Activation of cell surface receptors releases inside cells a number of inositol phosphates that regulate a range of cellular activities. We have identified an inositol tetrakisphosphate (Ins(3,4,5,6)P4) that inhibits calcium-dependent chloride secretion as part of the overall signalling process that regulates salt and water during a prolonged stimulation. We are currently purifying and characterizing the enzymes that regulate levels of Ins(3,4,5,6)P4 and investigating the effects upon chloride transport at the single channel level, in order to understand the molecular basis for these important effects. We are characterizing antagonists of the IP4 response with a view to improving therapy of cystic fibrosis. Another aspect of our work focuses on the fundamental problem of how proteins are delivered in vesicles to specific cellular sites - this vesicle trafficking process is central to secretion, neurotransmission, cell division and maintenance of cell polarity. We have discovered that another inositol polyphosphate, InsP6, interacts with a superfamily of proteins that regulate vesicle traffic, which has led our laboratory to propose that InsP6 is a """"""""fusion clamp"""""""". As a consequence we are purifying and characterizing and cloning important InsP6-metabolizing enzymes and binding-proteins and plan to use site- directed mutagenesis to probe the function of InsP6 at a molecular level. Not only do we aim to gain more insight into these important cellular processes, but we also intend to determine these processes are perturbed in disease and by environmental toxins. We are cloning and expressing a multi-functional phosphatase that in erythrocytes may be a pharmacological focus for therapy for stroke and sickle cell anemia.
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