Ca2+ and lipid control of vesicle fusion in ß-cells and neurons.
Information processing in the nervous system relies on chemical transmission across synapses where neuro- transmitters are released in a coordinated manner from the presynaptic neuron and detected by transmitter- receptors at the postsynaptic cell. For this, neurotransmitters are loaded into synaptic vesicles (SVs) which fuse at subsynaptic active zones (AZ) with the presynaptic plasma membrane. This reaction is triggered by an increase in the intracellular Ca2+ concentration upon action potentials (APs) that lead to the opening of voltage- gated presynaptic Ca2+ channels. The proteins operating in this fundamental reaction are among the most highly conserved throughout evolution and the relevance of proper function for the organism is highlighted by the severe defects of toxins or mutations that interfere with this machinery. Depending on the number of pre- synaptic SVs released per AP, transmission strength varies. Changes of this strength allow the nervous system to account for behavioral versatility, or to homeostatically prevent dysfunction when transmission is endan- gered. We will here study the local environment of synaptic vesicle release sites to deeper understand their in vivo regulation during homeostatic presynaptic plasticity.
To date we know that proper synaptic function relies on protein-protein, protein-lipid and protein-Ca2+ interac- tion. However, studying the relevance of lipids has been the most challenging due to technical limitations. Based on the progress made in this regard during the first funding period, we plan to investigate this in the context of homeostatic changes of synapse strength. We will focus on mechanisms that regulate the release sites themselves. We have previously recognized that the evolutionarily conserved vesicle priming protein Unc13 (Munc13 in mammals) essentially contributes to SV release site function (1), and more recently showed that Unc13 is a key target of homeostatic plasticity (2). We and others have recognized that transmitter release via Unc13 is modulated by binding to the signaling lipids PI(4,5)P2 and diacylglycerol (DAG), and that residual AZ Ca2+ controls Unc13 function by a Ca2+/Calmodulin complex (see work program)(3-7). These observations suggest that the activity of the synapse may be controlled by Ca2+/Calmodulin/lipid interactions with Unc13, thereby switching release sites on or off. Yet how this occurs exactly during homeostatic plasticity is unknown and shall be addressed in aim 1. In addition, the intra-AZ mobility of voltage gated Ca2+ channels, measured by single molecule imaging at intact AZs, is homeostatically regulated by Ca2+ and VGCCs contribute to ho- meostatic plasticity (8). In aim 2, we will therefore analyze the molecular basis of Ca2+-mediated changes of channel mobility within AZs. Importantly, preliminary data link both phenomena to presynaptic homeostatic plasticity, which warrants the analysis of their functional and molecular intersection.