LTRPC7 is a novel widely expressed divalent ion-permeable channel that appears to play an important role in Ca2+ and Mg2+ homeostasis and in cell viability. It also appears to represent a ubiquitous basal influx pathway for essential trace metals like Zn2+ and Fe2+ as well as toxic metals such as Al3. Given the known role of essential metals in metabolic processes vital for survival and mounting evidence implicating aberrations of metal homeostasis in neuropathology, a detailed study of metal ion permeation through LTRPC7 and its regulation in normal and pathophysiological conditions, is proposed. The study will be conducted as a collaboration between investigators at the University of Hawaii and Stanford University. An integrated approach using electrophysiological methods and fluorescence imaging of intracellular indicator dyes will be used to study LTRPC7 function and its regulation in the HEK-293 expression system, neuronal and glial cell lines, and primary cultures of zebrafish cerebellar neurons. Regulation of LTRPC7 channels by intracellular Ca2+ will be studied using fluorescence imaging of indicator dyes and flash photolysis of caged Cat+, as well as calcium imaging experiments in the neuronal cell line NIE-115 upon M1 muscarinic activation. The regulation of channel function by intracellular Mg 2+, and Mg-- nucleotides will be investigated in expressed LTRPC7 channels where potential Mg2+ coordination sites have been mutated. Experiments to study the effects of H2O2-induced oxidative stress on LTRPC7 function and metal ion permeation will be conducted to assess how the channel behaves during pathophysiological conditions. Finally, to determine whether susceptibility to neurodegeneration is conferred by genetic mutations, PCR-amplified fragments of the LTRPC7 gene will be sequenced and polymorphisms occurring with high incidence in brain tissue of Guamanian ALS and PD identified. These mutations will be then engineered in the expression system to assess resulting alterations in channel function.