SGER: Molecular Basis of Modulation of Neuronal Sodium Channels

主要研究人员：Satin，Jonathan抽象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部分通过抑制快钠电流来调节细胞兴奋性，但其对钠通道功能影响的分子机制尚不完全清楚。特别是，对慢钠电流成分的调制知之甚少。造成这种不完全理解的原因是两个因素使钠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认识不足；2)在许多天然制剂中表达了大量的钠通道亚型。我的长期研究目标是阐明电压门控离子通道的结构-功能关系。异源表达、克隆的电压门控钠通道是确定分子结构-功能约束的理想模型系统。这项提议的目的是测试我们对通道结构域相互作用的分子结构-功能假说，并评估促进从慢到快门控转变的条件。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通道的磷酸化导致激活门控的减慢，以及快速门控通道的部分移位。在第一个目标中，我们将对大鼠脑透明质酸通道进行定点突变，以确定PK-C的作用部位。用半胱氨酸残基取代PK-C靶标丝氨酸残基将使我们能够通过使用化学修饰剂来探索结构假说。甲硫磺酸盐类(MTS)中的化学改性剂将允许我们添加正电荷或负电荷，并创建已知分子尺寸的可逆交联剂。在第二个目标中，我们将检验我们的假设，即PK-C介导的失活结构域上丝氨酸的磷酸化有助于慢速-快速模式切换。我们还将测试激酶浓度的影响。最后，我们将使用第二种神经元亚型，它可能会表现出额外的慢门成分。我们的假设表明，具有不同慢门的通道对PK-C激动剂的调制可能有不同的反应。后一项实验设计将重点比较在相同表达系统中表达的两种不同亚型的神经元钠通道。我们将利用分子生物学和电生理学技术来了解与快速和缓慢失活以及模式门控相关的分子结构/功能。这一建议将促进我们对膜电学性质的生物物理基础的了解，以及细胞内第二信使对它们的调控。

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.