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

项目摘要 HERG(人EAG相关基因；KCNH2)编码一种电压激活的钾通道，表达于 大脑和心脏。HERG是在长时间作用的细胞中驱动复极化的延迟整流(IKR)电流 潜力。HERG基因的遗传突变会导致心律失常，这可能导致猝死和 HERG调节失调也与精神分裂症有关。药物抑制的脱靶效应 这是获得性LQT综合征的主要机制，这是一个常见的临床问题。尽管 HERG在众多生理功能中的重要性，它的电压感知能力是如何结构和 在功能上耦合到离子门控的机制还不是很清楚。这主要是因为 目前研究S4螺旋运动对电压的响应的方法存在局限性； HERG感测电压并将电压信号转换到通道的其余部分。要克服这些障碍 局限性需要新的技术来揭示S4和HERG门控的机制。我们假设 HERG的S4螺旋发生大的动态运动，间接调节通道关闭。至 检验我们的假设，我们直接结合了小的，结构上和功能上的非扰动， HERG基因S4螺旋中的荧光非正则氨基酸L-ANAP与琥珀密码子抑制。我们 实施L-ANAP作为强大的荧光记者，严格探测生物分子的短程构象变化 HERG通道具有过渡金属Förster共振能量转移(TmFRET)。我们将测量 同步电压和光谱技术研究S4螺旋在HERG通道中的动态运动 记录(TmFRET)，以测量S4螺旋运动的距离，以响应电压变化。第二, 我们将通过测量HERG失活(关闭)门控的运动来研究S4螺旋在HERG失活(关闭)门控中的作用 与野生型通道相比，快速失活HERG通道中带有tmFRET的S4螺旋，以及 经过化学处理的缓慢失活的通道。由于HERG在心脏兴奋性和 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.