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

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 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 Ca(2+)-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 > or = 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 Po is open probability. At negative membrane potentials (-60 to -130 mV), an estimate of the dependence of NPo on cytoplasmic Ca2+ yielded an apparent Kd of 150.2 mumol/L. At pCa 3, an average channel density of approximately equal to 3 microns-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 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 Ca2+ overload, these channels may generate transient inward currents, contributing to the development of triggered cardiac arrhythmias.

The Large Conductance Potassium Channel β-Subunit Can Interact with and Modulate the Functional Properties of a Calcium-activated Chloride Channel, CLCA1

“…The CLCA family of chloride channels shares a number of similarities with I Cl.Ca recorded from epithelial cells (14) but is less similar to I Cl.Ca recorded from smooth muscle cells (1,15), cardiac myocytes (16), or endothelial cells (17). Therefore, either mCLCA1 does not encode I Cl.Ca in these cell types, or auxiliary subunits are required to modulate the activity of mCLCA1.…”

“…First-strand cDNA was prepared from the RNA using the Superscript II TM Reverse Transcriptase kit (Invitrogen). 1 g of total RNA was reverse-transcribed with 200 units of reverse transcriptase in a 20-l reaction containing 25 ng of oligo(dT) [12][13][14][15][16][17][18] primer, 500 M each dNTP, 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl 2 , and 10 mM dithiothreitol. PCR was performed with gene-specific primers for mCLCA1, mKCNMB1, and ␤-actin using AmpliTaq Gold reagents (Applied Biosystems, Foster City, CA).…”

Role of Ca2+-Dependent Cl− Current on Delayed Afterdepolarizations. A Simulation Study

“…We used available published experimental data 5,12,16,26,[31][32][33] to obtain the different parameters of the model. Whenever possible, we used data directly measured on rabbit atrial cells at 35°C to derive model parameters.…”

“…Collier et al 5 formulated a Hill-type equation that describes the effect of pCa on Ca 2+ -dependent Clconductance channels. This formulation describes the effect of the [Ca 2+ ] i on I Cl,Ca current, by the f Ca factor:…”

“…12,16,26,31 When rabbit atrial data were not available, we used reliable data from rabbit and dog ventricular cells. 5,12,32 We have integrated the available data regarding I Cl,Ca dependencies on [Ca 2+ ] i , 5 voltage-dependent inactivation behavior 16,31,32 and diffusion-field relation 5 (following the Goldman-Hodking-Katz formulation) into a single set of equations. Temperature dependence has not been considered in the formulation but was used to obtain the p Cl value for rabbit atrial cell at 35°C from experimental data recorded from dog ventricular cells at 22°C.…”