Probing functional mechanisms in K+ channels using unnatural mutagenesis

电压门控K+(Kv)通道将K+的流量耦合到膜电位和Play键 在大脑和心脏中扮演的角色。Kv通道突变可导致人类严重疾病 例如癫痫和心律失常。在结构上取得了重大进展 Kv通道的测定。尽管有结构性信息可用，但有主要的 关于Kv通道功能机制的问题仍未得到回答。在这里我们 研究电压门控和C型失活调节K+流量的过程 通过KV频道。我们使用了以非天然氨基酸为中心的多学科方法 (UAA)在我们的研究中的突变。UAA诱变是一种非常有效的方法 蛋白质修饰，与传统的突变相比，因为它允许大量不同的 侧链修饰，还允许蛋白质骨架的修饰。我们用这个 探讨第四跨膜螺旋中主链氢键的作用 振荡器K+通道电压门控中的(TM4)与激活和循环的超极化 核苷酸门控离子通道HCN(目标1)。我们研究了离子结合位点在 振荡器通道在C型失活中的选择性过滤及功能补充 振荡器的研究与KvAP通道的结构研究 振动器通道(目标2)。我们还研究了HERG中C型失活的机制 K+通道，与振荡器中的C型失活有有趣的功能差异 在生理上对正常的心脏功能至关重要(目标3)。这项研究 这一建议具有重要意义，因为它为电压的作用机制提供了更深入的见解 Kv通道的门控和C型失活。这项研究也具有重要意义，因为它将提供一种 利用UAA诱变研究主链H-键和 离子结合部位，在许多膜蛋白家族中具有重要的功能。

Voltage gated K+ (Kv) channels couple the flux of K+ to the membrane potential and play key roles in the brain and heart. Mutations in Kv channels can cause severe diseases in humans such as epilepsies and cardiac arrhythmias. There have been major advances in the structure determination of Kv channels. In spite of the structural information available, there are major questions on the functional mechanisms in Kv channels that remain unanswered. Here we investigate the processes of voltage gating and C-type inactivation that regulate the flux of K+ through Kv channels. We use a multidisciplinary approach centered on unnatural amino acid (UAA) mutagenesis in our investigations. UAA mutagenesis is a very powerful method for protein modification, compared to traditional mutagenesis, because it allows a large variety of side chain modifications and also permits the modification of the protein backbone. We use this approach to investigate the role of the main chain H-bonds in the fourth transmembrane helix (TM4) in voltage gating of the Shaker K+ channel and the hyperpolarization activated and cyclic nucleotide gated ion channel HCN (aim 1). We investigate the role of ion binding sites in the selectivity filter of the Shaker channel in C-type inactivation and we complement the functional studies on Shaker with structural studies on the KvAP channel, an archaeal homolog of the Shaker channel (aim 2). We also investigate the mechanism of C-type inactivation in the hERG K+ channel, which has interesting functional differences from C-type inactivation in the Shaker channel and is physiologically critical for normal cardiac function (Aim 3). The research proposed is significant as it provides greater insight into the functional mechanisms of voltage gating and C-type inactivation in Kv channels. The research is also significant as it will provide a general strategy for using UAA mutagenesis to investigate the role of main chain H-bonds and ion binding sites, which are important for function in many families of membrane proteins.