: A large number of voltage-sensitive K+ channels are coded by the four genes in the Shaker family. In Drosophila, three of these genes undergo alternative splicing of their mRNA to generate multiple proteins with different biophysical properties. In addition, splice variants of a single gene can interact to form heteromultimeric channels. The physiological roles of this potassium channel diversity are not well understood. It is difficult to associate a particular gene with an ionic current in a cell, and to show how that gene product helps to shape the electrophysiological properties of the cell and the neural network within which the cell functions. This proposal is aimed at filling this gap using the small, well-defined pyloric network in the crustacean stomatogastric ganglion. This network's 14 neurons are easily identified, and their unique physiological properties are well known. We are cloning the cDNAs of the four genes in the Shaker family (shaker, shab, shaw and shal), and determining their functions in each of the 14 identified cells in this pyloric network. To understand the roles of Shaker family currents in the generation of the pyloric network's motor pattern, we propose the following goals: 1) Clone the alternative splice variants of shaker, shab, shaw and shal form lobster. 2) Express these variants in Xenopus oocytes and lobster neurons, and compare their biophysical properties to endogenous currents in the stomatogastric neurons. 3) Map the expression of these variants in the individual identified neurons using a single cell PCR method that we have recently developed. 4) Artificially alter the expression of Shaker family genes by injecting sense or antisense RNA into identified STG neurons, and determine the effect or raising or lowering specific currents on the cellular and network properties and cellular responses to monamine modulators. The ultimate goal of this work is to answer why there are so many genes and splice variants for voltage-dependent K+ channels. Our studies will allow us to assign physiological roles to specific K+ channel genes in determining both the intrinsic firing properties of identified neurons and the motor pattern generated by the pyloric network.
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