Localization and Regulation of Shal/Kv4 Channels

描述（由申请人提供）：离子通道和受体是神经系统中形成电和化学通信的基本组成部分。我们的长期兴趣是了解离子通道如何被运输和调节以执行其生理作用。在这个建议中，我们专注于高度保守的，电压门控Shal/Kv 4通道。在不同物种中，这些通道定位于体细胞-树突部位，在那里它们调节树突的兴奋性、突触输入的整合、mEPSC的形状、反向传播动作电位和长时程增强。由于这些重要的功能，具有减少/突变的Shal/Kv 4通道的动物模型显示空间学习缺陷、癫痫发作行为以及颞叶癫痫。Shal/Kv 4通道也被证明是Ito电流的基础，Ito电流负责心脏动作电位的初始复极化。因此，了解Shal/Kv 4通道定位和调节的机制对神经系统和心脏中重要过程的健康和功能具有重要意义。 使用果蝇作为我们的模型系统，我们提出的研究，以检查的Shal/Kv 4通道的体树定位的机制，并调查Shal/Kv 4通道在突触后位点发挥的新作用。在具体目标#1中，我们探索了Shal/Kv 4通道极化分布的两种机制。一种机制依赖于Shal/Kv 4通道C-末端上高度保守的二亮氨酸基序（LL-基序）。我们建议产生一个突变体的最近确定的蛋白质，Shal相互作用的二亮氨酸基序（SIDL），与这个LL基序相互作用，并检查GFP-Shal/Kv 4本地化是如何在体内受到影响。该SIDL突变体也将被工程化以允许我们敲入突变体SIDL构建体，并进行结构-功能研究。我们还将测试第二种机制是否涉及轴突起始段（AIS）的细胞骨架屏障，通过扰动AIS细胞骨架，跟踪单个Shal/Kv 4通道，并分析如何影响定位和移动性。 具体目标#2是基于强有力的初步研究，表明Shal/Kv 4通道响应于突触失活而上调。我们将测试这个模型，它是特定突触后受体的稳态上调， Shal/Kv 4通道表达增加，目的是调节突触后电位及其稳态调节。这种新的调节将为Shal/Kv 4通道在突触可塑性中的作用增加一个新的维度。使用果蝇作为我们的模型系统，我们结合联合收割机遗传，生物化学，电生理，细胞和分子生物学的方法，以获得独特的见解，这些问题将更难以解决在哺乳动物系统。1

DESCRIPTION (provided by applicant): Ion channels and receptors are the basic components that shape electrical and chemical communication in the nervous system. Our long-term interest is to understand how ion channels are trafficked and regulated to perform their physiological roles. In this proposal, we focus on the highly conserved, voltage-gated Shal/Kv4 channel. Across species, these channels are localized to somato-dendritic sites, where they regulate dendritic excitability, the integration of synaptic inputs, the shape of mEPSCs, backpropagating action potentials, and long-term potentiation. Because of these important functions, animal models with decreased/mutant Shal/Kv4 channels display spatial learning defects, seizure behavior, as well as temporal lobe epilepsy. Shal/Kv4 channels have also been shown to underlie the Ito current, which is responsible for initial repolarization of the cardiac action potential. Therefore, understanding the mechanisms of Shal/Kv4 channel localization and regulation have important implications for the health and functioning of vital processes in the nervous system and heart. Using Drosophila as our model system, we propose studies to examine mechanisms underlying the somato-dendritic localization of Shal/Kv4 channels, and investigate a new role Shal/Kv4 channels play at postsynaptic sites. In Specific Aim #1, we explore two mechanisms that underlie the polarized distribution of Shal/Kv4 channels. One mechanism depends on a highly conserved di-leucine motif (LL-motif) on the C-terminus of Shal/Kv4 channels. We propose to generate a mutant of a recently identified protein, Shal Interactor of Di-Leucine Motif (SIDL) that interacts with this LL-motif, and examine how GFP-Shal/Kv4 localization is affected in vivo. This SIDL mutant will also be engineered to allow us to knock-in mutant SIDL constructs, and perform structure-function studies. We will also test whether the second mechanism involves a cytoskeletal barrier at the axon initial segment (AIS) by perturbing the AIS cytoskeleton, tracking single Shal/Kv4 channels, and analyzing how localization and mobility are affected. Specific Aim #2 is based on strong preliminary studies showing that Shal/Kv4 channels are up-regulated in response to synaptic inactivity. We will test the model that it is the homeostatic up-regulation of specific postsynaptic receptors that triggers an increase in Shal/Kv4 channel expression, for the purpose of modulating postsynaptic potentials and their homeostatic regulation. This novel regulation will add a new dimension to the role Shal/Kv4 channels play in synaptic plasticity. Using Drosophila as our model system, we combine genetic, biochemical, electrophysiological, cell and molecular biological approaches to gain unique insight into these questions that would be more difficult to address in mammalian systems. 1