Voltage clamp of bull-frog cardiac pace-maker cells: a quantitative analysis of potassium currents

Abstract

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. A single suction micro-electrode technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981; Hume & 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 X 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. 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+]0 2.5 mM) is near -95 mV; and it shifts by 51 mV per tenfold increase in [K+]0, which strongly suggests that this current is carried by K+. We therefore labelled it IK. 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+]0 does not produce any significant artifacts. 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. 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. The steady-state activation variable (n infinity) 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. 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.(ABSTRACT TRUNCATED AT 400 WORDS)