The broad, long-term objectives of this project are to characterize the regulation and physiological roles of membrane-associated calcineurin. In brain, the equal distribution of calcineurin between the membrane and cytosol, and regulation of several ion channels and dynamin l by calcineurin implicates membrane-associated calcineurin in their regulation. The calcineurin B subunit is responsible for the Ca2+-dependent binding and activation of calcineurin by membrane phospholipids. A major goal of this project is to test the hypothesis that calcineurin binds to acidic phospholipids by a myristoyl-electrostatic switch composed of the B subunit N- terminal myristic acid and a basic residue cluster in the B subunit. Wild-type and site-directed mutant enzymes will be expressed and purified from bacteria or Sf9 cells. Structure/function studies of Ca2+/phospholipid-bound calcineurin will be carried out to identify the mechanism regulating the interaction between calcineurin and membrane phospholipids. Dynamin I plays a key role in synaptic vesicle recycling. Dynamin I is dephosphorylated and translocates to the plasma membrane during depolarization-induced Ca2+ influx. A primary objective of this project is to test the hypothesis that membrane-associated calcineurin dephosphorylates dynamin I. Using recombinant protein expression techniques, the in vitro phosphatase activity of calcineurin toward purified dynamin l in the presence of Ca2+/phospholipids will be characterized. The subcellular compartment where calcineurin dephosphorylates dynamin I will be identified by,examining calcineurin activity and distribution, and dynamin l dephosphorylation during Ca2+ influx into purified synaptosomes. Calcineurin plays a role in synaptic plasticity, and has been implicated in glutamate neurotoxicity following focal ischemia. In addition, the finding that calcineurin is the target of cyclosporin and FK5O6, the major drugs used to prevent tissue rejection following organ transplantation, indicates the importance of calcineurin in the immune system. The studies from this project will elucidate calcineurin regulation by membrane targeting, and further our understanding of its physiological roles in learning and memory, stroke, and immune function.
