| Research Abstract |
For synaptic transmission vesicles in the presynaptic terminal containing neurotransmitter have to fuse to the membrane and release the content to the synaptic cleft. Various proteins are known to be involved in this fusion process. A hypothesis, SNARE hypothesis, is proposed to account for the arrangement of those proteins. We have investigated the molecular mechanism of this process by using Drosophila mutant larvae which lack key molecules.
Neuronal synaptobrevin (n-syb) is vesicle membrane protein and a target of tetanus toxin. In mutant larvae which lack n-syb no nerve-evoked synaptic currents were observed. However, miniature synaptic currents (mSC) remained in lower frequencies. The frequency of mSG's was dependent on external Ca^<2+> concentrations in high potassium saline. Furthermore, the mSG frequency increased with black widow spider venom extract and Ca^<2+> ionophore, A23187.
These results indicate that the remaining mSC's in n-syb mutants are Ca^<2+> dependent. In wild typ
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e larvae an elevation of cAMP in the presynaptic nerve terminal either by an activator of adenylate cyclase, forskolin, or by a membrane permeable analog of cAMP, db-cAMP, resulted in an increase in msc's. However, in n-syb mutants neither of these drugs increased the mSG frequency. Thus we conclude that the n-syb independent pathway for vesicle fusion is insensitive to cAMP (Yoshihara et at., 1999).
Cysteine-string protein (csp) is also vesicle protein and supposed to interact with voltage-gated Ca^<2+> channels in the terminal membrane. In a temperature sensitive mutant of csp no nerve-evoked synaptic currents were observed in non-permissive temperature (32 ﾟC). This could be due to lack of function of voltage-gated Ca^<2+> channels at high temperature. To test this possibility we visualized terminal Ca^<2+> by a Ca^<2+> sensitive dye. At room temperature the Ca^<2+> concentration increase upon repetitive stimulation of nerve. However, at non-permissive temperature the Ca^<2+> signal decreased significantly, thus suggesting a failure of Ca^<2+> channel function.
Since csp is on the synaptic vesicle membrane it is possible that Ca^<2+> channels have to be associated with synaptic vesicles to operate properly. To test this.
possibility we used another mutant call shibire in which endocytosis is blocked at non-permissive temperature. We also found in shibire that Ca^<2+> signals were lower in non-permissive temperature. In conclusion, for voltage-gated Ca^<2+> channels in the presynaptic terminal to work properly vesicles might have to be docked to release sites (Umbach et al., 1998). Less
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