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


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Article Info

DATE

2014-10-26

AUTHORS

Krisztina Váczi, Bence Hegyi, Ferenc Ruzsnavszky, Kornél Kistamás, Balázs Horváth, Tamás Bányász, Péter P. Nánási, Norbert Szentandrássy, János Magyar

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 Ca2+ 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 (Vmax), but DIDS caused marked reduction of Vmax. 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. More... »

PAGES

87-100

References to SciGraph publications

  • 2013-10-27. TMEM16A knockdown abrogates two different Ca2+-activated Cl− currents and contractility of smooth muscle in rat mesenteric small arteries in PFLÜGERS ARCHIV - EUROPEAN JOURNAL OF PHYSIOLOGY
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  • 2008-08-24. TMEM16A confers receptor-activated calcium-dependent chloride conductance in NATURE
  • 1981-08. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches in PFLÜGERS ARCHIV - EUROPEAN JOURNAL OF PHYSIOLOGY
  • 1993-06. Anion and cation modulation of the guinea-pig ventricular action potential during β-adrenoceptor stimulation in PFLÜGERS ARCHIV - EUROPEAN JOURNAL OF PHYSIOLOGY
  • 1993-02. Effects of stilbenedisulfonic acid derivatives on the cAMP-regulated chloride current in cardiac myocytes in PFLÜGERS ARCHIV - EUROPEAN JOURNAL OF PHYSIOLOGY
  • Identifiers

    URI

    http://scigraph.springernature.com/pub.10.1007/s00210-014-1050-9

    DOI

    http://dx.doi.org/10.1007/s00210-014-1050-9

    DIMENSIONS

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    PUBMED

    https://www.ncbi.nlm.nih.gov/pubmed/25344201


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    30 schema:description 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 Ca2+ 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 (Vmax), but DIDS caused marked reduction of Vmax. 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.
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