MOLECULAR BASIS OF SODIUM CHANNEL INACTIVATION

这个建议的目标是阐明钠的分子基础 通道失活 钠通道是一种蛋白质， 膜通过支持快速电压诱导的变化而兴奋， Na离子的渗透性。 灭活在以下方面起着关键作用： 调节钠通道的可用性， 影响神经、骨骼肌和 心 膜片钳技术允许记录Na电流， 整个细胞或单个Na通道。 全细胞灭活 记录是明显的，因为钠电流的衰减， 膜去极化 在单通道记录中， 被看作是一个离散的过渡，从一个电流传导构象， 使通道蛋白变成一种持久的不导电构象。 的 向失活状态转变的动力学和电压依赖性 可以使用基于马尔可夫链的分析方法来明确描述 理论或Eyring率理论。 跨膜结构模型 Na通道的拓扑结构导致了关于功能的假设 几个高度保守的通道片段。 这些假设 现在可以通过技术联盟进行严格测试， 生物物理学和分子生物学。 该提案针对假定的 “失活门”，位于Na的内前庭附近 通道，并似乎摆动或滑动到一个位置，阻止 毛孔 具体目标是：（1）表征Na通道失活， 单通道记录;（2）调查推定的 失活门和Na通道失活;和（3）评估 失活门与Na+总体行为的关系 渠道 将首先在大鼠中表征和比较灭活 骨骼肌细胞和非洲爪蟾卵母细胞中表达功能性Na + Na通道α亚基的mRNA。 随后，委员会注意到， 定点诱变将用于合成突变的Na通道 具有改变的一级氨基酸序列的 失活门。 提出的突变包括：（1）切口突变， 划分失活门;（2）改变网络的点突变 失活门上的电荷;和（3）改变失活门上的电荷的点突变。 失活闸门的灵活性。 这些突变的通道， 在卵母细胞中，提供一种方法来探测 结构-功能关系。 总体而言，拟议的研究承诺 提高我们对基本电生理学的理解， Na通道的结构特性。

The goal of this proposal is to elucidate the molecular basis of Na channel inactivation. Na channels are integral proteins that render membranes excitable by supporting rapid voltage-induced changes in permeability to Na ions. Inactivation plays a critical role in modulating the availability of Na channels to open and therefore directly influences excitability and drug binding in nerve, skeletal muscle and heart. The patch-clamp technique allows recording of Na currents from whole cells or from single Na channels. Inactivation in whole-cell records is evident as the decay of the Na current during maintained membrane depolarization. In single-channel recordings, inactivation is seen as a discrete transition from a current-conducting conformation of the channel protein to a long-lasting nonconducting conformation. The kinetics and voltage-dependence of transitions to the inactivated state can be explicitly described using analytic methods based on Markov chain theory or Eyring rate theory. Structural models of the transmembrane topology of the Na channel have led to hypotheses regarding the function of several highly conserved segments of the channel. These hypotheses can now be rigorously tested through the alliance of techniques in biophysics and molecular biology. This proposal targets the putative "inactivation gate" which resides near the inner vestibule of the Na channel and appears to swing or slide to a position which blocks the pore. Specific aims are: (1) to characterize Na channel inactivation in single-channel recordings; (2) to investigate links between the putative inactivation gate and Na channel inactivation; and (3) to assess the relationship of the inactivation gate to the overall behavior of Na channels. Inactivation will first be characterized and compared in rat skeletal muscle cells and in Xenopus oocytes which express functional Na channels from mRNA encoding the Na channel a subunit. Subsequently, site-directed mutagenesis will be used to synthesize mutant Na channels with altered primary amino acid sequence in the region of the inactivation gate. Proposed mutations include: (1) nick mutations which divide the inactivation gate; (2) point mutations which change the net charge on the inactivation gate; and (3) point mutations which change the flexibility of the inactivation gate. These mutant channels, expressed in oocytes, provide a means to probe specific aspects of structure-function relationships. Overall, the proposed studies promise to enhance our understanding of fundamental electrophysiological and structural properties of the Na channel.