SGER: Molecular Basis of Modulation of Neuronal Sodium Channels

SGER：神经元钠通道调节的分子基础

• 批准号: 9977920
• 负责人: Jonathan Satin
• 金额: $ 4.25万
• 依托单位: University of Kentucky Research Foundation
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-04-15 至 2000-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9977920&HistoricalAwards=false
• 关键词: SGER; Molecular; Basis; Modulation; Neuronal

Principal Investigator: Satin, JonathanAbstractVoltage-gated sodium channels are key molecules for initiating and shaping neuronal excitability. It is known that protein kinase C modulates cellular excitability in part via attenuation of fast Na current, but there is an incomplete understanding of the molecular mechanism for effects of modulation on Na channel function. In particular, little is known regarding modulation of slow Na current components. The reason for this incomplete understanding is that two factors have complicated the study of Na channel modulation: 1) an inadequate understanding of the interaction between channel kinetics and channel modulation, and 2) the expression of numerous Na channel isoforms in many native preparations. My long-term research goal is elucidate structure-function relationships of voltage-gated ion channels. Heterologously expressed, cloned voltage-gated Na channels serve as an ideal model system to determine molecular structure-function constraints. The objectives of this proposal are to test our molecular structure-function hypothesis for the interaction of channel domains, and to evaluate conditions that promote slow to fast gating transitions. These studies are motivated by our preliminary data showing that protein kinase C mediated phosphorylation of the Na channel leads to slowing of activation gating, and shifting of the fraction of fast gating channels. In the first aim we will perform site-directed mutagenesis of the rat brain ha Na channel to define the site of action of PK-C. Replacement of the PK-C target serine residue with a cysteine residue will allow us to probe structural hypothesis by the use of chemical modifiers. Chemical modifiers in the methanethiosulfonate class (MTS) will allow us to add positive or negative charges, and to create reversible cross-linkers with known molecular dimensions. In the second aim we will test our hypothesis that PK-C mediated phosphorylation of the serine on the inactivation domain contributes to slow -- fast mode switching. We will also test the effects of concentration of kinase. Finally, we will use a second neuronal isoform that may exhibit additional slow gating components. Our hypothesis suggests that channels with different slow gating may respond differently to modulation by PK-C activators. The latter experimental design will stress comparison of two different isoforms of neuronal Na channels expressed in an identical expression system. We will utilize molecular biological and electrophysiological techniques to understand molecular structure/function correlates for fast and slow inactivation, and modal gating. This proposal will advance our knowledge of the biophysical basis of membrane electrical properties, and their regulation by intracellular second messengers.

电压门控钠通道是启动和塑造神经元兴奋性的关键分子。已知蛋白激酶C通过衰减快Na电流部分调节细胞兴奋性，但对Na通道功能调节作用的分子机制还不完全了解。特别是，很少有人知道关于慢钠电流成分的调制。这种不完全理解的原因是两个因素使Na通道调节的研究复杂化：1）对通道动力学和通道调节之间的相互作用的理解不足，以及2）在许多天然制剂中表达许多Na通道同种型。我的长期研究目标是阐明电压门控离子通道的结构与功能关系。异源表达，克隆的电压门控Na通道作为一个理想的模型系统，以确定分子结构功能的限制。这个建议的目的是测试我们的分子结构-功能假说的通道域的相互作用，并评估条件，促进慢到快的门控转换。这些研究的动机是我们的初步数据显示，蛋白激酶C介导的Na通道磷酸化导致激活门控减慢，和快速门控通道的分数的移位。在第一个目标中，我们将进行大鼠脑钠通道的定点突变，以确定PK-C的作用位点。用半胱氨酸残基替换PK-C靶丝氨酸残基将允许我们通过使用化学修饰剂来探测结构假设。甲硫基磺酸盐类（MTS）的化学改性剂将允许我们添加正电荷或负电荷，并产生具有已知分子尺寸的可逆交联剂。在第二个目标中，我们将测试我们的假设，PK-C介导的丝氨酸磷酸化的失活结构域有助于慢-快模式切换。我们还将测试激酶浓度的影响。最后，我们将使用第二个神经元亚型，可能会表现出额外的慢门控组件。我们的假设表明，不同的慢门控通道可能会对PK-C激活剂的调制产生不同的反应。后一种实验设计将强调在相同表达系统中表达的神经元Na通道的两种不同亚型的比较。我们将利用分子生物学和电生理学技术来了解快速和缓慢失活的分子结构/功能相关性，以及模式门控。这一建议将推进我们的知识的生物物理基础的膜电特性，其调节细胞内的第二信使。