movements of the S4 helix in hERG channels using simultaneous voltage and spectroscopic recordings (tmFRET) to measure the distance of S4 helix movement in response to voltage changes. Second, we will investigate the role of the S4 helix in hERG deactivation(closing) gating by measuring the movement of the S4 helix with tmFRET in fast deactivating hERG channels compared to wildtype channels, and in chemically treated slow deactivating channels. Due to the importance of hERG in cardiac excitability and arrhythmia, determining hERG gating mechanisms directly impacts health and disease.