DELAYED RECTIFIER CURRENT ASSAYS

hERG expressed in HEK293 cells

MEASUREMENT OF hERG TAIL CURRENTS IN HEK293 CELLS
UNDER WHOLE-CELL PATCH-CLAMP CONDITIONS


To study the effects of new chemical entities on IKr we use a stably transfected HEK 293 cell line expressing high levels of functional human ether-a go-go-related gene (hERG).  The hERG gene encodes a K+ channel with many characteristics of IKr. HERG has been shown to be a target for block by many drugs that cause QT prolongation and give rise to arrhythmias.

 


STANDARD CONDITIONS FOR THE MEASUREMENT OF MEMBRANE CURRENTS IN HEK293 CELLS

USING THE WHOLE-CELL PATCH-CLAMP TECHNIQUE

The conventional whole-cell patch-clamp configuration is used to record membrane currents (e.g. the calcium current or the delayed rectifier current) in single cells.    Membrane currents are measured using an AxoPatch 200B preamplifier in conjunction with a CV 203BU headstage at a recording frequency of 1 kHz.  Data acquisition and analysis is controlled using Clampex 8.2 and Clampfit 9.0 (Axon pClamp) software respectively.  Cells are continuously superfused with bath solution and measurements are made at room temperature.

Composition of the bath solution

The composition of the bath solution was (mM): NaCl 125; NaHCO3 25; KCl 5.4; CaCl2 1.8; MgCl2 1.0; NaH2PO4 1.2; D-glucose 5.5;  pH 7.4 when bubbled with 95% CO2 and 5% O2 mixture. 

Composition of the pipette solution

The pipette solutions are prepared in one batch prior to the start of the experimental phase of any study and stored in 0.5 mL aliquots at approximately ࠰C until the day of use. The composition of the pipette solution was (mM): 130 KCl, 1 MgCl2, 5 EGTA, 5 MgATP, 10 HEPES (pH 7.2 with KOH). When filled with this pipette solution electrode resistances are typically between 2ͼfont face="Symbol" size="2">W .

 

 

Figure 1  hERG tail current (IKr ) record in stably transfected HEK293 cell

 
   
 

Figure 1  shows typical data recorded by scientists at OCP. Membrane current was recorded under our standard conditions for the measurement of IKr in stably transfected HEK293 cells.

 


STANDARD PROTOCOL FOR THE MEASUREMENT OF NATIVE IK IN SINGLE VENTRICULAR MYOCYTES

The standard protocol for recording hERG tail currents is illustrated in Figure 1.
The cell was clamped at a holding potential of -80 mV. A step depolarized to +30 mV for 4 s is used to activate the hERG channel. On step repolarisation back to a potential of -50 mV the current flow through the hERG channel is recorded as a tail current. The amplitude of the tail current reflects the activity of the hERG channel and is measured as the difference between the peak of the current (measured just after repolarisation) and the steady-state current measured at -50 mV. hERG tail currents were elicited once every 15 seconds.

 

 
 

Figure 2   Stability of hERG tail current amplitude in the presence of 0.1% DMSO

 
 

Figure 2 shows mean data (mean 㥭) for the protocol used to elicit hERG tail currents in HEH293 cells.  The phenomenon of "rundown" of the amplitude of the membrane current can be a potential problem in a number of experimental preparations including cell lines expressing the hERG channel protein.  This can often lead to potential problems. However, these data show that the amplitude of the hERG tail current remains constant over this period of time.   After a 3 minute control recording period the cells are perfused with a solution containing 0.1% dmso (a widely used vehicle solution) which is then perfused for a further 13 minutes.  These data show that the vehicle solution did not greatly affect the amplitude of the hERG tail current.  In general, drug affects will have reached a plateau within 10 mins of application.  This can be demonstrated as compounds that block hERG tail current amplitude such as dofetilide and E-4031.

Using this protocol, we assess the effects of only one concentration of your test compound on hERG tail current in order to be sure of the integrity of the data.

 

 
   
  All data 㯰yright to OCP Ltd. 2006. All Rights Reserved. Design by OCP