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Abstract
Introduction
Protocol
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신경과학

全细胞贴片记录用于电泳生理测定通道视紫质中的离子选择性

Published: May 22, 2017
doi:
10.3791/55497
, , ,
1Experimental Biophysics, Institute of Biology,Humboldt-Universität zu Berlin

Summary

This article describes how the ion selectivity of channelrhodopsin is determined with electrophysiological whole-cell patch-clamp recordings using HEK293 cells. Here, the experimental procedure for investigating chloride selectivity of an anion-selective channelrhodopsin is demonstrated. However, the procedure is transferable to other channelrhodopsins of distinct selectivity.

Abstract

在过去十年中，通道观光素在神经科学研究中成为不可或缺的，它们被用作非侵入性操纵靶细胞电工作的工具。在这方面，通道视紫红质的离子选择性是特别重要的。本文介绍了通过HEK293细胞上的电生理膜片钳记录研究最近鉴定的蛋白质组培养基的阴离子导电通道视紫红质的氯选择性。用于测量光门控光电流的实验程序需要一个快速可切换的 – 理想的单色光源，耦合到另外传统的膜片钳设置的显微镜中。概述实验前的制备方法涉及缓冲溶液的制备，对液体接头电位的考虑，接种和细胞转染以及拉片移植管。实际记录电流 – 电压关系以确定不同氯化物浓度的逆转电位在转染后24小时至48小时。最后，关于氯化物传导的理论考虑分析电生理数据。

Introduction

通道视紫红质（Chr）是发生在运动性绿藻眼睛中的轻门控离子通道，可作为光敏感和恐惧反应的主要感光元件1 。自2002年第一次描述以来，ChR为新兴的光遗传学领域铺平了道路，可以应用于各种兴奋细胞， 如骨骼肌，心脏或大脑3,4,5 。 ChRs在靶细胞中的表达导致相应细胞的光可控离子渗透性。在神经元上下文中，这允许激活6,7,8或抑制作用电位（AP）的9,10（取决于导电离子）与空间和时间光的精度强调了ChR变体的离子选择性如何决定其光遗传应用。

第一个发现的Chlamydomonas reinhardtii和Volvox carteri的ChRs对质子是可渗透的，而且也可以是一钠阳离子，如钠，钾，二来阳离子如钙和镁11,12,13 。今天，超过70种天然阳离子导电通道视紫红质（CCR）14,15,16,17和几种具有不同性质的工程变体18,19,20具有 光电流尺寸，光谱灵敏度，动力学和阳离子选择性。而在神经科学中，CCRs a重新用于激活细胞并触发AP，光驱微生物泵是用于沉默神经元多年的唯一可用的拮抗剂。 2014年，两组同时表明，CCR可以通过分子工程9,21改变推定离子导电孔的极性，将其转化为阴离子导电通道视紫红质（ACR）。随后，在几种密类藻22,23,24中鉴定了自然ACR。最重要的是，ACR的光激活介导成年神经元中的氯化物电流，允许在比仅吸收光子仅传输单个电荷的微生物泵更低的光强度下抑制神经元活性。

ChR活性可以通过HEK293细胞中光诱导电流的电生理膜片钳记录直接解决。膜片夹技术最初是在20世纪70年代末开发的，而且由Hamill 等人进一步改进。允许从高电流分辨率的小电池（全电池模式）记录电流实体，并直接控制膜电压26 。应用于细胞培养，这种技术提供了离子和电记录条件的精确控制，并且能够研究离子选择性以及离子对总电流的相对贡献。这里我们举例说明通过记录各种细胞外氯化物浓度下的电流 – 电压关系来检测蛋白质组学（ Ps ACR1） 22,23的阴离子导电通道视紫红质的离子选择性，以证明高氯化物电导。

Protocol

图1：跳线钳设置。 （1）光源，（2）光纤，（3）可编程快门，（4）数字化仪，（5）快门驱动器，（6）放大器，（7）灌注系统，（8）个人计算机，（9） ，（10）法拉第笼，（11）显微镜台，（12）灌注入口，（13）灌注出口，（14）记录室，（15）液位传感器，（16）带琼脂桥的浴电极，（17）移液器支架，?…

Representative Results

图2示出了根据所述方案从测量获得的代表性结果。在用绿光照明时， Ps ACR1具有快速衰减到稳定电流水平的快速瞬态电流。光线关闭后，光电流在几毫秒内就会衰减到零（ 图2A ）。交换细胞外氯离子浓度会导致在获得的光电流迹线中可以直接看到的反转电位的偏移。从多次测量的反转电位的评估量化了由外部氯化物浓度的变化引起的?…

Discussion

在确定的离子和电气条件下的反转电位的测定提供了关于在光激活ChR之后传输的离子物种的信息。如果在复杂的生理介质中仅排除一种离子种类，并且根据理论能斯特电位，获得的反转电势发生变化，则该离子种类是唯一传播的离子。

然而，对于ChRs，由于对不同离子物质的渗透性以及导电离子之间的竞争，反向电位偏移通常不如从恩斯特方程所预期的那么明显。在这种情况?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

我们感谢Maila Reh，Tharsana Tharmalingam，特别是Altina Klein，为您提供卓越的技术帮助。这项工作得到德国研究基金会（DFG）（SFB1078 B2，FOR1279 SPP1665至PH）和催化，UniCat，BIG-NSE（JV）和E4（PH）的卓越统一概念支持。

Materials

HEK293 cells Sigma Aldrich 85120602 Human embryonic kidney cells
Retinal Sigma Aldrich R2500 all-trans retinal
FuGENE HD Promega E2312 Transfection reagent
DMEM Biochrome FG 0445 Dulbecco's Modified Eagle Medium
Agarose Roth 3810 Agar bridges
CaCl2 Roth 5239 CaCl2 2H2O
CsCl Biomol 2452
EGTA Roth 3054
FBS Biochrome S0615 Cell culture
Glucose Roth HN06 D(+)-Glucose
KCl Roth 6781
MgCl2 Roth 2189 MgCl2 6H2O
NaCl Roth 3957
NMG Sigma Aldrich M2004 N-Methyl-D-glucamine
Na-Aspartate Sigma Aldrich A6683 L-Aspartic acid sodium salt monohydrate
Citric acid Roth 6490
AgeI ThermoFischerScientific ER1462 Restriction enzyme
XhoI ThermoFischerScientific ER0695 Restriction enzyme
NheI ThermoFischerScientific ER0975 Restriction enzyme
XL1Blue E.coli/ Agilent Technologies 200249 Chemocompetent E.coli
Kanamycin Roth T832
Lysogeny broth medium Roth X964
Agar-Agar Roth 6494 Agar plates
Plasmid purification kit Marchery-Nagel 740727.25
Penicilin/Streptomycin Biochrome A 2213 Cell culture
Poly-D-lysine hydrobromide Sigma Aldrich P6407-5MG Cover slip coating
Microforge Custom made Fire polishing
Serological pipettes TPP Different sizes
Clean bench Kojair Biowizard SL130
Stirrer IKA RCT classic
Silver wire Science Products AG-T25; AG-T10 Electrodes, 0.64 mm (bath); 0.25 mm (electrode)
pH-meter Knick 765 Calimetric
Osmometer Vogel OM 815
Microscope Carl Zeiss ID03 Fire polishing
CO2 incubator Binder CB150
Cell culture dishes TPP 93040 34 mm internal diameter
Cover slips Roth P232 15 mm diameter
Thermometer Rössel Messtechnik MTM12
Beamsplitter Chroma 21011 90/10 transmission
Pipette holder ALA Scientific Instruments PPH-1P-AXU-0-1.5
Headstage Molecular Devices CV203BU
Amplifier Molecular Devices AxoPatch200B
Digitizer Molecular Devices DigiData1400 Digital analog converter
Lightsource TILL Photonics Polychrome V Set to 540 nm full intensity
Microscope Carl Zeiss Axiovert 100
Shutter Vincent Associates VS25
Shutter driver Vincent Associates VCM-D1
Glass capilarries Warner Instruments G150F-3 Boresilicate capillaries with fire polished ends OD 1.5 mm ID  0.86 mm
Micropipette puller Sutter Instruments P1000
Bath handler Lorenz Messgerätebau MPCU
Tripleband filterset Chroma 69008 Fluorescence filter  ECFP/EYFP/mCherry 
CCD camera Watec Wat-221SCCD
Optometer Gigahertz Optik P9710 Measure light intensities
Objective Carl Zeiss 421462-9900-000 W Plan-Apochromat 40X/1.0 DIC
Micromanipulator Scientifica PatchStar
Recording chamber Custom made
Power supply Manson HCS-3202 Avoids electrical noise from microscope built-in power supply
Vibration isolated table Newport M-VW-3636-OPT-01
Faraday cage Custom made or any commercial matching table
Hoses Any comercial; e.g. Roth Different sizes and materials for bath handling and application of pipette pressure; agar bridges
Linear shaker Sunlab Instruments SU 1000
Liquid junction potential calculator Molecular Devices or directly from Peter H. Barry Program is included in the Clampex aquisition software or can be obtained from  p.barry@unsw.edu.au
Data acquisition software Molecular Devices Clampex 10.X
Data evaluation software Molecular Devices Clampfit 10.X
PsACR1 GenBank or Addgene KF992074.1 or Addgene plasmid #85465 Gene encoding for PsACR1
Amplifier guide Molecular Devices The Axon Guide
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Tags

Keywords: Whole-cell Patch-clampElectrophysiologyIon SelectivityChannelrhodopsinsHEK CellsTransfectionPhotocurrentBiophysicsIntracellular BufferExtracellular BufferOsmolarityPatch PipetteFire PolishingRecording Chamber
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Grimm, C., Vierock, J., Hegemann, P., Wietek, J. Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins. J. Vis. Exp. (123), e55497, doi:10.3791/55497 (2017).
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