A new oil-gap method for internal perfusion and voltage clamp of single cardiac cells

(1.) We designed a new technique to achieve fast voltage clamp, combined with internal perfusion. The single guinea-pig cardiac cell, dissociated by collagenase treatment, was stretched across an oil-gap (30–40 m wide) from a pool of Tyrode solution to a pool of internal solution. Part of the cell membrane was disrupted in the internal solution by crushing on the cell, a tapered tip of a glass capillary. Through the open end, the intracellular medium was equilibrated with test solutions and electrical current was injected for the voltage clamp of the membrane in the Tyrode pool. (2.) The capacitive transient on stepping the membrane potential decayed with a time constant of 10–60 s, depending on the capacitive area (20–80 pF). The time course was a single exponential in 46% of the atrial cells and in 66% of the ventricular cells. In these tissues the series resistance, approximated by a ratio of the time constant andC_m, was 686±180 k (n=37) in the ventricular cells or 812±143 k (n=18) in the atrial cells. The stable seal resistance (R_seal) established in the oil-gap was around 33 M in the ventricular cells and 100 M in the atrial cells. (3.) A rapid increase in the inward current followed by a slow decay was observed on repolarization over the range negative to the potassium equilibrium potential. From the inward rectification of both peak and late currents and suppressive effects of Cs⁺ on the current, the current changes were atrributed to activation and inactivation of the inward rectifier K channel. (4.) The Na current was activated by depolarization from a holding potential of –100 mV across a threshold of about –60 mV. In normal external (145 mM Na⁺) and internal (15 mM Na⁺) solutions, peak amplitude was obtained around –25 mV. The maximum chord conductance was 6.2±1.6 mS/F in 15 ventricular cells and 3.0±0.90 mS/F in 9 atrial cells in normal Tyrode solution. The process of inactivation was fitted with a sum of two exponential functions. (5.) The reversal potential of the Na current agreed well with that predicted from the Nernst equation during perfusion of 15 and 100 mM Na⁺ internal solutions in the presence of external 140 mM Na⁺. The shift of the reversal potential was completed within 30 s of switching the internal solution. (6.) This oil-gap voltage clamp technique facilitates control of the composition of both the intra- and extra-cellular media and should prove suitable for use in studies of intracellular mechanisms controlling the membrane current of enzymatically dissociated elongated cells.