SLOW INACTIVATION OF SODIUM CHANNELS

这项建议的广泛、长期目标是(1)描述 钠通道部分的结构与功能关系 控制缓慢的失活和(2)评估潜在的贡献 钠通道在癫痫发病机制中的缓慢失活。 具体目标是： 1)测量缓慢失活过程中钠通道的特性 并测量缓慢失活的电压灵敏度和动力学， 2)表达具有重复序列M和IV的S4区突变的通道 为了确定钠通道分子的这一部分是否控制着减慢 失活，以及3)确定F(VH)曲线之间的差异 正常神经元和自发性癫痫神经元的测量 (摇摇欲坠)小鼠，并识别影响 电压依赖性和缓慢失活动力学。 这项建议与健康有关的是它对癫痫的应用；a 神经元过度兴奋的关键因素(可能导致癫痫) 是F(VH)曲线中点的右移，这将导致 休眠状态下可用于激活的通道数量增加 潜力。它的动力学、有效价态和电压依赖性 小龙虾巨型轴突的F(VH)曲线将用轴线测量 电压钳位。抗癫痫药物，如二苯基-2，4-二苯基-2，4，4，6-二苯基-2，4，4，4，6-二苯基-2，4，4，4，6，6-二苯基-2，4，4，4，6-二苯基-4，4，4，4，5，6-二苯基-2，4，4，4，5，6-二苯基-2，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4-三苯基-4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4，4-三苯基-4，4，4，4，4，4，4 海因、卡马西平和丙戊酸钠对F(VH)性质的影响 将研究小龙虾中的曲线。天然和突变的钠通道将 在非洲爪哇卵母细胞中表达，并记录宏观电流 采用双电极电压钳和外向外贴片结构。 我们将比较野生型通道的慢速失活特性 而带有S4的那些，重复III和/或IV氨基酸替换和 定点突变产生的缺失突变。属性 控制缓慢失活将在培养的神经元之间进行比较 使用Patch从非表型摇摇欲坠(自发性癫痫)的小鼠 钳夹技术。

The broad, long-term objectives of this proposal are to (1) characterize the structure-function relationship of the parts of sodium channels that control slow inactivation and to (2) assess the potential contribution of sodium channel slow inactivation in the mechanisms of epilepsy. The Specific Aims are: 1) to measure the properties of sodium channels during slow inactivation and to measure the voltage sensitivity and kinetics of slow inactivation, 2) to express channels with mutations of the S4 region of repeats M and IV to determine whether that part of the sodium channel molecule controls slow inactivation, and 3) to determine the differences between the F(Vh) curves measured from normal neurons and neurons from spontaneously epileptic (tottering) mice, and to identify anti-epileptic agents that affect the voltage dependence and kinetics of slow inactivation. The health-relatedness of this proposal is its application to epilepsy; a critical factor in neuronal hyperexcitability (that could lead to epilepsy) is a right-shift in the midpoint of the F(Vh) curve which leads to an increase in the number of channels available for activation from resting potential. The kinetics, effective valence, and voltage dependence of the F(Vh) curve in crayfish giant axons will be measured using axial-wire voltage clamp. The effects of anti-epileptic drugs such as diphenyl- hydantoin, carbamasepine and sodium valporate on properties of the F(Vh) curve in crayfish will be studied. Native and mutated sodium channels will be expressed in Xenopus oocytes, and macroscopic currents will be recorded using two-electrode voltage clamp and outside-out patch configuration. Slow inactivation properties will be compared between wild-type channels and those with S4, repeats III and/or IV amino acid replacement and deletion mutations produced by site-directed mutagenesis. Properties controlling slow inactivation will be compared between neurons cultured from non-phenotypic tottering (spontaneously epileptic) mice using patch clamp techniques.