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Department of Cardiovascular Diseases, Medical Research Institute, Tokyo Medical and Dental University, Japan.
1. The mechanism of activation of the Ca(2+)-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+ (Ca2+i) 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 > or = 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 C.- 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 microM Ca2+i, 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 'Ca(2+)-induced Ca(2+)-release' mechanism is essential for the activation of I(to)(Ca). 7. A steady-state Ca(2+)-activated Cl- current with a linear I-V relationship was observed at 1 microM 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 Ca2+i.
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