Presence of a calcium-activated chloride current in  mouse ventricular myocytes

Xu, Yancheng; Dong, Paul R; Zhang, Zhao; Ahmmed, Gias U.; Chiamvimonvat, Nipavan

doi:10.1152/ajpheart.00044.2002

Cited by 47 publications

(52 citation statements)

References 57 publications

(68 reference statements)

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“…We have previously documented the presence of a Ca 2ϩ -activated Cl Ϫ current (I Cl,Ca ) in mouse ventricular myocytes (22). However, the single channel activities identified here were insensitive to niflumic acid, in contrast to the previously identified I Cl,Ca (22) (Fig. 3B).…”

Section: Existence Of I Kca In Human Atrial and Mouse Atrial Andsupporting

confidence: 42%

Molecular Identification and Functional Roles of a Ca2+-activated K+ Channel in Human and Mouse Hearts

Tuteja

Zhang

et al. 2003

Journal of Biological Chemistry

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The repolarization phase of cardiac action potential is prone to aberrant excitation that is common in cardiac patients. Here, we demonstrate that this phase is markedly sensitive to Ca 2؉ because of the surprising existence of a Ca 2؉ -activated K ؉ currents in cardiac cells. The current was revealed using recording conditions that preserved endogenous Ca 2؉ buffers. The Ca 2؉ -activated K ؉ current is expressed differentially in atria compared with ventricles. Because of the significant contribution of the current toward membrane repolarization in cardiac myocytes, alterations of the current magnitude precipitate abnormal action potential profiles. We confirmed the presence of a small conductance Ca 2؉ -activated K ؉ channel subtype (SK2) in human and mouse cardiac myocytes using Western blot analysis and reverse transcription-polymerase chain reaction and have cloned SK2 channels from human atria, mouse atria, and ventricles. Because of the marked differential expression of SK2 channels in the heart, specific ligands for Ca 2؉ -activated K ؉ currents may offer a unique therapeutic opportunity to modify atrial cells without interfering with ventricular myocytes.Cardiac action potentials (APs) 1 are shaped predominantly by the interplay between transient inward Na ϩ , Ca 2ϩ , and outward K ϩ currents (1). While the repolarization phase of the AP can be wrought by the kinetics of the principal currents, small and sustained outward currents also define this phase, rendering this region prone to irregular membrane excitation.In humans, delineation of the outward currents that confer the late repolarization phase of the cardiac AP is crucial for our understanding of the etiology of arrhythmias. We provide a novel report that demonstrates that the repolarization phase of cardiac AP shows marked sensitivity toward apamin, an exclusive ligand for a small conductance Ca 2ϩ -activated K ϩ channel (2).Ca 2ϩ -activated K ϩ channels (K Ca ) are present in most neurons and mediate the afterhyperpolarizations following AP (3, 4). However, functional significance of K Ca in the heart has not previously been documented. K Ca channels can be divided into three main subfamilies (3, 5-7). These include the large-conductance Ca 2ϩ -and voltage-activated K ϩ channels (BK), the intermediate-conductance K Ca channels (IK), and the smallconductance K Ca channels (SK), which are sensitive to apamin and scyllatoxin. Among the SK channels, they are encoded by at least three genes, SK1, SK2, SK3 (4, 6), with differential sensitivity toward apamin. SK2 is highly sensitive to apamin, with a half-blocking concentration (IC 50 ) of 60 pmol/liter, whereas SK1 channels are not affected by 100 nmol/liter apamin (2). SK3 channels are intermediate.Here, we report for the first time, the presence of I K,Ca (Ca 2ϩ -activated K ϩ current) in cardiac myocytes that plays a crucial role in cardiac AP profile. Using a combination of electrophysiological recordings and biochemical and molecular biological techniques, we have identified the presence of SK2...

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Section: Existence Of I Kca In Human Atrial and Mouse Atrial Andsupporting

confidence: 42%

Molecular Identification and Functional Roles of a Ca2+-activated K+ Channel in Human and Mouse Hearts

Tuteja

Zhang

et al. 2003

Journal of Biological Chemistry

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243

292

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“…Another study performed again on rabbit ventricular myocytes described full inhibition of I Cl(Ca) by 0.1 mM DIDS (Zygmunt and Gibbons 1991). Similarly, in murine ventricular cells, 0.1 mM DIDS completely abolished the calcium-activated outward current (Xu et al 2002) as well as full reduction was found in swine ventricular myocytes by 0.15 mM DIDS (Li et al 2003). In case of 9-AC, much less information is available regarding its I Cl(Ca) inhibitory action in direct current measurements.…”

Section: Discussionmentioning

confidence: 85%

“…DIDS (0.1-2 mM) is applied more frequently for this purpose (Zygmunt 1994;Kawano et al 1995;Verkerk et al 2002;Hirayama et al 2002;Li et al 2003;Guo et al 2008) than 9-AC (0.5-1 mM) (Levesque et al 1993;Fülöp et al 2003). Many other compounds were used to inhibit chloride currents including another stilbene derivative 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS, 1-2 mM) (Zheng et al 2013;Guo et al 2008;Zygmunt and Gibbons 1992), diphenylamine-2-carboxylate (DPC, 0.5-1 mM) (Walsh and Wang 1996), 5-nitro-2-(3-phenylpropyl-amino) benzoic acid (NPPB) (Zheng et al 2013) and also the widely applied niflumic acid (NFA, 30-200 μM) (Xu et al 2002;Song et al 2009;Forrest et al 2012), just to list a few. These compounds should usually be used in a quite high concentration to achieve inhibition of the chloride channels.…”

Section: Introductionmentioning

confidence: 99%

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

Váczi

Hegyi

Ruzsnavszky

et al. 2014

Naunyn-Schmiedeberg's Arch Pharmacol

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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.

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“…In this study mBest3 currents displayed a K D for Ca 2ϩ of 175 nM; however, the Ca 2ϩ sensitivity of the native current has not been reported. Cardiac I ClCa is dependent on Ca 2ϩ influx via voltage-dependent Ca 2ϩ channels and Ca 2ϩ release from the SR stores (18,40,51). A more precise notion about the physiological role of mBest3 in the heart awaits the characterization of the phenotype of Best3 null mice.…”

Section: Discussionmentioning

confidence: 99%

Expression, localization, and functional properties of Bestrophin 3 channel isolated from mouse heart

O'Driscoll¹,

Hatton²,

Burkin³

et al. 2008

American Journal of Physiology-Cell Physiology

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Bestrophins are a novel family of proteins that encode calcium-activated chloride channels. In this study we establish that Bestrophin transcripts are expressed in the mouse and human heart. Native mBest3 protein expression and localization in heart was demonstrated by using a specific polyclonal mBest3 antibody. Immunostaining of isolated cardiac myocytes indicates that mBest3 is present at the membrane. Using the patch-clamp technique, we characterized the biophysical and pharmacological properties of mBest3 cloned from heart. Whole cell chloride currents were evoked in both HEK293 and COS-7 cells expressing mBest3 by elevation of intracellular calcium. mBest3 currents displayed a KD for Ca 2ϩ of ϳ175 nM. The calcium-activated chloride current was found to be time and voltage independent and displayed slight outward rectification. The anion permeability sequence of the channel was SCN Ϫ ϾI Ϫ ϾCl Ϫ , and the current was inhibited by niflumic acid and DIDS in the micromolar range. In addition, we generated a sitespecific mutation (F80L) in the putative pore region of mBest3 that significantly altered the ion conduction and pharmacology of this channel. Our functional and mutational studies examining the biophysical properties of mBest3 indicate that it functions as a poreforming chloride channel that is activated by physiological levels of calcium. This study reports novel findings regarding the molecular expression, tissue localization, and functional properties of mBest3 cloned from heart.

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Presence of a calcium-activated chloride current in mouse ventricular myocytes

Cited by 47 publications

References 57 publications

Molecular Identification and Functional Roles of a Ca2+-activated K+ Channel in Human and Mouse Hearts

Molecular Identification and Functional Roles of a Ca2+-activated K+ Channel in Human and Mouse Hearts

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

Expression, localization, and functional properties of Bestrophin 3 channel isolated from mouse heart

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