Two-suction-electrode voltage-clamp analysis of the sustained calcium current in cat sensory neurones

Abstract

1. The kinetics of the sustained calcium current were examined in cat dorsal root ganglion (DRG) neurones, using a two-suction-electrode voltage clamp. It was shown that this current could be examined with minimal contamination from other ionic currents. Experiments were performed at 20 degrees C, with a concentration of 10 mM-calcium externally. 2. The transient calcium current was eliminated by using a holding potential of -50 mV. The sustained calcium current showed no evidence of steady-state inactivation at potentials between -90 and -30 mV. 3. The activation and deactivation time course of the calcium current was described by a double-exponential function. The activation process was examined without interference from significant inactivation under the conditions used. 4. The steady-state activation of the calcium channels was approximated by a two-step activation process. Both reactions were voltage sensitive, the first having an equivalent valency of 3.4 +/- 0.1 electronic charges (e-), and the second with an equivalent valency of 1.0 +/- 0.2 e-. On average, half-maximal channel activation occurred at +10 mV. 5. The fast and the slow time constants of the exponential relaxations differed by a factor of 4-10 and both showed significant voltage dependence. Both the fast and slow time constants were greatest at potentials where approximately half the available channels were activated in the steady state. The slow time constant measured from activation and deactivation appeared to be independent of the starting potential. 6. The fractional amplitude of the slow exponential component of the tail currents was 0.09 +/- 0.01 at -70 mV and increased steadily at more positive potentials passing through a clear maximum of 0.59 +/- 0.03 at -10 mV. 7. Reducing the temperature decreased the magnitude of the peak inward current, with an apparent activation energy (Ea) of 67 kJ/mol. The slow time constants measured from activation and deactivation were also reduced at lower temperatures. The slow activation time constant had a higher temperature sensitivity (Ea = 85 kJ/mol) than the slow tail current time constant (Ea = 29 kJ/mol). The fast tail current time constant was reduced at lower temperatures with an apparent Ea of 79 kJ/mol. 8. Application of 10 microM-(+/-)Bay K 8644 prolonged the tail currents and shortened the activation time constant. The calcium current was activated at more negative potentials and the slope of the steady-state activation curve was reduced.(ABSTRACT TRUNCATED AT 400 WORDS)