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ION CHANNEL ASSAYS
VENTRICULAR MYOCYTE CELL PAIRS
JUNCTIONAL RESISTANCE MEASURED IN VENTRICULAR MYOCYTE CELL PAIRS
Figure 1
Junctional
conductance measured in isolated ventricular myocyte cell pairs.
Figure 1
shows typical data recorded by scientists at
OCP. Membrane current was
recorded under our standard conditions to investigate the
effects of the test substances on electrical
coupling through gap junctions measured by recording junctional
conductance in isolated ventricular myocyte cell pairs.
Top panel shows the voltage
protocol used to elicit junctional currents. The membrane
voltage of cell 1 is stepped from a holding potential of 0 mV to
test voltages ranging between -50 and +50 mV (10 mV increments,
200 ms duration).
Middle panel shows membrane
current recorded in cell 1.
Bottom panel shows membrane
current recorded in cell 2 where the membrane potential is kept
at 0 mV. The amplitude of the junctional current is measured
within the first 100 ms of the voltage step with respect to the
current measured just prior to the step at 0 mV.
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Junctional conductance was measured under whole-cell voltage-clamp
conditions. Cells were continuously superfused with bath solution
at 35 - 37î ”he conventional whole-cell patch-clamp
configuration was used to record membrane currents.
The composition of the bath solution was (mM):
NaCl 132, KCl 4.8, MgCl2 1.2, CaCl2 1.8, HEPES 10 and Glucose 5.
Blockers of non junctional membrane conductance were added to the
bath solution (BaCl2 0.5 mM to minimise the inward rectifier K+
current and and CdCl2 0.1 mM to minimise the inward Ca2+ current;
see Delau, 1998).
The composition of the pipette solution was (mM): KCl 120, NaCl 10,
MgCl2 3, EGTA 5, Mg2-ATP 5, HEPES 10, CsCl 20 and TEA-Cl 5 (the last
two are added to minimise the delayed rectifier K+ current, see
Delau, 1998), pH 7.2.
The pipette solution is prepared in one batch prior to the
start of the experimental phase of the study and is stored in 0.5 mL
aliquots at approximately à °C until the day of use.
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Ventricular myocyte cell pairs are continuously perfused with PSS at 35-37î Šunctional conductance
is measured using the double whole-cell patch-clamp technique
using two Axopatch 200B pre-amplifiers and two CV 203BU head stages
(Axon Instruments Inc).
When whole-cell patch-clamp is achieved on
each cell of a cell pair (using separate electrodes) the double
whole-cell configuration is achieved. In this configuration each
cell of the pair is independently maintained at a given membrane
potential which can be independently varied, but both electronic
systems are linked by the intercellular conductance and thus current
flow through the gap junctions can be measured. To elicit and thus
measure current flow through he gap junctions (Ij) both cells will
be clamped at the same holding potential (0 mV), then a voltage step
(-50 mV) will be applied to one cell thereby creating a voltage
gradient (Vj = V1 沩. Current flow can be measured in each cell
but the current flow in the cell to which the voltage step is not
applied corresponds to the current flowing through the gap junctions
(junctional current flow Ij).
Junctional conductance is calculated by Ij / Vj.
To obtain a current voltage relationship
voltage steps are applied between -50 and +50 mV in 10 mV
incremental steps (see below) at a frequency of 0.5 Hz.
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