We have comprehensively established that synaptic vesicles (SVs) release their neurotransmitters by two modes of exocytosis - Kiss-and-run and full fusion – and that there is switching between these two modes that is controlled by the level of intracellular calcium and by protein phosphorylation. Inhibition of protein phosphatase 2A (PP2A)induces a full fusion mode whilst inhibition of protein phosphatase 2B (calcineurin; PP2B) leads to a larger number of SVs undergoing kiss-and-run exocytosis. Activation of protein kinase C or inhibition of calcium-calmodulin dependent protein Kinase II leads to all SVs undergoing full fusion. Under some circumstances dynamin I and II regulate the mode of exocytosis whilst under other conditions myosin II regulates this: the phosphorylated state of these proteins is important in their action.
The phosphorylated state of phospho -proteins will be preserved by the addition of SDS-sample buffer to nerve terminals whose mode of exocytosis has been changed by their treatment with the various phosphatase/kinase inhibitors or other drugs. Such preparations will be subjected to Western blotting. A correlation between the specific phosphorylated state of certain proteins and the specific mode of exocytosis will be sought. We should be able to pinpoint the important phospho-proteins that play a role in this process by comparing the many different conditions. The use of material isolated from diabetic animals will also aid in this endeavour since it is known that these nerve terminals exhibit a larger amount of kiss-and –run. Following identification of relevant proteins, the specific amino acids that are phosphorylated will be determined. That such phospho-sites are important in defining the mode of exocytosis will be determined by transfection of neuronal cells with recombinant genes that code for the consitutively phosphorylated protein or for the protein whose specific phosphorylatable amino acids has been replaced so that the protein is no longer a substrate for the specific kinase. This should highlight which of the proteins identified play a direct role in the regulating the mode of exocytosis, and might reveal a requirement for the co-ordinated action of several proteins.
Such research is vitally important as it might establish the role of kiss-and-run in regulating synaptic plasticity, and memory and it could reveal that mode switching contributes to various neuronal disorders.
Project lead: Dr Anthony Christopher Ashton
Project staff: Dr Anthony Christopher Ashton
Dr Talvinder Sihra, University College London
2007 - present
One paper submitted 2013, and two other papers near completion.