Unitary Cl − Channels Activated by Cytoplasmic Ca 2+ in Canine Ventricular Myocytes

Abstract

Abstract Recent whole-cell studies have shown that Ca 2+ -activated Cl − currents contribute to the Ca 2+ -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 Ca 2+ 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 Ca 2+ -activated Cl − channels. Ca 2+ 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 Ca 2+ was held constant and was blocked by the anion transport blockers, DIDS and niflumic acid. Ca 2+ (ranging from pCa ≥6 to pCa 3) applied to the cytoplasmic surface of inside-out patches increased, in a dose-dependent manner, NP o , 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 NP o on cytoplasmic Ca 2+ yielded an apparent K d of 150.2 μmol/L. At pCa 3, an average channel density of ≈3 μm −2 was estimated. Calculations based on these estimates of cytoplasmic Ca 2+ 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 Ca 2+ within the physiological range. We suggest that these small-conductance Ca 2+ -activated Cl − channels underlie the transient Ca 2+ -activated 4-aminopyridine–insensitive current, which contributes to phase-1 repolarization, and under conditions of Ca 2+ overload, these channels may generate transient inward currents, contributing to the development of triggered cardiac arrhythmias.