Joint Study on the Molecular Biology of SA Channels

SA通道分子生物学联合研究

• 批准号: 07044245
• 负责人: SOKABE Masahiro
• 金额: $ 2.5万
• 依托单位: Nagoya University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for international Scientific Research
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07044245/
• 关键词: SA channel; me c-gene; Drosophila; volume regulation; antibody; opider venom; cGMP依存性チャネル; 遺伝子クローニング; 分子生物学; MEC遺伝子; A6細胞

Stretch activated (SA) ion channels are expressed in many types of cells and supposed to play an important role in fundamental cell functions including cell growth, division, morphogenesis, and volume regulation. However, because of the lack of information on their molecular structure, further exploration of SA channels is confronted with difficulties. Very recently a gene encoding an SA channel has been isolated from E.coli, however, its shows a peculiar sequence devoid of any homology with known sequences of ion channels in eucaryotic cels. The aim of the present study was to find SA channel genes from eucaryotic cells and to develop a specific blockers to the SA channel, by which we could explore structure-function and physiological roles of SA channels. We found a candidate gene in MDCK cells of which sequence has a significant homology with mec gene that is supposed to be a putative SA channel gene. We also found, in Drosophila, a gene encoding new type of cGMP gated ion channel that may contribute to mechanotransduction in this species. Expression and functional assay of these genes are in progress. A certain spider venom has been found to include peptides that specifically block the SA channel. We succeeded in purifying some of these peptides with defferent degree of affinity to the SA channel. The structure of a low a affinity one has been sccessfully determined. Determination of a high affinity one is now in progress. We are planning to prepare an affinity column by using the peptide to purify SA channel proteins. We are also going to prepare a fluorescent-conjugated peptide by which the distribution of SA channels on the cell surface could be studied.

牵张激活离子通道（stretchactivatedionchannels，SA）在多种细胞中均有表达，在细胞生长、分裂、形态发生和体积调节等细胞基本功能中发挥重要作用。然而，由于缺乏关于其分子结构的信息，SA通道的进一步探索面临困难。最近，编码SA通道的基因已从大肠杆菌中分离出来，然而，它显示出与真核生物中已知的离子通道序列没有任何同源性的特殊序列。本研究的目的是从真核细胞中寻找SA通道基因，并开发SA通道的特异性阻断剂，为进一步研究SA通道的结构功能和生理功能奠定基础。我们在MDCK细胞中发现了一个候选基因，其序列与推测的SA通道基因mec基因具有显著的同源性。我们还发现，在果蝇中，一个基因编码的新型cGMP门控离子通道，可能有助于在这个物种的机械转导。这些基因的表达和功能检测正在进行中。已经发现某些蜘蛛毒液包括特异性阻断SA通道的肽。我们成功地纯化了其中一些对SA通道具有不同程度亲和力的肽。一个低亲合力分子的结构已被严格确定。目前正在确定一种高亲和力的药物。我们正计划利用此肽制备亲和柱来纯化SA通道蛋白。我们还准备制备一种荧光结合肽，通过它可以研究SA通道在细胞表面的分布。

项目成果

曽我部 正博、成瀬 恵治、嶽本 和久、: "SAチャネルは細胞の機械センサか?" 蛋白質・核酸・酵素,. 41. 2-13 (1996)

Masahiro Sogabe、Keiji Naruse、Kazuhisa Takemoto：“SA 通道是细胞机械传感器吗？”，41. 2-13 (1996)

Kobuke, Y., Ueda.K., & Sokabe, M.: "Totally synthetic voltage dependent ion channels." Chem.Let.435-436 (1995)

小部，Y.，上田.K.，

Okada,H.,Yoshida,J.,Sokabe,M.,Wakabayashi,T.,Hagiwara,M.: "Suppression of CD44 expression decreases migartion and invasion of human glioma cells." Int J Cancer Res. 66. 255-260 (1996)

Okada,H.、Yoshida,J.、Sokabe,M.、Wakabayashi,T.、Hagiwara,M.：“抑制 CD44 表达可减少人胶质瘤细胞的迁移和侵袭。”

曽我部正博: "単一チャネルデータの処理と解析法 In パッチクランプ実験技術法(岡田編)pp50-62" 吉岡書店, 181 (1996)

Masahiro Sogabe：“膜片钳实验技术中的单通道数据处理和分析方法（冈田编）第50-62页”吉冈书店，181（1996）

曽我部正博: "イオンチャネルのゲートとゆらぎ In 生物物理別冊 「生体膜」 (葛西、田口編) pp47-66" 吉岡書店, 167 (1996)

曾我部正博：“生物物理学中的离子通道门和波动特别版“生物膜”（葛西和田口编辑）第47-66页”吉冈书店，167（1996）