Unitary Cl- channels activated by cytoplasmic Ca2+ in canine ventricular myocytes

Recent whole-cell studies have shown that Ca2+- activated Cl- currents contribute to the Ca2+-dependent 4-aminopyridine-insensitive component of the transient outward current and to the arrhythmogenic transient inward current in rabbit and canine cardiac cells. These Cl--sensitive currents are activated by Ca2+ release from the sarcoplasmic reticulum and are inhibited by anion transport blockers; however, the unitary single channels responsible have yet to be identified. We used inside-out patches from canine ventricular myocytes and conditions under which the only likely permeant ion is Cl- to identify 4-aminopyridine-resistant unitary Ca2+-activated Cl- channels. Ca2+ applied to the cytoplasmic surface of membrane patches activated small-conductance (1.0 to 1.3 pS) channels. These channels were Cl- selective, with rectification properties that could be described by the Goldman-Hodgkin-Katz current equation. Channel activity exhibited time independence when cytoplasmic Ca2+ was held constant and was blocked by the anion transport blockers, DIDS and niflumic acid. Ca2+ (ranging from pCa greater than or equal to 6 to pCa 3) applied to the cytoplasmic surface of inside-out patches increased, in a dose-dependent manner, NPo, where N is the number of channels opened and P-o is open probability. At negative membrane potentials (-60 to -130 mV), an estimate of the dependence of NOo on cytoplasmic Ca2+ yielded an apparent K-d of 150.2 mu mol/L. At pCa 3, an average channel density of approximate to 3 mu m(-2) was estimated. Calculations based on these estimates of cytoplasmic Ca2+ sensitivity and channel current amplitude and density suggest that these small-conductance Cl- channels contribute significant whole-cell membrane current in response to changes in intracellular Ca2+ within the physiological range. We suggest that these small-conductance Ca2+-activated Cl- channels underlie the transient Ca2+-activated 4-aminopyridine-insensitive current, which contributes to phase-1 repolarization, and under conditions of Ca2+ overload, these channels may generate transient inward currents, contributing to the development of triggered cardiac arrhythmias.