investigator's application): The long term goal of these studies is to understand how the diversity and cellular organization of ion channels contributes to neuronal function. Neurons consist of several regions, such as the cell body, the dendrites and the axon, each with a unique role. To perform their appropriate roles, the plasma membrane of each of these regions contains unique sets of ion channels. Nerve cells have special mechanisms that recognized signals in membrane proteins and target them to the required area. Epithelial cells also have a polarized membrane, use similar mechanisms to target membrane proteins, and have been used extensively to study membrane targeting signals of proteins of epithelial and neuronal origin. Potassium channels, a group of ligand- and voltage-gated channels, are present in most, if not all, eukaryotic cells. They play key roles in a wide range of cellular functions including excitability, secretion, differentiation, mitogenesis, and osmotic regulation, Defects in K+ channel genes have been shown to underlie two human genetic diseases (periodic ataxia and a cardiac arrhythmia, long QT syndrome). Most neurons express multiple K+ channel types. Their functional role varies depending on their precise location on the neuronal membrane. In this project, recombinant K+ channel protein will be used to discover and characterize the protein signals that dictate the localization of K+ channels to different parts of the plasma membrane. Preliminary studies demonstrate that three K+ channel subunit iosoforms encoded in alternatively spliced transcripts of a K+ channel gene (Kv3.2) are targeted to different membrane domains in polarized Madin-Darby Canine Kidney (MDCK) cells, suggesting that there are specific targeting signals in the part of the protein which is different among the isoforms, namely the alternatively-spliced C-terminal domains. MDCK cells are a classical model system to investigate membrane protein targeting mechanisms. Utilizing mutagenesis techniques and taking advantage of the large number of available Kv proteins, experiments are proposed to identify the targeting signals in Kv3.2 and other K+ channel proteins and to analyze the characteristics that are important for targeting. The relationship between targeting domains in epithelial cells and neurons will be explored by determining the localization of specific subunits in rat brain neurons with isoform specific antibodies, and by investigating the targeting behavior for foreign channel proteins introduced into cultured, polarized, hippocampal neurons. In addition to contributing to our understanding of K+ channel structure and neuronal organization, these studies are also important for cell biology in general, and specifically to the problem of how membrane proteins are targeted to their correct destinations in the cells.
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