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钾通道(Kv)的催化特性。Kv通道调节细胞的兴奋性，已被证明在氧气感觉、容量调节、记忆和学习中发挥重要作用。Kv？蛋白是与Kv通道的细胞质结构域相关的辅助蛋白，但这些蛋白的生理功能尚未明确。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蛋白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电流氧敏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蛋白在内源底物系列中的催化效率，这些底物系列包括前列腺素、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-？络合物的组装是否提高Kv？的酶活性，并确定其催化循环中的序列和限速步骤(目标1)。为了确定Kv？在生化实验中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细胞表达的Kva-？通道的电压敏感性和动力学的能力(目标2)。这些实验的结果将使我们能够区分核苷酸结合本身、催化循环或作为药物的配体结合是否调节Kv失活。综上所述，这项研究的结果将提供更好的了解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，以考察Kv？催化在体内的作用及其在调节表面兴奋性、氧感觉或编码记忆方面的假定作用。这些研究的结果也可以形成开发药物调节剂的Kv？介导的Kv电流变化的基础。

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.