PROJECT SUMMARY TMEM16A is a calcium-activated chloride channel that is required for numerous physiologic processes, including regulation of neuronal and cardiac excitability, uterine contractility, regulation of electrolyte balance, and sensory transduction. Their importance is supported by the embryonic lethality of TMEM16A knockout mice, and by the association of mutations in this channel with craniofacial, breast, and pancreatic cancers. In addition to intracellular calcium, TMEM16A channels are regulated by membrane lipids produced in the body. The long-term goal of this research is to understand the molecular mechanisms that enable TMEM16A channels to respond to cellular changes to alter channel activity. The overall objectives of the experiments outlined in this proposal is to uncover how the membrane phospholipid phosphatidylinositol 4,5-bisphosphate binds to the channel to enable chloride conduction, uncover the signaling events that turn on these channels in their native cells, and to delineate how polyunsaturated fatty acids regulate channel activity. We will ask the following questions: 1) How does the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) potentiate TMEM16A channels? 2) How does arachidonic acid and its metabolites inhibit TMEM16A? Finally, 3) How does fertilization open TMEM16A channels in eggs from the African clawed frog, Xenopus laevis? The proposed research includes conceptual and technical innovations and is significant because it is expected to provide detailed mechanisms by which TMEM16A responds to changing cellular environments. Ultimately, such knowledge has the potential to offer new opportunities for the development of innovative therapies to treat TMEM16A-associated diseases.

PROJECT NARRATIVE The proposed research is focused on studying molecular interactions regulating the calcium- activated chloride channel TMEM16A, which mediates diverse processes in humans ranging from regulating blood flow through the cardiovascular system to the excitability of neurons. The goal of this research is to uncover the mechanisms of how lipids created in the cell and acquired through diet, and physiologic processes regulate channel activity. It is relevant to public health because understanding how these molecular interactions are disrupted in disease will enable better therapeutic design for the wide array of pathologies associated with TMEM16A channels.