Activation in voltage-gated Na channels modified by the tarantula toxin ProTxll

描述（由申请人提供）：神经元电压门控离子通道允许神经系统根据最近的事件和局部环境的变化精确调节其兴奋性。单个通道响应于膜电位的波动而打开和关闭。激活是通道从封闭的非传导状态转变为开放的传导状态的过程，由这些通道的四个电压传感器的构象变化介导。先前的研究表明，激活门控特性的差异可以追溯到电压传感器运动的时间和协调的差异。因此，了解不同类型通道激活期间电压传感器如何移动和相互协调，对于设计针对特定通道的诊断、预防和治疗工具至关重要。在电压门控钠（Na）通道中，电压传感器运动发生在不同的时间过程中，并且可能表现出耦合的差异。ProTxIl是最近发现的狼蛛肽毒素，其修饰电压门控钠（Na）通道的激活。在这里，我建议使用这种毒素来探测激活门控期间通道的各个域内的电压传感器运动。全细胞、单通道和门控电流分析将首先用于确定ProTxIl的多种作用的机制基础。荧光标记技术结合靶向诱变，然后将用于系统地探索毒素与每个通道的电压传感器区域相互作用的位点。在这些突变体中ProTxIl对门控的影响的中断将揭示毒素相互作用和电压传感器运动在通道激活中的域特异性作用。钠通道门控的中断是许多严重神经病的原因，包括癫痫、睡眠障碍和神经源性疼痛。了解这些渠道的工作方式是开发预防和治疗这些疾病的预防和治疗工具的必要的第一步。

DESCRIPTION (provided by applicant): Neuronal voltage-gated ion channels allow the nervous system to precisely modulate its excitability according to recent events and changes in the local environment. Individual channels open and close in response to fluctuations in membrane potential. Activation, the process by which channels transition from a closed, non-conducting state to an open conducting state, is mediated by conformational changes in the four voltage sensors of these channels. Previous studies have shown that differences in activation gating properties can be traced to differences in the timing and coordination of voltage sensor movements. Therefore, understanding how the voltage sensors move and coordinate with one another during activation in different types of channels is crucial in designing diagnostic, prophylactic, and therapeutic tools that target specific channels. In voltage-gated sodium (Na) channels voltage sensor movements occur with different timecourses and likely exhibit differences in coupling. ProTxIl, a recently discovered tarantula peptide toxin, modifies activation in voltage-gated sodium (Na) channels. Here, I propose to use this toxin to probe voltage sensor movements within individual domains of the channel during activation gating. Whole cell, single channel, and gating current analyses will first be used to determine the mechanistic basis of the multiple effects of ProTxIl. Fluorescent labeling techniques combined with targeted mutagenesis will then be used to systematically explore the site(s) of toxin interaction with each of the channel's voltage sensor regions. Disruptions in the effect of ProTxIl on gating in these mutants will reveal the domain-specific role of toxin interactions and voltage sensor movements in channel activation. Disruptions in Na channel gating are the cause of many severe neuropathies including epilepsy, sleep disorders, and neurogenic pain. Understanding the way these channels work is the necessary first step towards developing prophylactic and therapeutic tools for the prevention and treatment of these conditions.