The overall goal of this project is to advance our understanding of the molecular basis of neuronal excitability. This proposal is focused on the analysis of KShIIIA, a gene encoding components of voltage-gated K channels and a member of a recently identified class of K channel genes (the ShIII subfamily). A recent finding of great interest is the highly localized nature of the expression of this gene in brain. Specifically, KShIIIA mRNAs are concentrated in certain thalamic nuclei, the red nucleus, and deep and intermediate layers of the cortex and the superior colliculus respectively. This distribution suggests that the functional role of this gene is very specific. Furthermore, the predicted amino acid sequence of products of this gene is over 99% identical in rats and humans, suggesting that their role is fundamental to the specific neurons that express them. The expression of unique sets of K channels is an essential factor influencing the intrinsic electrophysiological properties, in turn, have recently been demonstrated to profoundly affect the behavior of neuronal circuits in the CNS. For example, the firing patterns of thalamic neurons are thought to play pivotal roles in determining the processing of sensory information to the cortex, the modulation of global states such as wakefulness and sleep and to be associated with certain forms of epilepsy. Three approaches are proposed to further our understanding of the products of this gene: 1) Expression studies, including Northern-blot and RNAse- protection analysis, in-situ hybridization and immunocytochemistry will be used to elucidate the regional and cellular distribution of KShIIIA transcripts in rat brain. Three alternatively-spliced transcripts from this gene have been identified. Further specificity may be defined by the analysis of the localization of each of these subtypes. These techniques will also be used to study the distribution of transcripts from other ShIII genes, since heteromultimer formation between KShIIIA and other ShIII proteins could affect the properties of native channels containing KShIIIA. Electron microscopy will be used to localize KShIIIA proteins to specific regions of the cell. 2) The voltage-gated K channels expressed by KShIIIA transcripts will be characterized in Xenopus oocytes utilizing two- microelectrode voltage clamp and patch clamp techniques. The functional consequences of coexpression of KShIIIA transcripts with other ShIII RNAs will be examined as well. RNA isolated from brain regions expressing KShIIIA transcripts will be expressed in Xenopus oocytes to identify channels containing KShIIIA proteins encoded by native tissue RNA. Based on these studies functional assays will be developed which can be used to obtain via expression cloning unknown molecular components (such as novel subunits) which may affect the functional properties of KShIIIA channels. 3) Electrophysiological analysis of dissociated and cultured thalamic neurons will be carried out to identify and characterize native channels containing KShIIIA proteins.