Voltage clamp and internal perfusion of single rat heart muscle cells

1. Single cells from adult rat ventricle were dispersed using an enzymic dissociation technique. Electrical properties were measured with either suction pipettes or conventional glass micropipettes and the results were compared. 2. Suction pipette and micropipette measurements of resting membrane potentials and action potentials were comparable. Values were similar to those reported previously for both dispersed cardiac myocytes and whole tissue preparations from adult rat ventricle. 3. Voltage clamp with a single suction pipette was used in initial experiments, but the results were not sufficiently accurate. Consequently, voltage clamp of single cells was carried out using two suction pipettes (tip diameters 10-15 micrometers), one for passing current and the other for recording membrane potential. Dialysis of cell contents was performed by each suction pipette. A roving micropipette (tip diameter less than 1 micrometer) was used occasionally to measure membrane potential at selected sites. 4. Using the two-suction-pipette method, voltage-clamp steps rose with time constants of less than 10 microsec and the capacitative current transient decayed with a single time constant of less than 100 microsec. These values are more optimal than those observed in other voltage-clamped cardiac muscle preparations. 5. Single cardiac myocytes had membrane input resistances of 44.5 +/- 4.6 M omega, membrane time constants of 16.2 +/- 0.63 msec and membrane capacitances of 399.7 +/- 42.2 pF. (values are means +/- DS, n = 10-). The length constant, lambda, of a single myocte at ists resting potential and perfused with KH2PO4 was about 500 micrometers. For cells perfused with Cs aspartate solution commonly used in voltage-clamp experiments, the input resistance was approximately quadrupled and lambda was 1100 micrometers. The average length of a myocyte partially aspirated by two suction pipettes was 50 micrometers. At maximum -gNa in 50% extracellular Na, lambda was calculated to be about four times the actual cell length between voltage-recording and current-passing pipettes. 6. The half-time for the disappearance or recovery of outward Na currents, following removal or restitution of intracellular Na with two pipettes, was of the order 1 min, indicating that intracellular ionic composition of the cell could be readily controlled and modified.