DELAYED RECTIFIER CURRENT ASSAYS

SINGLE VENTRICULAR MYOCYTES

MEASUREMENT OF IKr IN VENTRICULAR MYOCYTES UNDER WHOLE-CELL PATCH-CLAMP CONDITIONS

 

In ventricular myocytes the delayed rectifier current IK consists of two components the rapidly activating component IKr, which corresponds to the hERG current in humans, and a more slowly activating component IKs.

Using the conventional whole-cell patch-clamp technique and the appropriate experimental conditions it is possible to record IKr in the native cardiac environment in isolation from other overlapping currents.

At OCP we can investigate the effects of compounds selectively on IKr by using the whole-cell patch-clamp technique and the appropriate conditions.


STANDARD CONDITIONS FOR THE MEASUREMENT OF MEMBRANE CURRENTS IN SINGLE VENTRICULAR MYOCYTES

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 all measurements are made at 36

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  IK tail current (IKr ) record in a single ventricular myocyte

 
   
 

Figure 1  shows typical data recorded by scientists at OCP. Membrane current was recorded under our standard conditions for the measurement of native IK in single left ventricular myocytes isolated from guinea pig.

 


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

The standard protocol for recording IKr is illustrated in Figure 1.
IKr is elicited by a brief (40 mS) step depolarisation to +40 mV from a holding potential of -40 mV. The outward tail current elicited on repolarisation back to the holding potential (-40 mV) accurately reflects activation of potassium channels.  Step depolarisations are at a frequency of 0.1 Hz (i.e. 10 sec between each depolarisation) to allow for recovery of IK.
The peak amplitude of IKr is measured with respect to the holding current prior to the step depolarisation.

 
 

Figure 2   Stability of IK tail current amplitude.

 
 

Figure 2 shows mean data for the protocol used to elicit native IKr in single ventricular myocytes.  In a number of experimental preparations (such a cell lines expressing hERG channel protein) "rundown" of the amplitude of the membrane current can often lead to potential problems. However, these data show that the amplitude of the IKr tail current remains constant over this period of time. In general, drug affects will have reached a plateau within 10 mins of application.  This can be demonstrated as compounds that block  IKr such as dofetilide and E-4031.
Using this protocol, we assess the effects of only one concentration of your test compound on IKr 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