Genetic Dissection of Neurotransmitter Release

Bernard Katz and colleagues established the hypothesis that Ca²⁺ influx into the presynaptic nerve terminal triggers neurotransmitter release. Current models for synaptic vesicle exocytosis propose that fusion requires assembly and activation of the SNARE complex. Although SNAREs are essential for synaptic transmission, fusion via reconstituted SNARE proteins occurs over slow timescales and is Ca²⁺ -independent, contrasting with synaptic transmission, where SNARE assembly and subsequent fusion is rapid and Ca²⁺ -triggered. A mechanistic understanding of how synaptic Ca²⁺ signals are transduced into membrane fusion in still lacking. The search for synaptic Ca²⁺ sensors that regulate SNARE-dependent fusion has largely focused on the synaptotagmins, transmembrane proteins containing tandem Ca²⁺ binding C2 domains (C2A and C2B).  Synaptotagmin1 (Syt 1) is by far the most studied isoform of the synaptotagmin family and contains a small N-terminal intravesicular domain, a single transmembrane domain and a large cytoplasmic region. Two domains homologous to the C2 regulatory domain of protein kinase C occupy the majority of the cytoplasmic region of Syt 1.  C2 domains are ~ 130 amino acid motifs that are found in an array of intracellular proteins, many of which bind Ca²⁺. To characterize the mechanisms that couple Ca²⁺ influx to rapid and synchronous synaptic vesicle fusion, we are analyzing Drosophila syt 1 mutants deficient in specific interactions mediated by its two Ca²⁺ -binding C2 domains.  In the absence of Syt 1, synchronous release is abolished and a kinetically distinct delayed asynchronous release pathway is uncovered.  Mutant synapses containing only the C2A domain of Syt 1 partially recover synchronous fusion, but have an abolished Ca²⁺ cooperativity.  Mutants that disrupt Ca²⁺ sensing by the C2B domain have synchronous release with normal Ca²⁺ cooperativity, but with reduced release probability. Our findings indicate Syt 1 is the major Ca²⁺ sensor for evoked release and functions to trigger synchronous fusion in response to Ca²⁺, while suppressing asynchronous release.

We have also characterized the six remaining synaptotagmin isoforms encoded in the Drosophila genome, including homologs of mammalian Syts 4, 7, 12 and 14.  Like Syt 1, Syt 4 is ubiquitously present at synapses but localizes postsynaptically rather than presynaptically. Consistent with their distinct localizations, overexpression of Syt 4 cannot functionally substitute for the loss of Syt 1 in synaptic transmission.  The remaining isoforms are not found at synapses (Syt 7) or are expressed within subpopulations of neurons.  Our results indicate that synaptotagmins are differentially distributed to unique subcellular compartments.  Current work is underway to determine the targeting signals that mediate trafficking of the synaptotagmin family to synaptic vesicles, postsynaptic vesicles, or large dense core vesicles. In addition, the function of the remaining synaptotagmin family members is currently being addressed by genetic analysis in Drosophila.  We are also interested in large-scale genetic screens to identify additional loci that function in synaptic transmission by directly screening for electrophysiological defects in mutant animals. Together with targeted genetic approaches, we expect these experiments to provide new insights into the molecular pathways that mediate synaptic communication between neurons.