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.

 

STANDARD CONDITIONS FOR THE MEASUREMENT OF JUNCTIONAL CONDUCTANCE IN VENTRICULAR MYOCYTES CELL PAIRS

 
 

Junctional conductance was measured under whole-cell voltage-clamp conditions. Cells were continuously superfused with bath solution at 35 - 37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 ࠰C until the day of use.

 

STANDARD PROTOCOL FOR THE MEASUREMENT OF JUNCTIONAL CONDUCTANCE IN VENTRICULAR MYOCYTES CELL PAIRS

Ventricular myocyte cell pairs are continuously perfused with PSS at 35-37unctional 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.

Figure 2  Current-voltage relationship of junctional conductance measured in isolated ventricular myocyte cell pairs.

Figure 1 shows the linear current-voltage relationship for the junctional current measured in the cell pair above. A slope conductance of 52 nS was calculated for these data.

References

Effects of Antiarrhythmic Agents on Junctional Resistance of Guinea Pig Ventricular Cell Pairs
PASCAL DALEAU JPET 284:1174᷹, 1998

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