Distribution and subunit composition of SK channels

In contrast to many other K+ channel families, small-conductance Ca2+-activated K+ (SK) channels are largely unexplored in mammals, both with respect to biochemical and functional properties. We have recently succeeded to raise a sequence-directed antibody against one SK channel isoform, the SK3 channel- (Science 289, 1942-1946 (2000)). This antibody was instrumental to establish key functional properties of this SK channel isoform using a novel transgenic approach. Based on this success, Aim 1 and 2 propose to raise and characterize sequence-directed antibodies against the two remaining SK channel isoforms, SK1 and SK2, in order to establish their distribution profile in rat and mouse brain through immunohistochemical distribution experiments. In collaboration with Dr. Adelman`s laboratory, Vollum Institute, Portland, Oregon, USA, a very similar genetic approach (mice that can be regulated in vivo for SK3 gene expression) is going to be taken thereby correlating the distribution of immunoreactivity of these SK1 or SK2 transgenic mice with the observed phenotype. Aim 3, 4 and 5 will introduce novel fluorescently-labeled, radio-labeled, and undecagold-labeled toxin analogues which specifically interact with SK channels. These tools will be used to expand our research on the subunit composition of SK channels in rodent brain, to confirm the distribution data obtained with the antibodies and to investigate the subcellular distribution profile of all three SK channels through immunogold labeling experiments as well as high-resolution laser scanning microscopy. Aim 6 will focus on the co-localization and coupling of SK channels to voltage-gated Ca2+ channels using a combined approach of immunoprecipitation experiments, immunogold labeling techniques and FRET studies. The obtained results are expected to provide significant contributions to our understanding of SK channel function in the mammalian CNS.