The purpose of this proposal is the development and use of new tools for the study and characterization of calcium channels. We will use for this purpose, toxins which are isolated from spider venom, and which are potent blockers of calcium currents and synaptic transmission in Drosophila melanogaster. The long term goal of this research is the characterization of calcium channels which serve various specific functions in Drosophila and other species. We have chosen to work in Drosophila because the well developed and uniquely accessible genetics of Drosophila allows detailed structure function studies of physiologically important molecules such as calcium channels, and current evidence suggests great similarity between various ion channels in Drosophila and their counterparts in vertebrates. Specifically in this study, we will preparatively purify toxins and determine the primary structure of one of the more carefully characterized toxins which we have previously isolated. We will perform voltage clamp studies and measure intracellular Ca with Ca sensitive dyes in order to better understand the specific mechanism of action. We will prepare labeled toxin analogs and use them to investigate the binding of toxin to receptors on Drosophila cells and membranes. Building on the binding studies, we will attempt to photoaffinity label and affinity purify toxin binding sites. If our affinity purifications are successful, we will attempt a partial structural characterization of the purified receptors, and make antibodies against them. This will provide us with the opportunity, in future studies, to attempt to clone and fully characterize the receptors. Since evidence now suggests that these receptors will be components of nerve (and perhaps muscle) calcium channels, and calcium channels play extremely important roles in neuronal activity and communication, their structural characterization win likely be a significant step in our understanding of the molecular basis of neuronal function in health and disease.
