Calcium currents play an important role in many cellular processes with the long term goal of understanding the mechanisms of calcium channel regulation in normal and pathological conditions, this project aims to identify molecular components involved in this regulation. Studies will be conducted in the fruit fly Drosophila which will allow easy genetic manipulation of such molecular components. In a first complete resolution of the Ca2+ current in Drosophila, the current has been resolved into two components, the equivalent of the vertebrate L-type and the T-type channels. Two-microelectrode voltage-clamp techniques will be used to analyze physiological properties of the two currents, such as kinetics of activation; kinetics of inactivation; voltage-dependence of activation and inactivation; and time course of recovery from inactivation and its dependence on the recovery potential. This will provide the first complete analysis of the Ca2+ currents of Drosophila in particular, and insects in general. It will also lay the basis for analyzing effects of mutations on the L- and the T-like currents. The role of second messenger system components, such as calcium/calmodulin responsive adenylyl cyclase, protein kinase A, protein kinase C, calcium/calmodulin dependent protein kinase and protein phosphatase 1 in regulating Ca2+ currents will be examined by analyzing currents from mutants that have lesions in these components. Since such components play a prominent role in regulating calcium channels in many organisms, these experiments will identify steps in the regulatory pathways of calcium channels in Drosophila muscles. As another strategy to identify mutations that disrupt Ca2+ currents, and thus provide an opportunity to study their regulation, mutations with the behavioral phenotype of temperature-induced paralysis will be used. The 30 mutants to be used were identified by an improved rapid method for obtaining autosomal mutations. This phenotype enriches for defects in ionic currents. These experiments will identify genes that may be involved in either specifying the structure of a calcium channel or regulating its function. Genetic analysis of calcium current mutations identified in this step will be undertaken to prepare these mutations for molecular analysis in subsequent studies. Because of a strong molecular homology between ion channels from different species, information on Drosophila calcium channels will also help in studying calcium channels in other organisms, including humans. calcium currents are regulated by many homologous and heterologous factors and in several diseases like hypertension, myocardial ischemia, cardiomyopathy, hyperthyroidism, Lambert-Eaton syndrome and Parkinson's disease. The studies proposed here will help in understanding the mechanisms underlying these regulatory phenomena and in designing better therapeutic strategies for such diseases.
