Regulation of Sodium Channel Density and Localization in Polarized Cells

电压门控钠通道是可兴奋细胞（如神经元、心肌细胞或骨骼肌肌细胞）中负责启动动作电位的膜蛋白。在神经元中，钠离子通道高密度分布于轴突起始节段和Ranvier节点。这种安排允许动作电位的有效、跳跃传导。本提案的长期目标是了解钠通道靶向和定位的分子基础，特别强调辅助b亚基在这一过程中的作用。提出钠通道亚基异构体在其氨基酸序列中含有一个内在的分子靶向结构域，该结构域将新合成的通道定向到特定的质膜位置。此外，假设极化细胞中的钠通道定位是由特定的a和b亚基组合调节的。提出了四个具体目标：1。目的探讨b亚基表达对极化犬肾（MDCK）细胞钠通道a亚基膜定位的影响。MDCK细胞已被证明是研究多种离子通道和受体的膜靶向性的强大模型系统。这一目的将揭示b亚基是否调节了a亚基定位，或者钠通道靶向信息是否仅编码在a亚基异构体cDNA中。2. 鉴定a和/或b亚基cdna编码的分子靶向信号。截断突变体和嵌合体将被用于定位编码在a和/或b亚基cdna中的分子靶向信号。3. 研究人NT-2神经元内源性表达的钠通道a和b亚基的定位。认识到使用上皮细胞模型研究神经元蛋白靶向轴突膜的局限性，还将研究在神经元细胞培养模型系统NT-2神经元中内源性表达的a和b亚基的定位。这些研究的结果将与MDCK细胞获得的结果相关联。4. 分析b亚基在胚胎干细胞向神经元分化过程中对a亚基定位的贡献。最近在实验室中建立了缺乏钠通道b2亚基基因的靶向小鼠胚胎干细胞系。其中一个细胞系在培养中也被双重靶向，使用高浓度的抗生素G418产生纯合子基因型。这些细胞现在缺少b2亚基基因的两个等位基因。一个类似的实验正在进行中，旨在敲除b1亚基基因。双靶胚胎干细胞将在培养中用视黄酸分化为神经元。将对分化神经元中的a亚基进行免疫细胞化学定位，以评估b亚基对a亚基膜靶向和定位的贡献。这些建议的研究结果将有助于离子通道生物学和大脑发育领域。对钠通道亚基组装和膜靶向机制的基本了解对于理解神经元极性的建立、Ranvier节点的形成和有效的动作电位传导至关重要。

Lorie Isom9975490LAY SUMMARYVoltage-gated sodium channels are the membrane proteins responsible for initiation of action potentials in excitable cells such as neurons, cardiac myocytes, or skeletal muscle myocytes. In neurons, sodium channels are localized at high density at axon initial segments and nodes of Ranvier. This arrangement allows for efficient, saltatory conduction of action potentials. The long-term goal of this proposal is to understand the molecular basis of sodium channel targeting and localization, with particular emphasis on the role of the auxiliary b subunits in this process. It is proposed that sodium channel a subunit isoforms contain an intrinsic molecular targeting domain in their amino acid sequence that directs newly synthesized channels toward specific plasma membrane locations. Furthermore, it is hypothesized that sodium channel localization in polarized cells is modulated by the combination of particular a and b subunits. Four specific aims are proposed: 1. To characterize the effects of b subunit expression on sodium channel a subunit membrane localization in polarized Madin-Darby canine kidney (MDCK) cells. MDCK cells have proven to be a powerful model system in which to study the membrane targeting of a variety of ion channels and receptors. This aim will reveal whether b subunits modulate a subunit localization or whether the information for sodium channel targeting is encoded in the a subunit isoform cDNA only. 2. To identify molecular targeting signals encoded in a and/or b subunit cDNAs. Truncation mutants and chimeras will be used in an attempt to localize the molecular targeting signals encoded in a and/or b subunit cDNAs. 3. To characterize the localization of sodium channel a and b subunits expressed endogenously by human NT-2 neurons. Recognizing the limitations of the use of epithelial cell models to study targeting of neuronal proteins to axonal membranes, the localization of a and b subunits that are endogenously expressed in a neuronal cell culture model system, NT-2 neurons, will also be studied. The results of these studies will be correlated with the results obtained from MDCK cells. 4. To analyze the contribution of b subunits to a subunit localization in targeted embryonic stem (ES) cells differentiated to neurons in culture. Targeted mouse ES cell lines that lack the gene for the sodium channel b2 subunit have recently been established in the lab. One of the cell lines was also doubly targeted in culture to produce the homozygous genotype using high concentrations of the antibiotic G418. These cells now lack both alleles of the b2 subunit gene. A similar experiment is underway that is designed to knockout the b1 subunit gene. The doubly targeted ES cells will be differentiated in culture to neurons using retinoic acid. Immunocytochemical localization of a subunits in the differentiated neurons will be performed to assess the contribution of b subunits to a subunit membrane targeting and localization. The results of these proposed studies will contribute to the fields of ion channel biology and brain development. A basic understanding of the mechanisms involved in sodium channel subunit assembly and membrane targeting is essential to understanding the establishment of neuronal polarity, formation of nodes of Ranvier, and efficient action potential conduction.