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.
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