Changes in membrane excitability occur during development, in response to neurotransmitters, hormones and drugs, and in pathological states. These changes are due to differences in not only the functional activity of specific ion channels, but also in the appropriate targeting of receptors and ion channels to specific domains of the surface membrane. This proposal is aimed at determining the fundamental mechanisms that establish and maintain the distribution of voltage-dependent K+ channels in the membranes of excitable cells. We have used a panel of polyclonal and monoclonal antibodies specific for the Kv2.1, and Kv1.5 K+ channel polypeptides in order to determine their distribution in rat central and peripheral neurons, and in the rat pheochromocytoma PC12 cell line. We find that the distinct subcellular distribution of these channels in neurons is recapitulated in the PC12 cell line expressing endogenous Kv2.1 and Kv1.5 channels. In addition, recombinant K+ channel polypeptides expressed in PC12 and MDCK cells from cDNA are sorted properly, indicating that we have established an excellent cell culture system for studying the molecular basis of sorting of these channels. We will used both wild type and transfected PC12 cells, and transfected MDCK cells, to characterize the protein- protein interactions important in determining the subcellular distribution of K+ channels, and the regions of the K+ channel polypeptides mediating these interactions. Together these studies will allow for an understanding of the cellular processes that regulate the distribution of ion channels in the membranes of nerve, heart and muscle cells. The information from these proposed studies complements studies in project 4, which will determine factors governing Na+ and K+ channel distribution in peripheral nerve in primary cultures and in vivo.
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