Oxidoreductase Activity of the Beta Subunit of Kv Channels

描述（由申请人提供）：本项目的总体目标是确定电压敏感性钾通道（Kv）的<$-亚基的催化特性。Kv通道调节细胞兴奋性，并已显示在氧感测、容量调节、记忆和学习中起重要作用。关于Kv蛋白质是与Kv通道的胞质结构域相关的辅助蛋白质，但这些蛋白质没有明确的生理功能。结构和序列分析表明，Kv？蛋白是醛酮还原酶超家族的成员。我们的中心假设是Kv ²蛋白质催化内源性羰基的还原，从而赋予Kv电流氧化还原敏感性。这种调节可能是Kv电流的氧传感或代谢依赖性的重要特征。为了验证这一假设，我们将测量Kv？蛋白与内源性底物系列的催化效率，所述内源性底物系列包括胡萝卜素、类固醇、神经递质的代谢物和脂质过氧化产物。我们还将测试Kva-<$$>复合物的组装是否增强Kv <$的酶活性，并确定其催化循环中的序列和限速步骤（目的1）。为了确定在生化实验中被Kv <$利用的内源性羰基是否也具有功能活性，我们将检查最有效的系列改变COS-7细胞中表达的Kva-<$通道的电压敏感性和动力学的能力（目的2）。这些实验的结果将使我们能够区分是否核苷酸结合本身，催化循环，或结合的配体，可以作为药理学试剂调节KV失活。总之，这项研究的结果将提供一个更好的理解Kv <$的催化和配体结合特性，并形成一个更深入的项目的基础，以检查Kv <$催化的体内作用及其在调节表面兴奋性，氧传感或编码记忆中的假定作用。这些研究的结果也可以形成开发Kv介导的Kv电流变化的药理学调节剂的基础。

DESCRIPTION (provided by applicant): The overall goal of this project is to define the catalytic properties of the ¿-subunit of the voltage-sensitive potassium channel (Kv). The Kv channels regulate cell excitability and have been shown to play an important role in oxygen-sensing, volume regulation, memory, and learning. The Kv¿ proteins are ancillary proteins that associate with the cytoplasmic domain of Kv channels, but no clear physiological function has been assigned to these proteins. Structural and sequence analyses show that Kv¿ proteins are members of the aldo-keto reductase superfamily. Our central hypothesis is that the Kv¿ proteins catalyze the reduction of endogenous carbonyls whereby they impart redox sensitivity to Kv currents. Such regulation may be an important feature of oxygen-sensing or metabolic dependence of Kv currents. To test this hypothesis we will measure the catalytic efficiency of Kv¿ proteins with endogenous substrate series consisting of prostaglandins, steroids, metabolites of neurotransmitters and lipid peroxidation products. We will also test whether assembly of Kva-¿ complexes enhance enzymatic activity of Kv¿ and determine the sequence and rate-limiting step in its catalytic cycle (Aim 1). To determine whether the endogenous carbonyls utilized by Kv¿ in biochemical experiments, are also functionally active, we will examine the ability of the most efficient series to alter the voltage-sensitivity and the kinetics of Kva-¿ channels expressed in COS-7 cells (Aim 2). Results of these experiments will allow us to distinguish whether nucleotide binding itself, catalytic cycle, or binding of ligands which can serve as pharmacologic agents modulate Kv inactivation. Taken together, the findings of this study will provide a better understanding of the catalytic and ligand binding properties of Kv¿ and form the basis of a more in-depth project to examine the in vivo role of Kv¿ catalysis and its putative role in regulating surface excitability, oxygen-sensing, or encoding memory. Results of these studies could also form the basis of developing pharmacological modulators of Kv¿-mediated changes in Kv current.