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Updated: Apr 21, 2026
Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents
Published on: July 3, 2013
9-Anthracene carboxylic acid is more suitable than DIDS for characterization of calcium-activated chloride current
Krisztina Váczi1, Bence Hegyi, Ferenc Ruzsnavszky
1Department of Physiology, Faculty of Medicine, University of Debrecen, 4012, Debrecen, Nagyerdei krt 98, Hungary.
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- (0.2 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 half-effective concentration of 9-AC was approximately 160 μM with a Hill coefficient of 2. The amplitudes of ICa,L, IKs, IKr and IK1 were not changed by 0.5 mM 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.
Insights
Researchers compared 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and 9-anthracene carboxylic acid (9-AC) for studying calcium-activated chloride current (ICl(Ca)). 9-AC is superior for action potential measurements due to its specificity.
Area of Science:
- Cardiovascular Physiology
- Electrophysiology
- Pharmacology
Background:
- Ionic currents critically shape cardiac action potentials (APs), and their dysfunction can cause sudden cardiac death.
- Inhibitors like DIDS and 9-AC are used to study calcium-activated chloride current (ICl(Ca)).
Purpose of the Study:
- To compare the efficacy and specificity of DIDS and 9-AC in studying ICl(Ca).
- To determine the preferable blocker for voltage-clamp and action potential measurements.
Main Methods:
- Whole-cell voltage-clamp technique to record ionic currents (ICa,L, IKs, IKr, IK1).
- Sharp microelectrode recordings for action potentials.
- Application of DIDS and 9-AC at varying concentrations.
Main Results:
- DIDS- and 9-AC-sensitive currents were similar in voltage-clamp conditions.
- Both drugs prolonged action potential duration (APD) similarly.
- 9-AC showed superior specificity in action potential measurements, not affecting sodium channels or other major ion currents, unlike DIDS.
Conclusions:
- DIDS and 9-AC are both useful for studying ICl(Ca) via voltage-clamp.
- 9-AC is the preferred agent for action potential studies due to its lack of off-target effects on sodium channels and other ion currents.
- Understanding ICl(Ca) role is crucial for preventing arrhythmias.
