过刊浏览

基于扫描离子电导显微镜负反馈扫描控制技术的高分辨率膜片钳技术

杨茜, 刘晓, 张晓帆, 卢虎杰, 张彦军

国家纳米技术与工程研究院，天津300457； 天津国家生物防护装备工程技术研究中心，天津300161

摘要

细胞膜表面精细结构中的离子通道具有重要的生理功能。为了克服目前利用光学显微镜进行微电极定位的传统膜片钳技术分辨率的不足，本实验室将扫描离子电导显微镜技术(scanning ion conductance microscopy, SICM)与商用膜片钳技术相结合，构建了基于SICM 负反馈扫描控制技术的高分辨率膜片钳技术。我们首先运用SICM 负反馈技术控制纳米尺度玻璃微探针进行活体细胞表面的非接触扫描，获得细胞膜表面微结构的高分辨率成像，而后运用SICM 负反馈控制技术操控该微探针在细胞膜表面非接触地移动并将其精确定位于扫描成像中感兴趣的膜表面纳米尺度微结构上方，最后利用该微探针作为膜片钳记录电极实现对此微结构的高分辨率电生理信号记录。为了检验该技术实现高分辨率离子通道记录的能力，分别在活体单层膜犬肾上皮(MDCK)细胞膜的微绒毛、细胞间的紧密连接等纳米尺度微结构上进行了细胞贴附式离子通道记录，结果显示MDCK 细胞膜微绒毛的离子通道在钳制电压(pipette holding potential)为-100、-60、-40、0、+40、+60、+100 mV 条件下处于开放状态，而MDCK 细胞间的紧密连接处在钳制电压为-100、-40、0、+40、+100 mV 条件下未检出有离子通道开放动作。结果提示，我们构建的高分辨率膜片钳技术实现了微探针的准确定位及特定纳米尺度微结构上的高分辨率膜片钳记录，为活体生物样品表面离子通道的空间分布及其功能研究提供了一种有效的工具。

关键词： 高分辨率膜片钳; 扫描离子电导显微镜; 离子通道; 犬肾上皮细胞

分类号：Q25

[High-resolution patch-clamp technique based on feedback control of scanning ion conductance microscopy.] [Ariticle in Chinese]

YANG Xi, LIU Xiao, ZHANG Xiao-Fan, LU Hu-Jie, ZHANG Yan-Jun

China National Academy of Nanotechnology and Engineering, Tianjin 300457, China； Tianjin National Bio-Protection Engineering Center, Tianjin 300161, China

Abstract

The ion channels located on the cell fine structures play an important role in the physiological functions of cell membrane.However, it is impossible to achieve precise positioning on the nanometer scale cellular microstructures by conventional patch-clamptechnique, due to the 200 nm resolution limit of optical microscope. To solve this problem, we have established a high-resolution patchclamptechnique, which combined commercial scanning ion conductance microscopy (SICM) and patch-clamp recording through ananopipette probe, based on SICM feedback control. MDCK cells were used as observation object to test the capability of thetechnique. Firstly, a feedback controlled SICM nanopipette (~150 MΩ) non-contactly scanned over a selected area of living MDCKcells monolayer to obtain high-resolution topographic images of microvilli and tight-junction microstructures on the MDCK cellsmonolayer. Secondly, the same nanopipette was non-contactly moved and precisely positioned over the microvilli or tight-junctionmicrostructure under SICM feedback control. Finally, the SICM feedback control was switched off, the nanopipette slowly contactedwith the cell membrane to get a patch-clamp giga-ohm sealing in the cell-attached patch-clamp configuration, and then performed ionchannel recording as a normal patch-clamp electrode. The ion channel recordings showed that ion channels of microvilli microstructure opened at pipette holding potential of -100, -60, -40, 0, +40, +60, +100 mV (n=11). However, the opening of ion channels of tightjunctionmicrostructure was not detected at pipette holding potential of -100, -40, 0, +40, +100 mV (n=9). These results suggest thatour high-resolution patch-clamp technique can achieve accurate nanopipette positioning and nanometer scale high-resolution patchclamprecording, which may provide a powerful tool to study the spatial distribution and functions of ion channel in the nanometerscale microstructures of living biological samples.

Key words: high-resolution patch-clamp; scanning ion conductance microscopy; ion channel; MDCK cells

收稿日期：2010-02-03　　录用日期：2010-05-07

通讯作者：张彦军　　E-mail: yanjun_zhang@cnane.com.cn

引用本文：

杨茜, 刘晓, 张晓帆, 卢虎杰, 张彦军. 基于扫描离子电导显微镜负反馈扫描控制技术的高分辨率膜片钳技术[J]. 生理学报 2010; 62 (3): 275-283.

YANG Xi, LIU Xiao, ZHANG Xiao-Fan, LU Hu-Jie, ZHANG Yan-Jun. [High-resolution patch-clamp technique based on feedback control of scanning ion conductance microscopy.] [Ariticle in Chinese] . Acta Physiol Sin 2010; 62 (3): 275-283 (in Chinese with English abstract).