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）抗性和感觉神经元特异性SNS（也称为PN 3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通道磷酸化增强电流可能有助于异位动作电位放电和神经病理性疼痛。然而，SNS通道的磷酸化如何导致通道功能的调节尚不清楚。 本研究采用多学科方法研究磷酸化介导的SNS通道功能改变的详细机制。具体地说，我们将测试的假设，PKA诱导的SNS通道的调制结果之间的电荷-电荷相互作用的LI-II环和环受体的细胞质域）的通道蛋白。该研究包括2-电极电压钳表征的SNS通道在非洲爪蟾卵母细胞中表达，由内而外的macropatch电流和单通道研究的通道在HEK 293细胞中表达，和2-杂交和免疫共沉淀研究的蛋白质-蛋白质之间的相互作用的LI-II环和其推定的环受体。定点突变广泛用于鉴定介导PKA作用的特定Ser残基。通过maniputations的离子强度和pH值的静电相互作用假说进行测试。概念上的创新在于我们利用的观察，PKA介导的磷酸化导致在一个增强的电流SNS，但抑制BIIA通道导致我们的新的假设。技术创新在于我们能够将钠通道蛋白质的最先进分子操作和生物化学方法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。 详细的分子水平上的SNS通道调制的理解是至关重要的，以获得更深入的了解神经性疼痛和发展的新的治疗策略的疾病过程具有重大的医疗和经济意义，但目前很少有有效的治疗方法。

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.