Ion channel dysfunction causes many inherited disease, including cystic fibrosis, cardiac dysfunction, neurodegeneration and deafness. A better understanding of how ion channels function will lead to better treatments for these diseases. The large-conductance, calcium-activated potassium (BK) channel is expressed in numerous cell types where it functions to repolarize membrane potential. A mutation in the human BK channel affects calcium activation and causes epilepsy. The molecular mechanism of calcium activation remains unknown. The goal of this study is to locate and evaluate regions of the protein that are important for BK channel activation. To discover such regions, we conducted a mutant screen in the nematode Caenorhabditis elegans. Thus far, this screen revealed twelve alleles of the slo-1 gene that encodes the BK channel and the first known splice variant-specific worm slo-1 mutant. This result indicates that biophysical differences may exist between BK channels isoforms and that these differences are physiologically relevant.
Aim 1 proposes to examine slo-1 splice variants. First, we will examine the expression pattern of the slo-1 c splice variant. We will specifically determine if slo-1 c functions pre- or post- synaptically at the NMJ to determine if splice variants have distinct or overlapping functions. Finally, we will determine whether the biophysical properties of splice variants are distinct by measuring macroscopic currents from BK channel isoforms in inside-out patches from Xenopus oocytes containing many channels.
Aim 2 proposes to sequence the remaining new BK channel alleles derived from our screen and to examine how these mutations affect channel biophysics in membrane patches. We predict that a subset of these slo-1 mutants will have defects in calcium sensitivity compared to wild-type channels. These results will be used to construct a model to evaluate how the mutated regions contribute to calcium activation and BK channel function. Ion channels are a class of proteins that function to generate electrical signals in the nervous sytem. A mutation in a human potassium channel causes epilepsy. We are examining how the structural features of this protein confer its biological function. We believe that the results of our work will contribute to the discovery of better treatments for epilepsy and other ion channel-related diseases. ? ? ?
