Analysis of subthreshold pace-maker currents in chick embryonic heart cells.

1. Small re-aggregates of cells dissociated from the ventricles of 7-day-old chick embryonic hearts beat spontaneously in low external potassium concentration (Ko = 1.3 mM) tissue culture medium. This activity was blocked by the addition of tetrodotoxin (TTX) or potassium ions to the external medium. 2. A two-micro-electrode voltage-clamp technique was used to analyse the subthreshold currents responsible for the pace-maker depolarization. 3. Voltage-clamp steps 6-10 sec in duration revealed a time-dependent current having first order kinetics. Its membrane potential range of steady-state activation was -90 to -60 mV. 4. The current kinetics were qualitatively similar to those of Hodgkin & Huxley (1952b) with a peak time constant of approximately 1 sec at V = -75 mV. The kinetics were independent of Ko. 5. The time-dependent current was attributed to gated membrane channels. The fully activated current-voltage (I-V) relation of the channels was determined from the ratio of the amplitudes of the time-dependent currents during and after voltage-clamp steps following the procedure of Noble & Tsien (1968). 6. The fully activated I-V relation displayed inward rectification with negative slope conductance at potentials more than 15 mV positive to its reversal potential. Changes of Ko shifted the I-V curve along the voltage axis like a potassium electrode. 7. The time-independent (background) current was obtained by subtracting the gated channel current from the steady-state I-V curve. This current also rectified in the inward direction. 8. The inwardly rectifying I-V relations were theoretically described by a channel having a row of ion-selective sites along which ions move in a single file (Hodgkin & Keynes, 1955), and a membrane-bound particle which blocked the channel in a voltage-dependent manner. 9. The relationship of the voltage-clamp results to spontaneous activity is discussed and comparisons are made with measurements from whole embryonic heart and other cardiac tissues.