Localization and Regulation of Shal/Kv4 Channels

描述(申请人提供)：离子通道和受体是神经系统中形成电子和化学通讯的基本组件。我们的长期兴趣是了解离子通道是如何被运输和调节以发挥其生理作用的。在这个方案中，我们关注高度保守的、电压门控的Shal/Kv4通道。在不同物种中，这些通道定位于躯体-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。由于这些重要功能，Shal/Kv4通道减少/突变的动物模型表现出空间学习缺陷、癫痫行为和颞叶癫痫。Shal/Kv4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。因此，了解Shal/Kv4通道的定位和调控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。以果蝇为模型系统，我们提出了研究Shal/Kv4通道体树突起定位的机制，并探讨了Shal/Kv4通道在突触后部位所起的新作用。在具体目标1中，我们探索了Shal/Kv4通道极化分布的两种机制。一种机制依赖于Shal/Kv4通道C-末端高度保守的二亮氨酸基序(LL-基序)。我们建议建立一个新发现的蛋白质的突变体，双亮氨酸基序的Shal相互作用元件(SIDL)，并研究GFP-Shal/Kv4在体内的定位如何受到影响。这个SIDL突变体还将被设计成允许我们敲入突变的SIDL结构，并进行结构功能研究。我们还将通过干扰轴突初始段(AIS)的细胞骨架，跟踪单个Shal/Kv4通道，并分析定位和移动性如何受到影响，来测试第二种机制是否涉及轴突初始段(AIS)的细胞骨架屏障。具体目的#2是基于强有力的初步研究，表明Shal/Kv4通道在突触不活跃时上调。我们将测试这一模型，即触发特定突触后受体的动态平衡上调 Shal/Kv4通道表达增加，目的是调节突触后电位及其动态平衡调节。这一新的调控将为Shal/Kv4通道在突触可塑性中所起的作用增加一个新的维度。以果蝇作为我们的模型系统，我们结合了遗传学、生化、电生理、细胞和分子生物学的方法，以获得对这些在哺乳动物系统中更难解决的问题的独特见解。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