ION CHANNEL ASSAYS

SINGLE VENTRICULAR MYOCYTES

CALCIUM CURRENT (I_Ca) MEASUREMENTS

Figure 1  The L-type calcium current ( I_Ca) 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  I_Ca in single left ventricular myocytes isolated from guinea pig.

STANDARD CONDITIONS FOR THE MEASUREMENT OF L-TYPE CALCIUM CURRENT IN SINGLE VENTRICULAR MYOCYTES

Using the whole-cell patch-clamp technique

Calcium current is measured under whole-cell patch-clamp conditions.
Cells are continuously superfused with bath solution at room temperature. The conventional whole-cell patch-clamp configuration was used to record membrane currents.

Composition of the bath solution

The composition of the bath solution is (mM): NaCl 125; NaHCO₃ 25; KCl 5.4; CaCl₂ 1.8; MgCl₂ 1.0; NaH₂PO₄ 1.2; D-glucose 5.5; CsCl 5; pH 7.4 when bubbled with 95% CO₂ and 5% O₂ mixture.

Composition of the pipette solution

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. The composition of the pipette solution is (mM): CsCl 120; TEACl 20; MgCl₂ 5.5; ethylene glycol-bis(ß­aminoethyl ether)-N,N,Nî’¼/i>-tetraacetic acid (EGTA) 5; ATP-_Na2 5, HEPES 20; phosphocreatine 5 pH 7.2 with 1M CsOH.

STANDARD PROTOCOL FOR THE MEASUREMENT OF I_CA IN SINGLE VENTRICULAR MYOCYTES

The voltage protocol for eliciting I_Ca is as follows:
membrane voltage is stepped from -40 mV to 0 mV for 400 ms, and then stepped back to the holding potential of -40 mV.  The voltage protocol is run at 5 s intervals (0.2 Hz stimulation frequency). After establishment of the whole-cell voltage-clamp configuration and before recordings are started, the cell is allowed to recover and I_Ca to stabilise.
The equilibration period usually lasts for about 5 minutes (a minimum of 3 minutes).
A control period of 3 minutes is then recorded while the cells are being superfused with normal bath solution after which there is a switch to perfusion of the test substance.  The test substance is then perfused through the bath for a pre-determined time (e.g. 10 minutes) or until the observed effects on I_Ca have reached a steady-state.

Calcium current amplitudes are subsequently measured offline.  Peak current amplitude (I_Ca) is measured relative to the holding current at à ­V.