Voltage clamp of bullfrog rana catesbeiana cardiac pace maker cells a quantitative analysis of potassium currents
Giles, W.R.; Shibata, E.F.
Journal of Physiology (Cambridge) 368: 265-292
ISSN/ISBN: 0022-3751 Accession: 006911163
1. Spontaneously active single cells have been obtained from the sinus venosus region of the bull-frog, Rana catesbeiana, using an enzymic dispersion procedure involving serial applications of trypsin, collagenase and elastase in nominally 0 Ca2+ Ringer solution. These cells have normal action potentials and fire spontaneously at a rate very similar to the intact sinus venosus. 2. A single suction micro-electrode technique (Hamill, Marty, Neher, Sakmann and Sigworth, 1981; Hume and Giles, 1983) has been used to record the spontaneous diastolic depolarizations or pace-maker activity as well as the regenerative action potentials in these cells. This electrophysiological activity is completely insensitive to tetrodotoxin (TTX;3 .times. 10-6 M) and is very similar to that recorded from an in vitro sinus venosus preparation. The present experiments were aimed at identifying the transmembrane potassium currents, and analysing their role(s) in the development of the pace-maker potential and the repolarization of the action potential. 3. Depolarizing voltage-clamp steps from the normal maximum diastolic potential (-75 mV) elicit a time- and voltage-dependent activation of an outward current. The reversal potential of this current in normal Ringer solution ([K+]o 2.5 mM) is near -95 mV; and it shifts by 51 mV per tenfold increase in [K+]o, which strongly suggests that this current is carried by K+. We therefore labelled it IK. 4. The reversal potential of IK did not shift in the positive direction following very long (20 s) depolarizing clamp steps to +20 mV, indicating that 'extracellular' accumulation of [K+]o does not produce any significant artifacts. 5. The fully activated instantaneous current-voltage (I-V) relationship for IK is approximately linear over the range of potentials -130 to -30 mV. Thus, the ion transfer mechanism of IK may be described as a simple ohmic conductance in this range of potentials. Positive relative to -30 mV, however, the I-V exhibits significant inward rectification. 6. A Hodgkin-Huxley analysis of the kinetics of IK, including a demonstration that the envelope of tails quantitatively matches the time course of the onset of IK during a prolonged depolarizing clamp step has been completed. 7. The steady-state activation variable (n.infin.) of IK spans the voltage range approximately -40 to +10 mV. It is well-fitted by a Boltzmann distribution function with half-activation at -20 mV. 8. The time course of decay of IK is a single exponential. However, the activation or onset of IK shows clear sigmoidicity in the range of potentials from the activation threshold (-40 mV) to 0 mV. This sigmoidicity may be accounted for by raising the activation variable, n, to the second power. 9. The kinetics of activation and deactivation of IK are strongly modulated by potentials negative to -60 mV, but are only weak functions of potential at more positive voltages. These results strongly suggest that IK is important in triggering repolarization and in controlling the rate of development of the first part of the pace-maker potential. 10. In a second series of experiments, the background K+ current in sinus venosus cells was compared and contrasted with that in adjacent atrial myocytes. 11. These data show that in single cells isolated from the atrium a conventional inwardly rectifying background current exists. The size of this current is very sensitive to changes in [K+]o, it is inhibited by relatively low doses of BaCl2 (50 .mu.M) and it exhibits very marked inward-going rectification. It therefore is very similar to IK1, in other cardiac preparations. In contrast, similar experiments in sinus venosus cells from the same animal show conclusively that there is no detectable inwardly rectifying background K+ current in this cardiac pace-maker cell type. 12. These findings are discussed in relation to three physiologically important phenomena: (1) the mechanism of repolarization of the action potential in cardiac pace-maker tissue; (2) the ionic basis of the development of the spontaneous diastolic depolarizat.