Slow Inactivation of Sodium Channels

描述（由申请人提供）：钠通道是神经元中电信号的关键组成部分。钠通道的可用性决定了神经元的电兴奋性。慢失活（SI）调节钠通道的可用性。我们的初步数据表明，SI的动力学受到钠通道磷酸化的调制。因此，由于通道磷酸化的变化而引起的SI水平的变化可能是神经元兴奋性通过激活触发磷酸化级联反应的受体来调节的候选者。这些事件伴随着细胞过程，而这些细胞过程是许多正常神经过程的基础。钠通道可用性的变化，以及因此神经元兴奋性的变化，也是兴奋性疾病如癫痫的基础。因此，我们的研究将阐明通过改变电兴奋性来控制大脑生理和病理生理过程的根本性重要机制。本研究的长期目标是确定钠通道SI对神经元兴奋性的贡献。这项研究建议的具体目标是：（1）基于我们的初步结果，检验如下假设：SI由经由蛋白激酶A的环AMP依赖性磷酸化的激活剂和经由蛋白激酶C的二酰基甘油依赖性磷酸化的激活剂调节，钠通道SI的磷酸化诱导的调节发生在神经元钠通道中鉴定的共有磷酸化位点，（2）检验SI受CaM激酶II调节的假设;（3）检验局麻药和抗惊厥药影响或模拟SI的假设，以及SI受β-1亚基共表达的影响;检验钠通道门控的药理学改变与门控电流和电荷移动的变化相关的假设。在追求这些特定目标的过程中，我们将记录离子和门控电流，以测量电压依赖性和缓慢失活速率，以及其他生物物理特性，来自（1）使用膜片钳在HEK 293细胞中表达的大鼠脑钠通道和（2）使用切开卵母细胞电压钳在非洲爪蟾卵母细胞中表达的钠通道。

DESCRIPTION (provided by applicant): Sodium channels are a critical component of electrical signaling in neurons. The availability of sodium channels determines neuronal electrical excitability. Slow inactivation (SI) regulates sodium channel availability. Our preliminary data demonstrate that the kinetics of SI is subject to modulation by sodium channel phosphorylation. Changes in the level of SI due to changes in channel phosphorylation is therefore a likely candidate by which neuronal excitability may be modulated by activation of receptors that trigger phosphorylation cascades. Events such as these accompany the cellular processes that underlie many normal neurological processes. Changes in sodium channel availability, and therefore neuronal excitability, also underlie diseases of excitability such as epilepsy. Our research, therefore, will elucidate fundamentally important mechanisms that control both physiological and pathophysiological processes in the brain through changes in electrical excitability. The long-term objective of this research is to determine the contribution of sodium channel SI to neuronal excitability. The specific aims of this research proposal are to: (1) test the hypothesis, based on our preliminary results, that SI is modulated by activators of cyclic AMP-dependent phosphorylation via protein kinase A and by activators of diacylglycerol-dependent phosphorylation via protein kinase C, that phosphorylation-induced modulation of sodium channel SI occurs at identified consensus phosphorylation sites in the neuronal sodium channel, and that modulation varies as a function of co-expression with WTor mutant beta-1 subunit; (2) test the hypothesis that SI is modulated by CaM kinase II; (3) test the hypothesis that local anesthetics and anticonvulsants affect or mimic SI, and that this varies as a function of beta-1 subunit co-expression; test the hypothesis that pharmacological alterations of sodium channel gating are correlated with changes in gating currents and charge movement. In pursuit of these specific aims, we will record ionic and gating currents to measure the voltage dependence and rates of slow inactivation, as well as other biophysical properties, from rat brain sodium channels expressed in (1) HEK 293 cells using patch clamp and (2) Xenopus oocytes using cut-open oocyte voltage clamp.