HEK293细胞中的钠电流测定

Helena  Riuró; Anna  Tarradas; Elisabet  Selga; Ramon  Brugada; Fabiana  Scornik; Guillermo  Pérez

doi:10.21769/BioProtoc.858

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Peer-reviewed

Sodium Current Measurements in HEK293 Cells

HEK293细胞中的钠电流测定

Fabiana Scornik email

GP Guillermo Pérez email

发布: 2013年08月20日第3卷第16期 DOI: 10.21769/BioProtoc.858 浏览次数: 13603

评审: Anonymous reviewer(s)

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Cover of PLOS ONE, featuring study using the protocol.

Jan 2013

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Abstract

This protocol is used to measure sodium currents from heterologously transfected cells lines, such as HEK293 cells. Standard whole cell patch clamp technique is used to assess ion channel function. This protocol has been used to study cardiac type Na_v1.5 sodium channel, and in particular to compare wild-type and mutant channels related to Brugada Syndrome (BrS). Mutations related to BrS provoke a loss of function of the cardiac sodium channel. This is evidenced by a reduction of sodium current density and/or alterations of the activation or inactivation kinetic parameters, such as a slow recovery from inactivation. These effects could explain the alteration of the cardiac action potential that leads to the characteristic ST elevation observed in the electrocardiogram of the Brugada Syndrome patients. This protocol, with some variations, is suitable for studying other cardiac arrhythmias related to alterations in the cardiac type sodium channel function as well as for studying other voltage-dependent sodium channels.

Keywords: Sodium current (钠电流)

Patch clamp (膜片钳)

Heterologous transfection (外源转染)

Ion channels (离子通道)

Whole cell current (全细胞电流)

Materials and Reagents

Human embryonic kidney HEK293 cells (Health Protection Agency Culture Collections, catalog number: 96121229 )
Vector/s: mammalian expression vectors harboring the cDNA of interest (i.e. pcDNA3 + SCN5A)
Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma-Aldrich, catalog number: D6546 )
Fetal Bovine Serum (Sigma-Aldrich, catalog number: F4135 )
Penicillin-streptavidin (Life Technologies, Gibco^®, catalog number: 15140-122 )
GlutaMAX^TM (Life Technologies, Gibco^®, catalog number: 35050-038 )
0.05% Trypsin-EDTA (Life Technologies, Gibco^®, catalog number: 25300-062 )
Dulbecco’s Phosphate Buffered Saline (Sigma-Aldrich, catalog number: D8662 )
GeneCellin^TMTransfection Reagent (BioCellChallenge, catalog number: GC-1000 )
Opti-MEM^® Reduced Serum Media + GlutaMAX^TM (Life Technologies, Gibco^®, catalog number: 31985-062 )
Sticky wax in bars (Cera de Reus, catalog number: 25005001 )
Sodium chloride (NaCl) (Sigma-Aldrich)
Potassium chloride (KCl) (Sigma-Aldrich)
Cesium chloride (CsCl) (Sigma-Aldrich)
Calcium chloride (CaCl₂) (Sigma-Aldrich)
Magnesium chloride (MgCl₂) (Sigma-Aldrich)
N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) (Sigma-Aldrich)
Ethylene glycol-bis (2-amino-ethylether)-N,N,N’,N’-tetra-acetic acid (EGTA) (Sigma-Aldrich)
Adenosine 5′-triphosphate magnesium salt (ATP-Mg²⁺) (Sigma-Aldrich)
Sodium hydroxide (NaOH) (Sigma-Aldrich)
Cesium hydroxide (CsOH) (Sigma-Aldrich)
Glucose (Sigma-Aldrich)
Bath solution (see Recipes)
Pipette solution (see Recipes)

Equipment

Inverted microscope Nikon Eclipse Ti fitted for epifluorescence (Nikon Instruments Inc.)
Glass capillary PC-10 Puller (Narishige International USA Inc.)
Micromanipulator MP-285 (Sutter Instrument Co.)
Vapor pressure Osmometer VAPOR 5520 (Wescor Inc.)
Axopatch 200B Capacitor Feedback Patch Clamp Amplifier (Molecular Devices)
Axon Digidata 1440A Data Acquisition System (Molecular Devices)
CO₂ incubator
Nunclon^® cell culture dishes 35 x 10 mm (Sigma-Aldrich, catalog number: D7804 )
Glass capillaries (Brand GmbH + CO KG, catalog number: 7493-21 )

Software

pCLAMP 10.2 Electrophysiology Data Acquisition and Analysis Software (Molecular Devices)
OriginPro 8 software (OriginLab Corporation)

Procedure

English

中文翻译

文章信息

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如何引用

Riuró, H., Tarradas, A., Selga, E., Brugada, R., Scornik, F. and Pérez, G. (2013). Sodium Current Measurements in HEK293 Cells. Bio-protocol 3(16): e858. DOI: 10.21769/BioProtoc.858.