Efficient and rapid transduction mechanisms at synapses are required for coherent oscillations and synchronization of activity of brain circuits. The presynapse transduces within <1 ms action potentials into a Ca2+-triggered synchronous fusion of neurotransmitter containing vesicles. Two protein families, the Complexins and Synaptotagmins, are key players in tuning the generic SNARE protein-based membrane fusion apparatus into a high speed transducer. Both proteins are known to interact with the SNARE complex. The goal of this study is to understand how these proteins accomplish fast fusion, and to determine which interactions are relevant for their function. We hypothesize that Complexin and Synaptotagmin 1 act by reducing the energy barrier of the fusion reaction, but they accomplish this using different molecular mechanisms. Following binding to the SNARE complex, Complexins seem to cause stabilization of a profusion complex, or alternatively, serve as an adaptor to enable Synaptotagmin 1 binding to the SNARE complex. Synaptotagmin 1 may trigger fast neurotransmitter release by Ca2+-dependent binding of its C2A and C2B domains to the plasma membrane, and this membrane penetration determines fusion rates by destabilizing the profusion membrane complex. To decipher the function of Complexin and Synaptotagmin, we propose an integrated approach using electrophysiological, structural, and biochemical experiments. First, we will analyze synaptic properties in neurons derived from Complexin 1/2/Synaptotagmin 1 knockout mice. We will then explore the structure-function relationship of protein domains that are putatively involved in synchronous release using a rescue approach. Finally, we will probe whether both proteins act independently or in conjunction with each other, and whether they act sequentially. A better understanding of the mechanism of synchronous release may help to find the cause and treatment of diseases that show disturbed oscillations and synchronization patterns in brain circuits, like Attention Deficit Syndrome, Autism, Huntingdon's Disease or Schizophrenia.
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