REGULATION OF SODIUM CHANNEL DISTRIBUTION IN NEURONAL CELLS

神经细胞钠通道分布的调节

• 批准号: 6112562
• 负责人: Simon R LEVINSON
• 金额: $ 13.51万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-01 至 1999-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6112562
• 关键词: chimeric proteins; embryo /fetus; immunocytochemistry; laboratory rabbit; laboratory rat; peripheral nervous system; protein isoforms; protein sequence; protein structure function; recombinant DNA; sodium channel; tissue /cell culture; voltage gated channel

Recombinant DNA approaches have revealed that voltage gated sodium channels exist as a number of distinct isoforms. There is evidence that some excitable cells express multiple channel isoforms that are targeted to different cellular areas. This project addresses the hypothesis that peripheral nervous system (PNS) channel isoforms have distinct structural properties that allow them to be differentially targeted to appropriate membrane domains in peripheral neurons and their processes. There are three specific aims: 1) The cellular and subcellular distribution of PNS-abundant sodium channel isoforms will be established using immunocytochemical techniques. Isoform specific antibodies will be produced and used to establish the protein distribution of co- expressed isoforms in the nervous system of the rat. Of particular interest will be the identification of the predominant channel isotypes in the different cell types of peripheral ganglia and those that inhabit nodes of Ranvier in myelinated fibers of the PNS. 2) A peripheral neuron paradigm will be developed that will allow molecular manipulation of isoform expression and structure, The PC12 cell line will be studied as a possible model for selective targeting of channel isoforms to cell bodies and processes. Primary cultures of dorsal root and superior cervical ganglion neurons will similarly be investigated for their suitability in these studies. Each system will be assessed for the ability to control selectively wild-type isoform expression and to express mutant channel isoforms. 3) Channel isoform domains that specify targeting properties and the moieties that interact with them will be characterized. Candidate targeting domains will be identified by comparing primary sequences among channels whose targeting differs. The role of these domains in targeting will be tested through mutagenesis and chimeric construct alteration of their structure. These domains will then serve as the basis for identification of accessory molecules that assist in the targeting process. In a health context, this project will address cellular and molecular processes that strongly relate to the treatment of chronic pain and the dysfunction experienced in demyelinative disorders of the CNS and PNS.

重组DNA方法已经揭示了电压门控钠 通道以许多不同的亚型存在。有证据 一些可兴奋细胞表达多种通道亚型， 针对不同的细胞区域。该项目涉及 外周神经系统（PNS）通道 同种型具有不同的结构特性， 差异靶向适当的膜结构域， 外周神经元及其过程。有三个具体的 目的：1）研究富含PNS的细胞和亚细胞分布 将使用以下方法建立钠通道亚型 免疫细胞化学技术。同种型特异性抗体将是 生产并用于建立共- 在大鼠的神经系统中表达同种型。特别 感兴趣的将是主要信道的识别 外周神经节不同细胞类型中的同种型， 那些栖息在有髓纤维的朗维尔结中的细胞， PNS。2)将开发一种外周神经元范例， 允许同种型表达和结构的分子操作， 将研究PC 12细胞系作为以下的可能模型： 通道同种型选择性靶向细胞体， 流程.脊髓背根和上级颈神经原代培养 神经节神经元也将同样被研究， 在这些研究中的适用性。每个系统都将被评估， 选择性控制野生型同种型表达的能力， 表达突变的通道亚型。3)通道亚型结构域， 指定靶向性质和与靶向性质相互作用的部分， 他们将被描述。候选靶向域将是 通过比较信道之间的主序列来识别， 目标不同。这些结构域在靶向中的作用将是 通过诱变和嵌合构建体改变测试 他们的结构。这些领域将作为 鉴定辅助靶向的辅助分子 过程在健康方面，该项目将解决细胞 和分子过程密切相关的治疗 慢性疼痛和功能障碍经历的脱髓鞘 CNS和PNS的疾病。