STRUCTURAL STUDIES OF K CHANNEL COMPLEX

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 KV1家族电压依赖性钾通道在心脏中高度表达，对心脏动作电位的复极化起重要作用。在人类中，KV1通道功能丧失突变与房颤直接相关。KV1通道由一个相关的胞浆β亚基(Kv？)调节，该亚基有一个类似氧化还原酶的保守核心区，~330个氨基酸。某些Kv？S也有一个灵活的N-末端，~70个氨基酸，通过阻断离子传导孔来关闭通道。我们已经证明，Kv？的保守核心是一个以NADPH为辅因子的功能性醛酮还原酶，而Kv？结合的辅因子无论是被底物酶催化氧化，还是被过氧化氢或NADP+非酶氧化，都会导致通道电流的大幅增加。为了了解NADPH氧化如何增加通道电流，我们开展了一项系统的工作，以确定KV1和Kv？上需要进行调制的区域。这些研究的结果使我们提出了这样的假设，即氧化还原调节是由Kv的N末端介导的。为了进一步研究这一新机制，我们提出了以下三个目标来进一步研究这一机制： 1)确定Kv？N端改变氧化还原调制的区域； 2)检测N末端是否以氧化还原依赖的方式与Kv？的酶核心相互作用； 3)获得具有Kv保守核心区的复合体的N-末端结构。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Kv1 family voltage-dependent potassium channels are highly expressed in heart and important for repolarization of a cardiac action potential. In humans, loss-of-function mutations of Kv1 channels are directly linked to atrial fibrillation. Kv1 channels are modulated by an associated cytosolic beta subunit (Kv¿), which has a conserved core domain, ~330 amino acids, that resembles an oxidoreductase. Certain Kv¿s also have a flexible N-terminus, ~70 amino acids, that closes the channel by blocking the ion conduction pore. We have shown that the conserved core of Kv¿ is a functional aldo-keto reductase that utilizes NADPH as cofactor, and that oxidation of the Kv¿-bound cofactor, either enzymatically by a substrate or non-enzymatically by hydrogen peroxide or NADP+, induces a large increase in channel current. To understand how NADPH oxidation increases channel current, we have mounted a systematic effort to identify regions on Kv1 and Kv¿ that are required for the modulation. Results from these studies have led us to the hypothesis that redox modulation is mediated by the N-terminus of Kv¿. To further examine the novel mechanism, we propose the following three aims to further examine the mechanism: 1) To identify regions on the N-terminus of Kv¿ that alter the redox modulation; 2) To examine if the N-terminus interacts with the enzymatic core of Kv¿ in a redox dependent manner; 3) To obtain a structure of the N-terminus in complex with the conserved core domain of Kv¿.