Modulation of SNS Sodium Channel by Phosphorylation

描述(由申请人提供)： 最近发现的河豚毒素(TTX)抗性感觉神经元特异性SNS(或称为PN3或NaV1.8)电压门控钠通道在神经病理性疼痛中起关键作用。蛋白激酶A(PKA)介导的SNS通道的磷酸化增强了可能参与异位动作电位放电和神经病理性疼痛的电流。然而，SNS通道的磷酸化如何导致通道功能的调制尚不清楚。 本研究从多学科的角度研究了SNS通道功能磷酸化改变的具体机制。具体地说，我们将检验这样的假设，即PKA诱导的SNS通道的调制是由通道蛋白的Li-II环和细胞质结构域上的环-受体之间的电荷相互作用引起的。这项研究包括非洲爪哇卵母细胞表达的SNS通道的双电极电压钳特性，HEK293细胞表达的通道的内向外宏膜电流和单通道研究，以及Li-II环与其可能的环受体之间蛋白质-蛋白质相互作用的双杂交和免疫共沉淀研究。定点突变被广泛用于鉴定介导PKA作用的特异性丝氨酸残基(S)。通过对离子强度和pH的模拟验证了静电相互作用假说。概念上的创新在于我们利用了PKA介导的磷酸化导致SNS电流的增强而对BIIA通道的抑制这一观察结果，从而得出了我们的新假设。技术创新在于我们能够将最先进的钠通道蛋白质和生化方法的分子操作与使用第一潜伏期和条件概率技术对通道功能进行严格的生物物理分析相结合。 在分子水平上详细了解SNS的通道调节对于深入了解神经病理性疼痛和开发新的治疗策略至关重要，这种疾病具有重要的医学和经济意义，但目前几乎没有有效的治疗方法。

DESCRIPTION (provided by applicant): The recently discovered tetrodotoxin (TTX)-resistant and sensory neuron specific SNS (alternatively called the PN3 or NaV1.8) voltage gated sodium channel plays a critical role in neuropathic pain. Protein kinase A(PKA)-mediated phosphorylation of the SNS channel enhances the current possibly contributing to ectopic action potential firing and neuropathic pain. However, how phosphorylation of the SNS channel results in the modulation of channel function is not known. This study takes a multidisciplinary approach to study the detailed mechanisms responsible for the phosphorylationmediated alteration of the SNS channel function. Specifically, we will test the hypothesis that PKAinduced modulation of the SNS channel results from a charge-charge interaction between the LI-II loop and the loop-receptor on the cytoplasmic domain) of the channel protein. The study consists of 2-electrode voltage clamp characterization of SNS channels expressed in Xenopus oocytes, inside-out macropatch current and single channel study of channels expressed in HEK293 cells, and a 2-hybrid and co-immunoprecipitation study of protein-protein interaction between the LI-II loop and its putative loop receptor. Site-directed mutagenesis is used extensively to identifity the specific Ser residue(s) mediating the PKA-action. The electrostatic interaction hypothesis is tested through maniputations of the ionic strength and the pH. The conceptual innovation lies in our capitalizing on the observation that PKA-mediated phosphorylation results in a potentiation of current for SNS but an inhibition for BIIA channels leading to our novel hypothesis. The technical innovation lies in our ability to integrate state-of-art molecular manipulation of the sodium channel protein and biochemical approaches with a rigorous biophysical analysis of the channel function using first-latency and conditional probability techniques. A detailed molecular-level understanding of SNS channel modulation is critical to gain a deeper understanding of neuropathic pain and for the development of novel therapeutic strategies for a disease process with major medical and economic implications but with few effective treatments at present.