STRUCTURAL STUDIES OF K CHANNEL COMPLEX

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 Kv 1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非常重要。在人类中，Kv 1通道的功能丧失突变与房颤直接相关。Kv 1通道由相关的胞质β亚基（Kv <$）调节，其具有类似于氧化还原酶的保守核心结构域，约330个氨基酸。某些KV也有一个灵活的N-末端，约70个氨基酸，通过阻塞离子传导孔关闭通道。我们已经表明，Kv <$的保守核心是一个功能性醛酮还原酶，利用NADPH作为辅因子，并且Kv <$结合辅因子的氧化，无论是通过底物的酶促氧化还是通过过氧化氢或NADP+的非酶促氧化，都会诱导通道电流的大幅增加。为了了解NADPH氧化如何增加通道电流，我们进行了系统性的努力，以确定Kv 1和Kv？上调节所需的区域。这些研究的结果使我们假设氧化还原调节是由Kv的N-末端介导的。为了进一步研究新机制，我们提出以下三个目标来进一步研究该机制： 1)确定改变氧化还原调节的Kv <$N-末端区域; 2)检查N-末端是否以氧化还原依赖性方式与Kv <$的酶核心相互作用; 3)为了获得与Kv的保守核心结构域复合的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¿.