KV CHANNEL FUNCTIONING IN MACROMOLECULAR COMPLEXES

描述（由申请人提供）：电压门控K+ (Kv)通道是神经元兴奋性的关键调节器，在控制静息膜电位、动作电位波形、重复放电特性以及调节对突触输入和突触可塑性的反应方面发挥作用。分子克隆通过鉴定大量Kv通道成孔(1)和辅助(2)亚基，为神经元Kv通道多样性的基础提供了见解。越来越多的证据表明，神经元Kv通道作为大分子蛋白复合物的组成部分发挥作用，包括(4)Kv 1和多个Kv2亚基和调节蛋白，尽管辅助蛋白和调节蛋白在控制神经元Kv通道表达、分布和功能中的作用尚不清楚。这项新的R21提案将测试新的假设，即电压门控Na+ (Nav)通道附属（Nav2）亚单位调节电压门控K+ (Kv)通道的功能表达，这些通道是皮层锥体神经元中快速激活和失活的Kv4.2编码的“A”电流（IA）的基础，而不是调节电压门控Na+ (Nav)通道。这一假设反映了最近的生化研究结果，表明在Kv4.2编码的IA通道大分子蛋白复合物中，Nav21 （SCN1b）和Nav22 （SCN2b）亚基与大脑中的Kv4.2共同免疫沉淀。这项建议有两个相关的目标，这两个目标将同时实现。具体来说，这里的研究将测试Nav21调节皮质锥体神经元中kv4.2编码的IA通道（目的1）的假设，而不是或除了Nav通道（目的2），并直接确定Nav21的作用。调节这些细胞的放电特性（目标2）。为了实现这些目标，内源性Nav21的表达将在体外使用小干扰rna （sirna）的靶向基因“敲低”策略来操纵，这些操作对（小鼠）皮质锥体神经元IA（和Nav）通道的特性和细胞表面表达的功能后果将被确定。平行实验将在小鼠皮质锥体神经元（SCN1b-/-）上完成，这些神经元含有SCN1b （Nav21）位点的靶向破坏。预计这些研究将为调控大分子神经元Kv通道复合物的表达和功能的机制提供重要的新见解。此外，这里的研究结果将指导未来在本实验室和其他实验室的研究，重点是描述参与神经元膜兴奋性动态调节的分子、细胞和系统机制，以及定义与神经元兴奋性紊乱相关的SCNxb亚基突变的功能后果。公共卫生相关性：电压门控钾（Kv）通道是神经元膜兴奋性的关键决定因素，在控制静息膜电位、动作电位波形、重复放电特性、对突触输入的反应和突触可塑性方面发挥作用。尽管越来越多的证据表明，神经元Kv通道作为大分子蛋白复合物的组成部分发挥作用，包括孔形成亚基和各种影响通道稳定性、运输和/或特性的辅助亚基，但目前对辅助亚基在神经元Kv通道生理调节中的作用知之甚少。利用分子遗传策略在体内和体外操纵通道亚基，这个新的研究项目的重点是确定Nav2 （SCNxb）副亚基在调节皮质神经元兴奋性中的生理作用，并测试Nav2副亚基实际上调节Kv通道而不是调节电压门控Na+ (Nav)通道的新假设。这些研究将为Nav2亚基在神经元Kv通道调控中的生理作用以及控制神经元功能和可塑性的分子机制提供新的和根本性的重要见解。

DESCRIPTION (provided by applicant): Voltage-gated K+ (Kv) channels are key regulators of neuronal excitability, functioning in the control of resting membrane potentials, action potential waveforms, repetitive firing properties, and in modulating the responses to synaptic inputs and synaptic plasticity. Molecular cloning has provided insights into the basis of neuronal Kv channel diversity with the identification of large numbers of Kv channel pore-forming (1) and accessory (2) subunits. Accumulating evidence suggests that neuronal Kv channels function as components of macromolecular protein complexes, comprising (four) Kv 1 and multiple Kv2 subunits and regulatory proteins, although the roles of accessory and regulatory proteins in controlling neuronal Kv channel expression, distribution and functioning are poorly understood. This new R21 proposal will test the novel hypothesis that voltage-gated Na+ (Nav) channel accessory (Nav2) subunits regulate the functional expression of the voltage- gated K+ (Kv) channels underlying the rapidly activating and inactivating, Kv4.2--encoded "A" currents (IA) in cortical pyramidal neurons rather than, or in addition to, regulating voltage-gated Na+ (Nav) channels. This hypothesis reflects recent biochemical findings demonstrating the Nav21 (SCN1b) and Nav22 (SCN2b) subunits co-immunoprecipitate with Kv4.2 from brain in Kv4.2--encoded IA channel macromolecular protein complexes. There are two related aims in this proposal, and these will be pursued simultaneously. Specifically, the studies here will test the hypothesis that Nav21 functions to regulate Kv4.2-encoded IA channels (aim #1), rather than, or in addition to, Nav channels (aim #2) in cortical pyramidal neurons and determine directly the role of Nav21.in regulating the firing properties of these cells (aim #2). To achieve these aims, the expression of endogenous Nav21 will be manipulated in vitro using targeted gene "knockdown" strategies with small interfering RNAs (siRNAs), and the functional consequences of these manipulations on the properties and the cell surface expression of IA (and Nav) channels in (mouse) cortical pyramidal neurons will be determined. Parallel experiments will be complete on cortical pyramidal neurons from mice (SCN1b-/- ) harboring a targeted disruption of the SCN1b (Nav21) locus. It is anticipated that these studies will provide fundamentally important new insights into the mechanisms that regulate the expression and the functioning of macromolecular neuronal Kv channel complexes. In addition, the results of the studies here will guide future investigates in this and other laboratories focused on delineating the molecular, cellular and systemic mechanisms involved in the dynamic regulation of neuronal membrane excitability and on defining the functional consequences of mutations in SCNxb subunits linked to derangements in neuronal excitability. PUBLIC HEALTH RELEVANCE: Voltage-gated potassium (Kv) channels are key determinants of neuronal membrane excitability, functioning in the control of resting membrane potentials, action potential waveforms, repetitive firing properties, the responses to synaptic inputs and synaptic plasticity. Although accumulating evidence suggests that neuronal Kv channels function as components of macromolecular protein complexes, comprising pore- forming (1) subunits and a variety of accessory (2) subunits that affect channel stability, trafficking and/or properties, very little is presently known about the roles of accessory subunits in the physiological regulation of neuronal Kv channels. Exploiting molecular genetic strategies to manipulate channel subunits in vivo and in vitro, this new research program is focused on defining the physiological role(s) of the Nav2 (SCNxb) accessory subunits in regulating the excitability of cortical neurons and on testing the novel hypothesis that the Nav2 accessory subunits actually function to regulate Kv channels rather than, or in addition to, regulating voltage-gated Na+ (Nav) channels. These studies will provide new and fundamentally important insights into the physiological roles of Nav2 subunits in the regulation of neuronal Kv channels and into the molecular mechanisms controlling neuronal functioning and plasticity.