9–Anthracene carboxylic acid is more suitable than DIDS for characterization of calcium-activated chloride current during canine ventricular action potential

Abstract

Understanding the role of ionic currents in shaping the cardiac action potential (AP) has great importance as channel malfunctions can lead to sudden cardiac death by inducing arrhythmias. Therefore, researchers frequently use inhibitors to selectively block a certain ion channel like 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) and 9-anthracene carboxylic acid (9-AC) for calcium-activated chloride current (I Cl(Ca) ). This study aims to explore which blocker is preferable to study I Cl (Ca) . Whole-cell voltage-clamp technique was used to record I Ca,L, I Ks, I Kr and I K1 , while action potentials were measured using sharp microelectrodes. DIDS-(0.2 mM) and 9-AC-sensitive (0.5 mM) currents were identical in voltage-clamp conditions, regardless of intracellular Ca 2+ buffering. DIDS-sensitive current amplitude was larger with the increase of stimulation rate and correlated well with the rate-induced increase of calcium transients. Both drugs increased action potential duration (APD) to the same extent, but the elevation of the plateau potential was more pronounced with 9-AC at fast stimulation rates. On the contrary, 9-AC did not influence either the AP amplitude or the maximal rate of depolarization (V max ), but DIDS caused marked reduction of V max . Both inhibitors reduced the magnitude of phase-1, but, at slow stimulation rates, this effect of DIDS was larger. All of these actions on APs were reversible upon washout of the drugs. Increasing concentrations of 9-AC between 0.1 and 0.5 mM in a cumulative manner gradually reduced phase-1 and increased APD. 9-AC at 1 mM had no additional actions upon perfusion after 0.5 mM. The halfeffective concentration of 9-AC was approximately 160 μM with a Hill coefficient of 2. The amplitudes of I Ca,L, I Ks, I Kr and I K1 were not changed by 0.5 mM 9-AC. These results suggest that DIDS is equally useful to study I Cl(Ca) during voltageclamp but 9-AC is superior in AP measurements for studying the physiological role of I Cl(Ca) due to the lack of sodium channel inhibition. 9-AC has also no action on other ion currents (I Ca,L , I Kr , I Ks , I K1 ); however, I Ca,L tracings can be contaminated with I Cl(Ca) when measured in voltage-clamp condition.

Ca2+-activated Cl− current is antiarrhythmic by reducing both spatial and temporal heterogeneity of cardiac repolarization

“…I Cl(Ca) started to activate at −20 mV and had its highest value at +60 mV in all studied cell types (Fig. 3B, C) similarly to our earlier studies [28,31]. …”

“…9-AC prolonged APD in both ENDO and MID cells (Fig. 1A, B and F) in line with previous findings [18,31]. In EPI cells, however, there was no AP prolongation (Fig.…”

“…Similarly to our previous study [31] 9-AC did not modify V max in any studied cell type (Tables 1 and 2) indicating its lack of action on fast Na + current. This makes the drug suitable for studying the role of I Cl(Ca) even during AP measurements.…”

“…Moreover, two other factors might contribute to the 9-AC-induced, rate-dependent AP prolongation. One is the rate-dependent variation of [Ca 2+ ] i as it was shown that [Ca 2+ ] i is larger upon the increase of stimulation rate both during steady-state [31] as well as without steady-state [34]. Moreover, [Ca 2+ ] i correlates well with I Cl(Ca) measured as I 9-AC with square pulses [31].…”

“…1). 9-AC is suitable to study I Cl(Ca) as it evoked maximal effect on AP in 0.5 mmol/L concentration and did not alter L-type Ca 2+ current (I Ca,L ), I Kr , the slow component of delayed rectifier K + current (I Ks ), and the inward rectifier K + current (I K1 ) [31]. Moreover, neither the transient outward K + current (I to1 ) nor the intracellular Ca 2+ concentration ([Ca 2+ ] i ) was influenced by 0.5 mmol/L 9-AC (Supplementary Fig.…”