Developing an E. coli-expression-function platform for study of 3-D structure-function relationships of channel molecules

开发大肠杆菌表达功能平台以研究通道分子的 3D 结构功能关系

• 批准号: 13680737
• 负责人: OIKI Shigetoshi
• 金额: $ 2.3万
• 依托单位: Fukui Medical University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13680737/
• 关键词: Ion channel; KcsA channel; patch clamp technique; planar lipid bilayer method; single-channel recording; E. coli; whole-cell recording; liposome

To elucidate structure-function relationships of ion channels a novel experimental platform is developed. So far only ion channels originated from prokaryotes were successfully crystallized. To study such targets more effectively, electrophysiological recording system for ion channels from prokaryotes is critically important. Based on Yabe's method for current recordings from giant E. coli, channel recording techniques were explored. Many technical problems arose for recording whole-cell current from giant bacteria. These problems have been mostly solved, although a few problems still remain.KcsA channel was expressed and recorded by inside-out excised patch methods from giant E. coli. Reconstituted KcsA channel into liposome was also recorded by a modified tip-dip method. On the other hand, whole-cell current of KcsA channel could not be observed from giant bacteria. The discrepancy between successful single channel recordings and unsuccessful whole-cell recordings poses fundamental issue on functional role of ion channels on bacteria.

为了阐明离子通道的结构-功能关系，开发了一种新的实验平台。到目前为止，只有来源于原核生物的离子通道被成功地结晶。为了更有效地研究这些靶点，原核生物离子通道的电生理记录系统至关重要。根据Yabe的巨E. coli中，对通道记录技术进行了探索。记录巨型细菌的全细胞电流存在许多技术问题。本研究采用由内而外的切除斑片法对巨噬细胞KcsA通道进行了表达和记录。杆菌重组KcsA通道进入脂质体也记录了一个改进的tip-dip方法。另一方面，在巨型细菌中不能观察到KcsA通道的全细胞电流。成功的单通道记录和不成功的全细胞记录之间的差异提出了离子通道对细菌的功能作用的基本问题。

项目成果

Maeser P, Hosoo K, Nakayama H, Oiki S, Schroeder J I, Uozurni N: "Glycine residues in potassium channel-like selectivity filters determine Potassium selectivity in four-loop-per-subunit HKT"Proc.Natl.Acad.Sci.USA. 99. 6428-6433 (2002)

Maeser P、Hosoo K、Nakayama H、Oiki S、Schroeder J I、Uozurni N：“钾通道样选择性过滤器中的甘氨酸残基决定了每个亚基四环 HKT 中的钾选择性”Proc.Natl.Acad.Sci.USA

Maeser, P., Hosoo, Y., Goshima, S., Horie, T., Eckelman, B., Yamada, K., Yoshida, K., Bakker, E.P., Shinmyo, A., Oiki, S., Schroeder, J.I., and Uozumi, N.: "Glycine residues in potassium-channel-like selectivity filters determine potassium selectivity in

Maeser, P.、Hosoo, Y.、Goshima, S.、Horie, T.、Eckelman, B.、Yamada, K.、Yoshida, K.、Bakker, E.P.、Shinmyo, A.、Oiki, S.、Schroeder

Maser P, Hosoo K, Nakayama H, Oiki S, Schroeder JI, Uozumi N: "Glycine residues in potassium channel-like selectivity filters determine potassium selectivity in four-loop-pet-subumit HKT"Prou Natl-Acad, Sci USA. (in press).

Maser P、Hosoo K、Nakayama H、Oiki S、Schroeder JI、Uozumi N：“钾通道样选择性过滤器中的甘氨酸残基决定了四环 pet-subumit HKT 中的钾选择性”Prou Natl-Acad，Sci USA。

W.Uozumi, K.Yamada, S.Goshima, T.Ona, S.Oiki: "Sodium blocking induced by a point mutation at the C-terminal end of the pore helix of KAT1"J. Membrane Biol. 181. 163-170 (2001)

W.Uozumi、K.Yamada、S.Goshima、T.Ona、S.Oiki：“KAT1 孔螺旋 C 末端点突变诱导的钠阻断”J。

Berthomieu, P., Nublat, A., Brackenbury, W.J., Lambert, C., Savio, C., Uozumi, N., Oiki, S., Yamada, K., Conejero, G., Cellier, F., Gosti, F., Simonneau, T., Very, A.-A., Sentenac, H. and Casse, F.: "Functional analysis of AtHKT1 in Arabidopsis shows that

Berthomieu, P.、Nublat, A.、Brackenbury, W.J.、Lambert, C.、Savio, C.、Uozumi, N.、Oiki, S.、Yamada, K.、Conejero, G.、Cellier, F.、Gosti