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DELAYED
RECTIFIER CURRENT ASSAYS
SINGLE VENTRICULAR MYOCYTES
MEASUREMENT AND DIFFERENTIATION OF BOTH COMPONENTS OF IK (IKr & IKs)
IN VENTRICULAR MYOCYTES
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. Both IKr and IKs can influence the timing of repolarisation. At OCP we can investigate the effects of compounds on both IKr and IKs
measured simultaneously in the same cell.
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STANDARD CONDITIONS FOR THE MEASUREMENT OF NATIVE IK 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.
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Figure 1
The delayed rectifier current ( IK )
record in a single ventricular myocyte
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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.
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STANDARD PROTOCOL FOR THE MEASUREMENT OF NATIVE IK IN SINGLE
VENTRICULAR MYOCYTES
Total IK
recorded in ventricular myocytes comprises a rapidly activating IKr
which corresponds to the hERG channel current in humans, and a
slowly activating IKs. 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 IK.
The peak amplitude of IK are measured
with respect to the holding current prior to the step
depolarisation.
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Figure 2
IK tail current protocol.
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Figure 2
shows mean data for the protocol used to
elicit native IK in single ventricular
myocytes. These data show that the tail current (IK)
amplitude increases as pulse duration lengthens. The benefit of
using the above protocol is that the rapidly activating IKr is
exclusively activated at the shorter pulse durations (e.g. 40 ms)
whilst the more slowly activating IKs will
predominate at the longer pulse durations (e.g. 400 ms) though IKr
will also contribute to this current. This
can be demonstrated as compounds that block IKr
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 IKs 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 IKs.
Using this protocol therefore, we can assess the effects of your test compounds on both IKr
and IKs quickly and accurately.
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