Roles of SNARE Proteins and Synaptotagmin I in Synaptic Transmission: Studies at the Drosophila Neuromuscular Synapse

Abstract

The roles of SNARE proteins, i.e. neuronal Synaptobrevin (n-Syb), SNAP-25 and Syntaxin 1A (Syx 1A), and Synaptotagmin I (Syt I) in synaptic transmission have been studied in situ using mutant embryos or larvae that lack these molecules or have alterations in them. Because of the ease of genetic manipulation, the Drosophila neuromuscular synapse is widely used for these studies. The functional properties of synaptic transmission have been studied in mutant embryos using the patch-clamp technique, and in larvae by recording with microelectrodes. A major vesicular membrane protein, n-Syb, is indispensable for nerve-evoked synaptic transmission. Spontaneous synaptic currents (minis), however, are present even in embryos totally lacking n-Syb (n-syb). Furthermore, Ca²⁺-independent enhancement of mini frequency induced by hypertonic sucrose solutions (hypertonicity response) is totally absent in n-syb. Embryos that have defects in SNAP-25 (SNAP-25) have similar but milder phenotypes than n-syb. The phenotype in synaptic transmission was most severe in the synapse lacking Syx 1A. Neither nerve-evoked synaptic currents nor minis occur in embryos lacking Syx 1A (syx 1A). No hypertonicity response was observed in them. Syt I binds Ca²⁺ in vitro and probably serves as a Ca²⁺ sensor for nerve-evoked synaptic transmission, since nerve-evoked synaptic currents were greatly reduced in embryos lacking Syt I (syt I). Also, Syt I has a role in vesicle recycling. Interestingly, the Ca²⁺-independent hypertonicity response is also greatly reduced in syt I. Minis persist in mutant embryos lacking any of these proteins (n-Syb, SNAP-25 and Syt I), except Syx, suggesting that minis have a distinct fusion mechanism from that for fast and synchronized release. It appears that these SNARE proteins and Syt I are coordinated for fast vesicle fusion. Minis, on the other hand, do not require SNARE complex nor Syt I, but Syx is absolutely required for vesicle fusion. The SNARE complex and Syt I are indispensable for the hypertonicity response. None of these molecules seem to serve for selective docking of synaptic vesicles to the release site. For further studies on synaptic transmission, the Drosophila neuromuscular synapse will continue to be a useful model.