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[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

Received: 2010-02-03　　Accepted: 2010-05-07

Corresponding author: 张彦军　　E-mail: yanjun_zhang@cnane.com.cn

Citing This Article：

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).