DELAYED RECTIFIER CURRENT ASSAYS

SINGLE VENTRICULAR MYOCYTES

MEASUREMENT AND DIFFERENTIATION OF BOTH COMPONENTS OF I_K (I_Kr & I_Ks) IN VENTRICULAR MYOCYTES

In ventricular myocytes the delayed rectifier current I_K consists of two components the rapidly activating component I_Kr, which corresponds to the hERG current in humans, and a more slowly activating component I_Ks. Both I_Kr and I_Ks can influence the timing of repolarisation.
At OCP we can investigate the effects of compounds on both I_Kr and I_Ks measured simultaneously in the same cell.

STANDARD CONDITIONS FOR THE MEASUREMENT OF NATIVE I_K IN SINGLE VENTRICULAR MYOCYTES

Using the discontinuous single-electrode voltage-clamp technique

The conventional single electrode discontinuous switch-clamp configuration is used to record membrane currents (e.g. the calcium current or the delayed rectifier current) in single cells.    An Axoclamp 2B (Axon Instruments Inc) amplifier will be used for electrical recording and measurement of membrane current and voltage and will be used in discontinuous single-electrode voltage-clamp mode. in conjunction with a Digidata 1322A.  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 basic 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

Conventional glass microelectrodes will be used to measure membrane currents and are  filled with 1 M KMeSO₄ and 10 mM KCl.   Electrode resistances are in the range of 40 to 60 MW.  The switching frequency of the electrode (routinely between 2-4 kHz) is continuously monitored on an oscilloscope so that optimal voltage-clamp conditions can be achieved. 

Figure 1 The delayed rectifier current ( I_K ) 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 I_K in single left ventricular myocytes isolated from guinea pig.

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

Total I_K recorded in ventricular myocytes comprises a rapidly activating I_Kr which corresponds to the hERG channel current in humans, and a slowly activating I_Ks.  The standard tail current protocol for recording these currents in ventricular myocytes is illustrated in Figure 1 which shows a superimposed á­©lies of current⥣ords elicited by a series of step depolarisations (to +40 mV) of progressively increasing duration (from 10 to 600 ms). Outward tail currents elicited on repolarisation to the holding potential (-40 mV) accurately reflect 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 I_K.
The peak amplitude of I_K are measured with respect to the holding current prior to the step depolarisation.

Figure 2   I_K tail current protocol.

Figure 2 shows mean data for the protocol used to elicit native I_K in single ventricular myocytes.  These data show that the tail current (I_K) amplitude increases as pulse duration lengthens. The benefit of using the above protocol is that the rapidly activating I_Kr is exclusively activated at the shorter pulse durations (e.g. 40 ms) whilst the more slowly activating I_Ks will predominate at the longer pulse durations (e.g. 400 ms) though I_Kr will also contribute to this current. This can be demonstrated as compounds that block  I_Kr such as sotalol and dofetilide completely abolish tail currents elicited after short (40 ms) step depolarisations and only partially attenuate currents elicited after longer (400 ms) step depolarisations.
However, compounds which selectively block I_Ks do not to effect  tail currents elicited after short (40 ms) step depolarisations but do inhibit the increase in tail current amplitude elicited by increasing the duration of the step depolarisation that is associated with activation of I_Ks. 
Using this protocol therefore, we can assess the effects of your test compounds on both I_Kr and I_Ks quickly and accurately.