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.
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