A genetic analysis of complexin function in neurotransmitter release and synaptic plasticity

Abstract

Information transfer at neuronal synapses requires rapid fusion of docked synaptic vesicles in response to calcium influx during action potentials. The molecular nature of the fusion clamp machinery that prevents exocytosis of synaptic vesicles in the absence of a calcium signal is still unclear. Here we show that complexin, a small alpha-helical protein that binds fully assembled SNARE complexes, functions as the synaptic vesicle fusion clamp in vivo. Drosophila has a single complexin homolog that is abundantly expressed in presynaptic nerve terminals. Animals lacking complexin die throughout development, with adult escapers showing severe locomotion defects and a loss of visual function. Electrophysiological analysis at neuromuscular junctions in complexin null mutants reveals a dramatic increase in spontaneous synaptic vesicle fusion that is independent of nerve stimulation or extracellular calcium. High frequency stimulation at high calcium concentrations shows that the readily releasable pool in complexin mutants is severely depleted. Thus, complexin is required for maintenance of the readily releasable pool of vesicles at the synapse, and without it vesicles exocytose directly after priming. These data indicate that complexin interacts with assembled SNARE complexes to prevent premature vesicle fusion in the absence of calcium entry. In addition, a preliminary analysis of synaptotagmin 1; complexin double mutants reveals that the elevated mini frequency in complexin single mutants is dependent on synaptotagmin 1. This finding suggests that the dominant function of complexin at the synapse is to prevent synaptotagmin 1 from triggering fusion in the absence of calcium. Further analysis of synaptotagmin 1; complexin double mutants may reveal new aspects of the mechanism of the calcium-regulated vesicle fusion reaction. Minis have long been thought to represent background noise at the synapse, but there is now growing evidence that mini frequency is important in synaptic maintenance and plasticity. Complexin mutants display a substantial synaptic overgrowth phenotype. We hypothesized that the enhanced mini frequency in complexin mutants drives synaptic overgrowth and that complexin is phosphorylated by PKA to regulate mini frequency at Drosophila synapses in an activity-dependent retrograde signaling pathway that mediates a large increase in mini frequency and a concomitant induction of synaptic growth. Like complexin mutants, a syntaxin mutant with elevated mini frequency also displays enhanced synaptic growth, providing further evidence that an increase in mini frequency drives synaptic plasticity. S126 in complexin is phosphorylated by PKA in vitro. Future results may reveal that S126 is phosphorylated by PKA in vivo to regulate mini frequency in an activity-dependent manner. These results have the potential to reveal a new role for minis in local synaptic plasticity in response to neuronal activity.