SAKAI Takaomi Gunma University, Graduate School of Medicine, Assistant Professor, 大学院・医学系研究科, 助手 (50322730)
UENO Kohei Gunma University, Graduate School of Medicine, Assistant Professor, 大学院・医学系研究科, 助手 (40332556)
|Budget Amount *help
¥15,300,000 (Direct Cost: ¥15,300,000)
Fiscal Year 2004: ¥7,400,000 (Direct Cost: ¥7,400,000)
Fiscal Year 2003: ¥7,900,000 (Direct Cost: ¥7,900,000)
Function of Synaptotagmin I(Syt I) ; Identification of Ca^<2+> binding sites
Syt I is considered to be a major Ca^<2+> sensor for fast synaptic transmission. Syt I has two Ca^<2+> binding domains, C2A and C2B. It is still debated which of these domains is sensing Ca^<2+>. In this study, we attempted to identify the Ca^<2+> binding domain for fast synaptic transmission using Drosophila syt I mutants.
1)Syt I is a major Ca^<2+> sensor for fast synaptic transmission at the Drosophila neuromuscular junction We used three syt I mutants : syt I^<AD4> ; a null mutant, syt I^<AD1> ; lacks C2B while C2A remains intact, syt I^<AD3> ; has one amino-acid substitution in C2B. All of these mutants have severe impairment in synaptic transmission and embryonic lethal. In syt I^<AD4> synaptic transmission was severely impaired but not abolished. Remaining synaptic currents were Ca^<2+>-dependent. The apparent cooperativity, N, was 0.95. In syt I^<AD1> synaptic transmission was also severely impaired but
slightly better than syt I^<AD4>. N was also 1.06, suggesting that the remaining C2A does not work as a Ca^<2+> sensor by itself. In syt I^<AD3> synaptic transmission was better than other two mutants but the mean amplitude was about 1/20 of the control, and N was 1.54 which was significantly smaller than 3.01 in the control. Since the AD3 mutation is known to block Ca^<2+>-dependent oligomerization of Syt I, this defect in syt I^<AD3> could be due to lack of oligomerization (Okamoto et al., 2005).
2)Ca^<2+> binding sites, Ca1 and Ca2, in the Syt I C2B domain are sensing Ca^<2+> for fast synaptic transmission For this study we used two transformants in which two aspartates at Ca1 or Ca2 was changed to arginines to block Ca^<2+> binding. In the Ca1 mutant no fast synaptic transmission was detected, while in the Ca2 mutant synaptic transmission was strongly reduced but remained. Remaining synaptic currents were Ca^<2+>-dependent with smaller N, 1.86, suggesting that at least two Ca^<2+> molecules bind to induce fast vesicle fusion. Since in this mutant there is only one Ca^<2+> binding site remained, we had to look for another Ca^<2+> binding site. At this moment, we Ca^<2+>-dependent oligomerization is providing another Ca^<2+> binding site in this mutant.
3)Ca^<2+>-dependent oligomerization is essential for fast synaptic transmission It has been reported that Sr^<2+> does not induce Ca^<2+>-dependent oligomerization of Syt I (Chapman et al.,1996). Taking advantage of this property of Sr^<2+> we tested whether Ca^<2+>-dependent oligomerization is required for synaptic transmission. Synaptic transmission in Sr^<2+> was strongly reduced and the cooperativity, N, was 2.01, which is significantly smaller than that, 3.01, in Ca^<2+>. Thus we conclude that Ca^<2+>-dependent oligomerization is essential for fast synaptic transmission. Less