IN VIVO EXPRESSION OF SCHWANN CELL NA CHANNEL

实验建议(1)测量在体内的表达 雪旺细胞钠通道在mRNA水平，(2)表征 C DNA水平上雪旺细胞钠通道的类型； 以定义这些通道的可能功能。我们的长期合作 目的是检验目前的一个假说，即雪旺细胞可能 充当Na频道的当地工厂，成为 结合在节膜中。这一假设的证明 将对两国之间的相互关系产生巨大的影响 轴突和它们的卫星细胞。 在这项提案中，将采用分子方法来 解决钠通道是否由Schwann在体内合成 细胞。将确定坐骨神经中的钠通道mRNAs 用大鼠脑进行Northern印迹分析或S1核酸酶定位 NA通道基因克隆。在肯定识别后 雪旺细胞钠通道mRNAs的克隆技术 应用于获得雪旺细胞钠通道基因克隆。 几个钠通道特异的cDNA克隆将通过 杂交筛选。然后，分离的cdna克隆可以 以限制性内切酶图谱和部分基因为特征 测序，这样雪旺细胞钠通道的类型就可以 与大鼠的大脑相比较。随后， 雪旺细胞钠通道的功能将在 细胞水平。C DNA-m RNA原位杂交将是 用来证实Na通道在体内的表达 雪旺细胞与大的有髓神经纤维相关。 此外，针对雪旺细胞钠产生的寡肽的抗血清 通道基因将被用来定位雪旺细胞钠 合并到Ranvier节点中的频道。 卫星细胞含有类似于钠通道的可能性 可兴奋组织的电压依赖性钠通道具有较长的 是有人建议的。钠在体内表达的证明 雪旺细胞中的通道及其功能定位 现在迫切需要发挥作用。通过cdna克隆方法，这些 问题可以直接在分子水平上得到回答。

Experiments are proposed (1) to measure the in vivo expression of Schwann cell Na channels at the mRNA level, (2) to characterize the type of Schwann cell Na channels at the cDNA level, and (3) to define the possible function of these channels. Our long-term objective is to test a current hypothesis that Schwann Cells may be acting as local factories for Na channels that become incorporated in the nodal membrane. The proof of this hypothesis will have immense implications on the interrelationship between axons and their satellite cells. In this proposal, a molecular approach will be undertaken to resolve whether Na channels are synthesized in vivo by Schwann cells. The Na channel mRNAs in sciatic nerve will be identified by Northern blot analysis or S1 nuclease mapping using rat brain Na channel cDNA clones. After positive identification of Schwann cell Na channel mRNAs, cloning techniques will be applied to obtain the Schwann cell Na channel cDNA clones. Several Na channel specific cDNA clones will be selected by hybridization screening. The isolated cDNA clones can then be characterized by restriction mapping and partial cDNA sequencing, so that the type of Schwann cell Na channels can be compared with the rat brain counterparts. Subsequently, the function of Schwann cell Na channels will be explored at the cellular level. In situ cDNA-mRNA hybridization will be performed to confirm the in vivo expression of Na channels in Schwann cells associated with large myelinated nerve fibers. Also, antisera against oligopeptide deduced from Schwann cell Na channel cDNA will be used to localize the Schwann cell Na channel incorporated in the node of Ranvier. The possibility that satellite cells contain Na channels resembling the voltage-dependent Na channels of excitable tissue had long been suggested. The proof of the in vivo expression of Na channels in Schwann cells and the assignment of their functional role are now urgently needed. With cDNA cloning approach, these questions can be answered directly at the molecular level.