Patch clamp study of mechanism of membrane depolarization induced by a pulse of blue light in pulvinar motor cells of Phaseolus vulgaris

蓝光脉冲诱导菜豆小球运动细胞膜去极化机制的膜片钳研究

• 批准号: 10640642
• 负责人: OKAZAKI Yoshiji
• 金额: $ 1.22万
• 依托单位: Osaka Medical College
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10640642/
• 关键词: blue light; Phaseolus vulgaris; phototropism; proton pump; turgor pressure; pulvinus; パッチクランプ

1. Phaseolus vulgaris L. was cultivated under light regime of 12h light/12h dark at relative humidity of 70% and 23℃. Materials of 2-3 weeks were usually used for preparing protoplasts. 2. Measurements of both apoplastic concentration of KィイD1+ィエD1 and apoplastic potential with double-barreled KィイD1+ィエD1 microelectrodes revealed close relationship between depolarization of membrane potential and efflux of KィイD1+ィエD1 from motor cells of pulvinus. 3. Protoplasts of pulvinar motor cells were prepared mainly according to methods taken by Wang & Iino (1998) and Vogelzhang & Prins (1992). 4. Seal tests of patch electrodes against tonoplast prepared from protoplasts with hypotonic treatment were tried and successful (more than 1GΩ). That showed the system for performing patch-clamping works without problems. 5. Seal tests of patch electrodes against plasmalemma of protoplasts were not successful (less than 0.1GΩ) though following points were tried to get giga seal. Days for cultivating materials, osmotic and potential differences between patch pipettes and external media, quality of glass of pipettes, resistances of electrodes, preparing protoplasts without debris of cell walls on the surface of plasmalemma. The last point gave maximum seal resistance of patch electrode (0.1GΩ). As a result, patch clamp study of involvement of plasmalemma HィイD1+ィエD1-pump on blue-light-induced membrane depolarization could not be performed. 6. Biochemical assay of HィイD1+ィエD1-pump activity using suspension of protoplasts is now being undertaken indirectly to confirm whether or not HィイD1+ィエD1-pump of plasmalemma is inactivated during irradiation of blue light.

1.菜豆在23℃、相对湿度70%、12 h光照/12 h黑暗的光照条件下培养。原生质体的制备一般采用2-3周的材料。2.用双管K β D1 + β D1微电极测定细胞质外体K β D1 + β D1浓度和细胞质外体电位，发现细胞膜电位的去极化与K β D1+ β D1从枕部运动细胞的流出密切相关。3.主要参照Wang & Iino（1998）和Vogelzhang & Prins（1992）的方法制备了枕运动细胞的原生质体。4.用低渗处理的原生质体制备液泡膜，进行了贴片电极的密封试验，并获得成功（大于1GΩ）。这表明执行膜片钳工作的系统没有问题。5.尽管尝试了以下几点以获得千兆密封，但贴片电极对原生质体质膜的密封测试不成功（小于0.1GΩ）。培养材料的天数、贴片移液器与外部培养基之间的渗透压差和电势差、移液器玻璃的质量、电极的电阻、制备质膜表面无细胞壁碎片的原生质体。最后一点给出了贴片电极的最大密封电阻（0.1GΩ）。因此，无法进行质膜H β D1+ H β D1-泵参与蓝光诱导的膜去极化的膜片钳研究。6.目前正在利用原生质体悬浮液间接进行H β D1+ H β D1-泵活性的生化测定，以确认质膜的H β D1+ H β D1-泵在蓝光照射期间是否失活。

项目成果

Okazaki, Y.: "A pulse of blue light induces a transient increase in activity of apoplastic K^+in laminar pulvinus of Phaseolus vulgaris L"Plant and Cell Physiology. 41.2. 230-233 (2000)

Okazaki, Y.：“蓝光脉冲诱导菜豆层状叶粒中质外体 K 活性的短暂增加”植物和细胞生理学。

Okazaki,Y.: "A pulse of blue light induces a transient increase in activity of apoplastic K in laminar pulvinus of Phaseolus vulgaris L."Plant & Cell Physiology. 41-2. 230-233 (2000)

Okazaki,Y.：“蓝光脉冲可诱导菜豆层状叶粒中质外体 K 活性的短暂增加。”植物

Okazaki,Y.: "A pulse of blue light induces a transient increase in activity of apoplastic K^+ in laminar pulvinus of Phaseolus vulgaris L"Plant & Cell Physiology. 41・2. 230-233 (2000)

Okazaki, Y.：“蓝光脉冲诱导菜豆层状叶粒中质外体 K^+ 活性的短暂增加”《植物与细胞生理学》41·2 (2000)。