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神経伝達物質・修飾物質
Neurotransmitters and Modulators

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開催日
2010年09月02日(木)
時 間
11:00 - 12:00
会 場
Poster Room 1

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8-nitro-cGMPによるSNAREcomplex形成の調節
Regulation of SNARE complex formation by 8-nitro-cGMP

演題番号 : P1-b07

國枝 恒兵 / Kouhei Kunieda:1 井田 智章 / Tomoaki Ida:1 澤 智裕 / Tomohiro Sawa:2 赤池 孝章 / Takaaki Akaike:2 板倉 誠 / Makoto Itakura:3 高橋 正身 / Masami Takahashi:3 居原 秀 / Hideshi Ihara:1 

1:大阪府大院・理・生物科学 / Dept. of Biol. Sci., Grad. Sch. of Sci., Osaka Pref. Univ., Osaka 2:熊本大院・医薬・微生物学 / Dept. of Microbiol., Grad. Sch. Med. Sci., Kumamoto Univ., Kumamoto 3:北里大・医・生化学 / Dept. of Biochem., Kitasato Univ. Sch. of Med., Kanagawa 

 

[Background] Exocytosis of the neurotransmitter is triggered by the formation of a stable trimer complex of v-SNARE(synaptobrevin on synaptic vesicle) and t-SNAREs(SNAP25 and syntaxin on the presynaptic membrane). Formation of the SNARE complex is a crucial step in neurotransmitter release, and this process is regulated by several factors. We detected 8-nitro-cGMP, which is formed in an NO-dependent manner, as a novel second messenger in the cells. In addition to functioning as a cGMP analog, 8-nitro-cGMP plays an important role in membrane permeability and protein S-guanylation. We found that 8-nitro-cGMP regulates exocytosis. In this study, we examined the effect of 8-nitro-cGMP on the formation of the SNARE complex and the S-guanylation of SNARE proteins.
[Method] Rat synaptosome was incubated with 8-nitro-cGMP to perform S-guanylation reaction. The SNARE complex was detected using a low-temperature SDS-PAGE. S-guanylation of SNARE proteins was detected by immunoprecipitation using anti-S-guanylated protein antibody, and western blotting was performed using the anti-SNARE proteins antibody. The amount of SNAP25 and S-guanylated SNAP25 in the SNARE complex was analyzed by immunoprecipitation using anti-syntaxin antibody, followed by western blotting using anti-SNAP25 antibody and anti-S-guanylated protein antibody.
[Result] Low-temperature SDS-PAGE revealed that the amount of the SNARE complex in the synaptosome was increased after treatment with 8-nitro-cGMP. SNAP25 was detected in the immunoprecipitation reaction with anti-S-guanylated protein antibody. It was shown that SNAP25 in the synaptosome could be S-guanylated by trearment with 8-nitro-cGMP. Coimmunoprecipitation studies with the anti-syntaxin antibody revealed that S-guanylated SNAP25 was concentrated in the complex together with a syntaxin. These results suggest that 8-nitro-cGMP causes S-guanylation of SNAP25 and increased formation of the SNARE complex.

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