ACTIVATION MECHANISM OF CA2+-SENSITIVE TRANSIENT OUTWARD CURRENT IN RABBIT VENTRICULAR MYOCYTES

1. The mechanism of activation of the Ca2+-sensitive and 4-aminopyridine (4-AP)-insensitive transient outward current, I-to(Ca) was examined in single rabbit ventricular myocytes using the whole-cell patch-clamp technique. 2. When the steady-state intracellular Ca2+ (Ca-i(2+)) concentration ([Ca2+](i)) was < 1 nM, I-to(Ca) could not be activated by applying pulses at 0.1 Hz. When [Ca2+](i) was increased to greater than or equal to 10 nM, I-to(Ca) was activated by 0.1 Hz depolarizing pulses in all control experiments. 3. I-to(Ca) was completely blocked by an anion transport blocker, DIDS, or by replacement of NaCl with sodium aspartate. Upon changing extracellular [Cl-], the reversal potential was shifted as predicted for a chloride-selective conductance. When intracellular K+ was replaced with Cs+, I-to(Ca) was also observed. From these results it was concluded that I-to(Ca) was carried by Cl-. 4. Anion selectivity of I-to(Ca) was investigated by the replacement of Cl- with various anions. The sequence of permeability was SCN- > I- > Br- > Cl-. 5. The amplitude of I-to(Ca) was enhanced in a [Ca2+](i)-dependent manner between 10 nM and 1 mu M Ca-i(2+) while steady-state inactivation curves and the voltage-dependent activation curves were unchanged. The half-inactivation and half-activation potentials were -35 mV and +37 mV, respectively, at all [Ca2+](i). 6. I-to(Ca) was inhibited by blocking Ca2+ influx or Ca2+ release from sarcoplasmic reticulum, suggesting that a 'Ca2+-induced Ca2+-release' mechanism is essential for the activation of I-to(Ca). 7. A steady-state Ca2+-activated Cl- current with a linear I-V relationship was observed at 1 mu M Ca2+ while the current activated by depolarization was strictly dependent on Ca2+ entry or Ca2+ release from the sarcoplasmic reticulum. These results suggest that the I-to(Ca)channel is purely ligand (Ca2+) gated and its time course reflects the concentration of Ca-i(2+).