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 (ICl(Ca)). This study aims to explore which blocker is preferable to study ICl(Ca). Whole-cell voltage-clamp technique was used to record ICa,L, IKs, IKr and IK1, while action potentials were measured using sharp microelectrodes. DIDS- (0.2mM) and 9-AC-sensitive (0.5mM) 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 0.5mM in a cumulative manner gradually reduced phase-1 and increased APD. 9-AC at 1 mM had no additional actions upon perfusion after 0.5mM. The half-effective concentration of 9-AC was approximately 160μM with a Hill coefficient of 2. The amplitudes of ICa,L, IKs, IKr and IK1 were not changed by 0.5mM 9-AC. These results suggest that DIDS is equally useful to study ICl(Ca) during voltage-clamp but 9-AC is superior in AP measurements for studying the physiological role of ICl(Ca) due to the lack of sodium channel inhibition. 9-AC has also no action on other ion currents (ICa,L, IKr, IKs, IK1); however, ICa,L tracings can be contaminated with ICl(Ca) when measured in voltage-clamp condition.
Highlights
Sudden cardiac death as a result of cardiac arrhythmias is a major cause of death in western civilizations (Fishman et al 2010)
Our results suggest that DIDS is useful to study ICl(Ca) during voltage-clamp, but 9anthracene carboxylic acid (9-AC) is superior in action potential (AP) measurements for studying the physiological role of ICl(Ca) due to the lack of INa inhibition
Calcium-activated chloride current measured as either DIDSor 9-AC-sensitive current is identical during conventional voltage-clamp recording
Summary
Sudden cardiac death as a result of cardiac arrhythmias is a major cause of death in western civilizations (Fishman et al 2010). These arrhythmias are often due to malfunctions of cardiac ion channels (Schmitt et al 2014). Understanding the role of ionic currents in shaping the cardiac action potential (AP) has great importance. This could be achieved by several approaches. One is to use expression systems to evaluate the biophysical properties of the current.
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