Comprehensive Physiology Wiley Online Library
For Librarians For Authors Editors About

Phenomics of Cardiac Chloride Channels

Dayue Darrel Duan

10.1002/cphy.c110014

Source: Volume 3, Issue 2, April 2013

Published online: April 2013

Full Article on Wiley Online Library



Abstract

Forward genetic studies have identified several chloride (Cl−) channel genes, including CFTR, ClC‐2, ClC‐3, CLCA, Bestrophin, and Ano1, in the heart. Recent reverse genetic studies using gene targeting and transgenic techniques to delineate the functional role of cardiac Cl− channels have shown that Cl− channels may contribute to cardiac arrhythmogenesis, myocardial hypertrophy and heart failure, and cardioprotection against ischemia reperfusion. The study of physiological or pathophysiological phenotypes of cardiac Cl− channels, however, is complicated by the compensatory changes in the animals in response to the targeted genetic manipulation. Alternatively, tissue‐specific conditional or inducible knockout or knockin animal models may be more valuable in the phenotypic studies of specific Cl− channels by limiting the effect of compensation on the phenotype. The integrated function of Cl− channels may involve multiprotein complexes of the Cl− channel subproteome. Similar phenotypes can be attained from alternative protein pathways within cellular networks, which are influenced by genetic and environmental factors. The phenomics approach, which characterizes phenotypes as a whole phenome and systematically studies the molecular changes that give rise to particular phenotypes achieved by modifying the genotype under the scope of genome/proteome/phenome, may provide more complete understanding of the integrated function of each cardiac Cl− channel in the context of health and disease. © 2013 American Physiological Society. Compr Physiol 3:667‐692, 2013.

Comprehensive Physiology offers downloadable PowerPoint presentations of figures for non-profit, educational use, provided the content is not modified and full credit is given to the author and publication.

Download a PowerPoint presentation of all images


Figure 1. Figure 1.

Schematic representation of Cl− channels in cardiac myocytes. Cl− channels and their corresponding molecular entities or candidates are indicated. ClC‐3, a member of voltage‐gated ClC Cl− channel family, encodes Cl− channels that are volume‐regulated (ICl,vol) and can be activated by cell swelling (ICl,swell) induced by exposure to hypotonic extracellular solutions or possibly membrane stretch. ICl,b is a basally activated ClC‐3 Cl− current. ClC‐2, a member of voltage‐gated ClC Cl− channel family, is responsible for a volume‐regulated and hyperpolarization‐activated inward rectifying Cl− current (ICl,ir). Membrane topology models (α‐helices a‐r) for ClC‐3 and ClC‐2 are modified from Dutzler et al. (63). ICl,acid is a Cl− current regulated by extracellular pH and the molecular entity for ICl,acid is currently unknown. ICl,Ca is a Cl− current activated by increased intracellular Ca2+ concentration ([Ca2+]i); Molecular candidates for ICl,Ca include CLCA1, a member of a Ca2+‐sensitive Cl− channel family (CLCA), bestrophin‐2, a member of the Bestrophin gene family, and TMEM16, transmembrane protein 16. CFTR, cystic fibrosis transmembrane conductance regulator, encodes Cl− channels activated by stimulation of cAMP‐protein kinase A (PKA) pathway (ICl,PKA), protein kinase C (PKC) (ICl,PKC), or extracellular ATP through purinergic receptors (ICl,ATP). CFTR is composed by two membrane spanning domains (MSD1 and MSD2), two nucleotide‐binding domains (NBD1 and NBD2), and a regulatory subunit (R). P, phosphorylation sites for PKA and PKC; PP, serine‐threonine protein phosphatases; Gi, heterodimeric inhibitory G protein; A1R, adenosine type 1 receptor; AC, adenylyl cyclase; H2R, histamine type II receptor; Gs, heterodimeric stimulatory G protein; β‐AR, β‐adrenergic receptor; P2R, purinergic type 2 receptor; proposed intracellular signaling pathway for purinergic activation of CFTR. VDAC, voltage‐dependent anion channels (porin); mito, mitochondrion (50). (Copyright Request: Duan D. J Physiol 587: 2163‐2177, 2009.)
Figure 2. Figure 2.

Modulation of cardiac electrical activity by activation of Cl− channels in heart. Changes in action potentials (top), membrane currents (middle), and ECG (bottom) due to activation of CFTR or volume‐regulated ClC‐3 Cl− channels are depicted. Top panel: numbers illustrate conventional phases of a prototype ventricular action potential under control conditions (black) and after activation of ICl (red). Range of estimates for normal physiological values for Cl− equilibrium potential (ECl) is indicated in blue. Middle panel: range of zero‐current values corresponding to ECl is shown in grey. Activation of CFTR or ClC‐3 channels generates both inward (indicated by green) and outward (indicated by red) currents and cause both depolarization as well as repolarization during the action potential. Activation of ICl, therefore, induces larger membrane depolarization and induction of early afterdepolarizations under conditions where resting K+ conductance is reduced (dotted red lines in top panel). Bottom panel: the letters (P, Q, R, S, and T) indicate the conventional waves of electrocardiograph (ECG) complex under control conditions (black) and after activation of ICl (red). Corresponding to the shortening of action potential in ventricular myocytes activation of ICl causes a shortening of Q‐T interval. See text for details (50). (Copyright Request: Duan D. J Physiol 587: 2163‐2177, 2009.)
Figure 3. Figure 3.

Effects of CFTR gene knockout (FABPCFTR) on ischemic preconditioning in isolated working mouse heart. (A) Experimental protocol. (B‐D) Recovery of left ventricular contractile (B and C.) and relaxation (D) function of wild‐type WT (FVB/NJ), CFTR+/−, and CFTR−/− (FABPCFTR) mice after 45 min ischemia and 40 min reperfusion. (E) IPC on infarct size of ventricles. Representative ventricle transverse slices after ischemia (Isch) or IPC. (F). Mean infarct size measured from age‐matched WT, CFTR+/−, or CFTR−/− mouse heart after ischemia or IPC (n = 6 for each group). *, P < 0.05; **, P < 0.01; ***, P < 0.001, NS: not significant (23). (Copyright Request: Chen H, et al. Circulation 110: 700‐704, 2004.)
Figure 4. Figure 4.

The cystic fibrosis transmembrane conductance regulator (CFTR) interactome. All components comprising the CFTR interactome are depicted as nodes (ovals) in the network. Components identified in previous studies as CFTR interactors are highlighted with bold lines surrounding the ovals. Straight blue lines are edges in the network that show direct or indirect protein interactions between CFTR and the indicated component identified by MudPIT. Straight red lines illustrate edges that define interactions based on the BIND (http://www.bind.ca/Action) and DIP (http://dip.doe‐mbi.ucla.edu/) protein interaction databases and the Tmm coexpression database (http://microarray.cpmc.columbia.edu/tmm/), which were accessed using the Cytoscape platform (http://www.cytoscape.org/). Proteins involved in folding and export from the ER are illustrated as gray nodes; green nodes highlight protein interactions involved in postendoplasmic reticulum trafficking and activity. Yellow nodes indicate interactors with unknown function. See the Supplementary Discussion for a more complete description of proteins defined by green and yellow nodes. The network includes proteins involved in the modulation of CFTR folding and function (168). (Copyright request: Wang et al. Cell 127: 803‐815, 2006.)
Figure 5. Figure 5.

Model of the mechanotransduction process coupling β1 integrin stretch to activation of Cl‐ channels in ventricular myocytes. Integrin stretch triggers the phosphorylation and activation of focal adhesion kinase and Src, and the release of angiotensin II (Ang II) from secretory vesicles. Ang II binds to the AT1 receptor (AT1R) and activates the AT1R signaling cascade. Components of the AT1R signaling cascade, possibly in concert with components of integrin signaling, induce the activation of p47phox, p67phox, and rac, which translocate to the membrane and assemble with gp91phox and p22phox to form the active NADPH oxidase complex. NADPH oxidase recruits NAD(P)H as an electron donor and catalyzes the transmembrane transfer of electrons to molecular O2 to form superoxide (O2−). Extracellular O2− is rapidly converted to membrane‐permeant H2O2 by ecSOD. H2O2 may activate Cl− stretch‐activated channels (SAC) either directly or via reactive oxygen species (ROS)‐sensitive signaling pathways (18). (Copyright Request: Browe and Baumgarten. J Gen Physiol 124: 273‐287, 2004.)
Figure 6. Figure 6.

Comparison of pressure overload‐induced remodeling of wild‐type and Clcn3−/− mouse hearts. Hearts from age‐matched wild‐type (WT, Clcn3+/+) and Clcn3−/‐ mice were excised 1 week (top panel) or 10 weeks (bottom panel) after minimally invasive transverse aorta binding (MTAB) or sham operation are shown. Hearts were cleaned of blood and connective tissues and then fixed in 4% paraformaldehyde. Bar = 5 mm. Compared to WT mice disruption of ClC‐3 gene significantly changed the remodeling process after MTAB. Both left ventricle and atrium were extremely enlarged after 10 weeks of MTAB. [Adapted, with permission, from Duan (50)]. (Copyright Request: Duan D. J Physiol 587: 2163‐2177, 2009)
Figure 7. Figure 7.

Schematic representation of ClC‐3 Cl− channels in VSMCs. ClC‐3, a member of voltage‐gated ClC Cl− channel family, encodes Cl− channels in vascular smooth muscle cells that are volume regulated (ICl,vol) and can be activated by cell swelling (ICl,swell) induced by exposure to hypotonic extracellular solutions or possibly membrane stretch. ICl,b is a basally activated ClC‐3 Cl− current. α‐helices of ClC‐3 are shown as a‐r. ClC‐3 proteins are expressed on both sarcolemmal membrane and intracellular organelles including mitochondria (mito) and endosomes. The proposed model of endosome ion flux and function of Nox1 and ClC‐3 in the signaling endosome is adapted from Miller Jr. et al. (120). Binding of IL‐1β or TNF‐α to the cell membrane initiates endocytosis and formation of an early endosome (EEA1 and Rab5), which also contains NADPH oxidase subunits Nox1 and p22phox, in addition to ClC‐3. Nox1 is electrogenic, moving electrons from intracellular NADPH through a redox chain within the enzyme into the endosome to reduce oxygen to superoxide. ClC‐3 functions as a chloride‐proton exchanger, required for charge neutralization of the electron flow generated by Nox1. The ROS generated by Nox1 result in NF‐κB activation. Both ClC‐3 and Nox1 are necessary for generation of endosomal reactive oxygen species (ROS) and subsequent NF‐κB activation by IL‐1β or TNF‐α in VSMCs. Statins block ClC‐3 channels, which causes hyperpolarization of the cell membrane, closure of Ca2+ channels and vasorelaxation, and inhibition of cell proliferation. PKC, protein kinase C; PP, serine‐threonine protein phosphatases; α−AR, α‐adrenergic receptor; Gi, heterodimeric inhibitory G protein. Nox: NADPH oxidase (61). (Copyright Request: Duan, Hypertension, 2010)
Figure 8. Figure 8.

Comparative two‐dimensional (2D) electrophoresis analysis of protein expression patterns in membranes of cardiac cells from Clcn3+/+ and Clcn3−/− mice. (A) representative 2D gel depicts Coomassie‐stained proteins from wild‐type (Clcn3+/+) mouse heart. (B) Representative 2D gel depicts Coomassie‐stained proteins from Clcn3−/− mouse heart. (C) Spot sets created from images of 2D gels of both wild‐type and Clcn3−/− mouse heart run under the same conditions as the gels in A and B and compared using Bio‐Rad PDQuest version 7.1.1 software. Three gels were run for each mouse heart type; two hearts were pooled to provide proteins for each gel. The filled symbols indicate changes in protein patterns in Clcn3−/− compared to wild type. A total of 35 proteins consistently changed (minimum criteria: more than twofold change) in membranes from Clcn3−/− mouse heart in all 3 experiments (6 missing proteins, 2 new proteins, 9 upregulated proteins, 15 downregulated proteins, and 2 translocated proteins). The open squares (□) in A, B, and C indicate the location (molecular mass 85 kDa and pI 6.9) of the ClC‐3 protein spot (No. 3812) in the 2D gels, which was independently confirmed by Western blotting using a specific anti‐ClC‐3 C670∼687 antibody (180). [Copyright Request: Yamamoto‐Mizuma et al. (180) with permission from Blackwell Publishing].
Figure 9. Figure 9.

Echocardiographic evaluation of cardiac function. (A) Representative M‐mode echocardiography from wild‐type (Clcn3+/+; left) and heart‐specific ClC‐3 knockout (hsClcn3−/−; right) mice. (B) Echocardiographic measurements in Clcn3+/+ and hsClcn3−/− mice. IVSd, interventricular septum thickness at the end of diastole; LVDd, left ventricular (LV) dimension at the end of diastole; LVPWd, LV posterior wall thickness at the end of diastole; IVSs, interventricular septum thickness at the end of systole; LVDs, LV dimension at the end of systole; LVPWs, LV posterior wall thickness at the end of systole; LVEP, calculated LV ejection fraction; %FS, LV fractional shortening; estimated LV mass, LVM (mg) = 1.05[(IVS + LVID + LVPW)3 − (LVID)3], where 1.05 is the specific gravity of the myocardium. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with Clcn3+/+ mice. C. Single longitudinal section (μm) of hearts to demonstrate all four heart chambers. Longitudinal were stained with hematoxylin and eosin (Bar = 2 mm) (Duan D. et al. unpublished data.)
Figure 10. Figure 10.

Effects of inducible heart‐specific ClC‐3 knockout on cardiac volume‐regulated Cl− current (VRCC) and heart function. (A) Representative current traces in isotonic condition and under hypotonic challenge recorded in freshly isolated atrial myocytes from the inducible heart‐specific ClC‐3 knockout (doxyhsClC‐3−/−) mice with doxycycline (on Doxy) in the diet (panel a), or after withdraw of doxycycline (off Doxy) from the diet for 3 weeks (panel b). (c) Summary of VRCC current densities in isotonic and hypotonic solutions, recorded at +80 mV and −80 mV. (B) Representative M‐mode echocardiography from on Doxy (a) and off Doxy (b) mice. (C) Time‐dependent changes in M‐mode echocardiogram of age matched on Doxy or off Doxy for 1.5 and 3 weeks. *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus off Doxy 0 week; #, P < 0.05; ##, P < 0.01; ###, P < 0.001 versus on Doxy at the same time point. (D) Comparison of hearts isolated from age‐matched (11‐week old) doxyhsClcn3−/− mice on Doxy or off Doxy for 3 weeks. Hearts were cleaned up blood and connective tissues and fixed in 4% paraformaldehyde. [Adapted, with permission, from Xiong et al. (174).]
Figure 11. Figure 11.

Effects of inducible heart‐specific ClC‐3 knockout on cardiac volume‐regulated Cl− current (VRCC) and heart function. (A) Representative current traces in isotonic condition and under hypotonic challenge recorded in freshly isolated atrial myocytes from the inducible heart‐specific ClC‐3 knockout (doxyhsClC‐3−/−) mice with doxycycline (on Doxy) in the diet (panel a), or after withdraw of doxycycline (off Doxy) from the diet for 3 weeks (panel b). (c) Summary of VRCC current densities in isotonic and hypotonic solutions, recorded at +80 mV and −80 mV. (B) Representative M‐mode echocardiography from on Doxy (a) and off Doxy (b) mice. (C) Time‐dependent changes in M‐mode echocardiogram of age matched on Doxy or off Doxy for 1.5 and 3 weeks. *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus off Doxy 0 week; #, P < 0.05; ##, P < 0.01; ###, P < 0.001 versus on Doxy at the same time point. (D) Comparison of hearts isolated from age‐matched (11‐week old) doxyhsClcn3−/− mice on Doxy or off Doxy for 3 weeks. Hearts were cleaned up blood and connective tissues and fixed in 4% paraformaldehyde. [Adapted, with permission, from Xiong et al. (174).]
Figure 12. Figure 12.

Modulation of cardiac electrical activity by activation of ClC‐2 channels in cardiac pacemaker cells and myocytes. Changes in action potentials (top panels) and membrane currents (bottom panels) of cardiac pacemaker cells (A) or atrial and ventricular myocytes (B) due to activation of ClC‐2 channels are depicted. ICl,ir is activated by hyperpolarization, cell swelling, and acidosis. Top panels: numbers illustrate conventional phases of a prototype ventricular action potential under control conditions (black) and after activation of ICl (red). Range of estimates for normal physiological values for Cl− equilibrium potential (ECl) is indicated in blue. Bottom panels: range of zero‐current values corresponding to ECl is shown in grey. (A) Activation of ICl,ir in pacemaker cells during hyperpolarization causes acceleration of phase 4 depolarization and automaticity, shortening of action potential duration, and decrease in cycle length and action potential amplitude (dashed red line in top panel). (B) Activation of ICl,ir in atrial and ventricular myocytes during hyperpolarization causes depolarization of resting membrane potential and induction of phase 4 auto depolarization and abnormal electrical impulse (trigger activity) and automaticity (dotted red line in top panel). [Adapted, with permission, from Duan (50)]. (Copyright Request: Duan D. J Physiol 587: 2163‐2177, 2009)
Figure 13. Figure 13.

Whole‐cell currents recorded from SAN cells of guinea‐pig heart. (A) An example of single SAN cells (arrows) isolated from the SAN region of guinea pig heart by enzymatic dispersion. (B) Whole‐cell currents recorded from SAN cells. When cations (Na+ and K+) were included in the extracellular solutions, inward currents were slowly activated upon hyperpolarization under isotonic (a) conditions. Exposure of the same cell to hypotonic extracellular solution caused cell swelling and an increase in the inward current amplitude (b). The difference current caused by hypotonic cell swelling is shown in panel e. Subsequent replacement of 20 mmol/L of NaCl with CsCl caused a significant inhibition of the inward current (c). The Cs+‐sensitive current is shown in panel f. Subsequent addition of 0.2 mmol/L of Cd2+ to the hypotonic solution caused an inhibition of the inward current (d). The Cd2+‐sensitive currents are shown in panel g (91).
Figure 14. Figure 14.

Molecular expression of ClC‐2 in SAN cells. (A) Localization of ClC‐2 chloride channels in guinea‐pig SAN tissue. (a) Section labeled with anti‐Connexin 43 (red) to illustrate the adjacent atrial (AT) septum was positively labeled while the SAN was negative (dark region), which clearly delineates the SAN region from the AT septum (dashed white line). (b) Section stained with 4′,6‐diamidino‐2‐phenylindole (DAPI) (blue) to compare nuclei density in the SAN region and in AT. The SAN region had a higher DAPI staining density (higher nuclei density) than the adjacent AT. (c) Section stained with anti‐ClC‐2 (green). ClC‐2 immunoreactivity is evident in both SAN and AT regions. (d) Merged images of a, b, and c illustrate that ClC‐2 is expressed in the densely nucleated and Cx43 negative SAN region. (B) Agarose gel depicting real time polymerase chain reaction product of ClC‐2 amplified from mRNA prepared from enzymatically dispersed guinea‐pig SA nodal cells. (C) Images of ClC‐2‐like immunofluorescence in a representative SAN cell visualized using fluorescent microscopy. Phase contrast (a) and fluorescent micrographs (b) of a single SAN cell.
Figure 15. Figure 15.

Effects of Anti‐ClC‐2 Ab on ICl,ir in SAN cells. (A) Representative whole‐cell currents recorded from SAN cells under isotonic (panel a) and hypotonic (panel b) conditions in the presence of anti‐ClC‐2 Ab in the pipette solutions. SAN cells were exposed to isotonic solution for at least 10 min before whole‐cell recordings. Currents shown in panel a were recorded right after successful whole‐cell configuration under isotonic conditions. Currents shown in panel b were recorded after exposure to hypotonic solution for 20 min. Pipette and bath solutions were identical to those described in Figure 1B except the pipette solution contained 3 μg/mL anti‐ClC‐2 Ab. (d) Mean I‐V from 5 SAN cells under the same conditions. (B) SAN cells were exposed to hypotonic solution for 20 min to fully activate ICl,ir before whole‐cell recordings. Bath and pipette solutions were the same as in panel A. Representative current traces recorded by voltage‐clamp (protocol is shown in inset) from the SAN cell immediately after membrane rupture (a) and after 20 min of anti‐ClC‐2 Ab dialysis (b). The anti‐ClC‐2 Ab‐sensitive current (a)‐(b) is shown in (c) (current traces) and (d) (mean I‐V, n = 5). Notice the anti‐ClC‐2 Ab‐sensitive current (c) was similar to ICl,ir shown in Figure 1 and the typical ICa and If (b) were not affected by anti‐ClC‐2 Ab.
Figure 16. Figure 16.

Effects of Anti‐ClC‐2 Ab on pacemaker action potential in SAN cells. (A) Representative spontaneous action potentials recorded from an SAN cell by current‐clamp (no current injection) with pipette solution containing no anti‐ClC‐2 Ab under isotonic (a) and hypotonic (b) conditions. SAN cells were exposed to isotonic solution for at least 10 min before action potential recordings. Action potentials shown in panel a were recorded right after successful whole‐cell configuration under isotonic conditions. Action potentials shown in panel b were recorded after exposure to hypotonic solution for 20 min. For comparison, the action potentials recorded under these conditions were superimposed with an expanded time scale in panel c. The dotted lines indicate zero voltage. (B) Spontaneous action potentials recorded from a SAN cell by current clamp using a pipette solution containing pre‐absorbed anti‐ClC‐2 Ab (control) and cell was exposed to isotonic solutions for 10 min (a) and hypotonic solutions for 20 min (b). For comparison, the action potentials recorded under these conditions were superimposed with an expanded time scale in panel c. (C) SAN cells were perfused with isotonic solutions for 20 min before whole‐cell recordings. Action potentials were recorded immediately after membrane rupture (a) and after dialysis of anti‐ClC‐2 Ab for 20 min (b) under the same isotonic conditions. Panel c shows the expanded and superimposed action potentials as shown in panel a and panel b. Note that after 20 min dialysis of anti‐ClC‐2 Ab in to the cell the spontaneous action potential rate was not significantly altered. (D) SAN cells were exposed to hypotonic solution for 20 min to fully activate ICl,ir before whole‐cell recordings. Action potentials were recorded immediately after membrane rupture (a) and after dialysis of anti‐ClC‐2 Ab for 20 min (b). Panel c shows the expanded and superimposed action potentials as shown in panel a and panel b. Note that the spontaneous action potential rate significantly decreased after 20 min dialysis of anti‐ClC‐2 Ab in to the cell, which corresponds with the decrease in inward
Figure 17. Figure 17.

Telemetry electrocardiogram (ECG) recordings in Clcn2−/− mice and their Clcn2+/+ and Clcn2+/− littermates during treadmill exercises. (A) Representative ECG (Lead II) recordings in Clcn2+/+, Clcn2+/−, and Clcn2−/− mice while they were subjected to treadmill exercise at (a) rest period: acclimation at 0 m/min, incline 0o for 5 min; (b) walk period: walking at 5m/min, incline 0o for 5 min; (c) run period: running at 15 m/min, uphill incline 8o for 5 min. (B) Mean heart rate during the last minute of each treadmill exercise segment for the Clcn2+/+ (n = 6), Clcn2+/‐ (n = 5), and Clcn2−/− (n = 7) mice. (C) Mean heart rate of the Clcn2+/+ (n = 5) and Clcn2−/− (n = 4) mice before (Control, Cont) and after the intraperitoneal injection of atropine (Atro), propranolol (Prop), or atropine plus propranolol (Atro + Prop) during the last minute of each treadmill exercise segment (rest, walk, and run). *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus Clcn2+/+; #, P < 0.05; ##, P < 0.01; ###, P < 0.001 versus Clcn2+/−; $, P < 0.05; $$, P < 0.01; $$$, P < 0.001 versus control (Cont); d, P < 0.05 versus rest (91).
Figure 18. Figure 18.

Modulation of cardiac electrical activity by activation of Ca2+‐activated Cl− channels in heart. Changes in action potentials (top) and membrane currents (bottom) due to activation of Ca2+‐activated Cl− channels are depicted. Top panel: numbers illustrate conventional phases of a prototype ventricular action potential under control conditions (black) and after activation of ICl (red). Range of estimates for normal physiological values for ECl is indicated in blue. Bottom panel: Range of zero‐current values corresponding to ECl is shown in grey. Activation of ICl,Ca during [Ca2+]i overload results in oscillatory transient inward current (ITI) and induction of delayed afterdepolarization (DAD) (dotted red lines). [Adapted, with permission, from Duan (50)]. (Copyright Request: Duan D. J Physiol 587: 2163‐2177, 2009.)


Figure 1.

Schematic representation of Cl− channels in cardiac myocytes. Cl− channels and their corresponding molecular entities or candidates are indicated. ClC‐3, a member of voltage‐gated ClC Cl− channel family, encodes Cl− channels that are volume‐regulated (ICl,vol) and can be activated by cell swelling (ICl,swell) induced by exposure to hypotonic extracellular solutions or possibly membrane stretch. ICl,b is a basally activated ClC‐3 Cl− current. ClC‐2, a member of voltage‐gated ClC Cl− channel family, is responsible for a volume‐regulated and hyperpolarization‐activated inward rectifying Cl− current (ICl,ir). Membrane topology models (α‐helices a‐r) for ClC‐3 and ClC‐2 are modified from Dutzler et al. (63). ICl,acid is a Cl− current regulated by extracellular pH and the molecular entity for ICl,acid is currently unknown. ICl,Ca is a Cl− current activated by increased intracellular Ca2+ concentration ([Ca2+]i); Molecular candidates for ICl,Ca include CLCA1, a member of a Ca2+‐sensitive Cl− channel family (CLCA), bestrophin‐2, a member of the Bestrophin gene family, and TMEM16, transmembrane protein 16. CFTR, cystic fibrosis transmembrane conductance regulator, encodes Cl− channels activated by stimulation of cAMP‐protein kinase A (PKA) pathway (ICl,PKA), protein kinase C (PKC) (ICl,PKC), or extracellular ATP through purinergic receptors (ICl,ATP). CFTR is composed by two membrane spanning domains (MSD1 and MSD2), two nucleotide‐binding domains (NBD1 and NBD2), and a regulatory subunit (R). P, phosphorylation sites for PKA and PKC; PP, serine‐threonine protein phosphatases; Gi, heterodimeric inhibitory G protein; A1R, adenosine type 1 receptor; AC, adenylyl cyclase; H2R, histamine type II receptor; Gs, heterodimeric stimulatory G protein; β‐AR, β‐adrenergic receptor; P2R, purinergic type 2 receptor; proposed intracellular signaling pathway for purinergic activation of CFTR. VDAC, voltage‐dependent anion channels (porin); mito, mitochondrion (50). (Copyright Request: Duan D. J Physiol 587: 2163‐2177, 2009.)



Figure 2.

Modulation of cardiac electrical activity by activation of Cl− channels in heart. Changes in action potentials (top), membrane currents (middle), and ECG (bottom) due to activation of CFTR or volume‐regulated ClC‐3 Cl− channels are depicted. Top panel: numbers illustrate conventional phases of a prototype ventricular action potential under control conditions (black) and after activation of ICl (red). Range of estimates for normal physiological values for Cl− equilibrium potential (ECl) is indicated in blue. Middle panel: range of zero‐current values corresponding to ECl is shown in grey. Activation of CFTR or ClC‐3 channels generates both inward (indicated by green) and outward (indicated by red) currents and cause both depolarization as well as repolarization during the action potential. Activation of ICl, therefore, induces larger membrane depolarization and induction of early afterdepolarizations under conditions where resting K+ conductance is reduced (dotted red lines in top panel). Bottom panel: the letters (P, Q, R, S, and T) indicate the conventional waves of electrocardiograph (ECG) complex under control conditions (black) and after activation of ICl (red). Corresponding to the shortening of action potential in ventricular myocytes activation of ICl causes a shortening of Q‐T interval. See text for details (50). (Copyright Request: Duan D. J Physiol 587: 2163‐2177, 2009.)



Figure 3.

Effects of CFTR gene knockout (FABPCFTR) on ischemic preconditioning in isolated working mouse heart. (A) Experimental protocol. (B‐D) Recovery of left ventricular contractile (B and C.) and relaxation (D) function of wild‐type WT (FVB/NJ), CFTR+/−, and CFTR−/− (FABPCFTR) mice after 45 min ischemia and 40 min reperfusion. (E) IPC on infarct size of ventricles. Representative ventricle transverse slices after ischemia (Isch) or IPC. (F). Mean infarct size measured from age‐matched WT, CFTR+/−, or CFTR−/− mouse heart after ischemia or IPC (n = 6 for each group). *, P < 0.05; **, P < 0.01; ***, P < 0.001, NS: not significant (23). (Copyright Request: Chen H, et al. Circulation 110: 700‐704, 2004.)



Figure 4.

The cystic fibrosis transmembrane conductance regulator (CFTR) interactome. All components comprising the CFTR interactome are depicted as nodes (ovals) in the network. Components identified in previous studies as CFTR interactors are highlighted with bold lines surrounding the ovals. Straight blue lines are edges in the network that show direct or indirect protein interactions between CFTR and the indicated component identified by MudPIT. Straight red lines illustrate edges that define interactions based on the BIND (http://www.bind.ca/Action) and DIP (http://dip.doe‐mbi.ucla.edu/) protein interaction databases and the Tmm coexpression database (http://microarray.cpmc.columbia.edu/tmm/), which were accessed using the Cytoscape platform (http://www.cytoscape.org/). Proteins involved in folding and export from the ER are illustrated as gray nodes; green nodes highlight protein interactions involved in postendoplasmic reticulum trafficking and activity. Yellow nodes indicate interactors with unknown function. See the Supplementary Discussion for a more complete description of proteins defined by green and yellow nodes. The network includes proteins involved in the modulation of CFTR folding and function (168). (Copyright request: Wang et al. Cell 127: 803‐815, 2006.)



Figure 5.

Model of the mechanotransduction process coupling β1 integrin stretch to activation of Cl‐ channels in ventricular myocytes. Integrin stretch triggers the phosphorylation and activation of focal adhesion kinase and Src, and the release of angiotensin II (Ang II) from secretory vesicles. Ang II binds to the AT1 receptor (AT1R) and activates the AT1R signaling cascade. Components of the AT1R signaling cascade, possibly in concert with components of integrin signaling, induce the activation of p47phox, p67phox, and rac, which translocate to the membrane and assemble with gp91phox and p22phox to form the active NADPH oxidase complex. NADPH oxidase recruits NAD(P)H as an electron donor and catalyzes the transmembrane transfer of electrons to molecular O2 to form superoxide (O2−). Extracellular O2− is rapidly converted to membrane‐permeant H2O2 by ecSOD. H2O2 may activate Cl− stretch‐activated channels (SAC) either directly or via reactive oxygen species (ROS)‐sensitive signaling pathways (18). (Copyright Request: Browe and Baumgarten. J Gen Physiol 124: 273‐287, 2004.)



Figure 6.

Comparison of pressure overload‐induced remodeling of wild‐type and Clcn3−/− mouse hearts. Hearts from age‐matched wild‐type (WT, Clcn3+/+) and Clcn3−/‐ mice were excised 1 week (top panel) or 10 weeks (bottom panel) after minimally invasive transverse aorta binding (MTAB) or sham operation are shown. Hearts were cleaned of blood and connective tissues and then fixed in 4% paraformaldehyde. Bar = 5 mm. Compared to WT mice disruption of ClC‐3 gene significantly changed the remodeling process after MTAB. Both left ventricle and atrium were extremely enlarged after 10 weeks of MTAB. [Adapted, with permission, from Duan (50)]. (Copyright Request: Duan D. J Physiol 587: 2163‐2177, 2009)



Figure 7.

Schematic representation of ClC‐3 Cl− channels in VSMCs. ClC‐3, a member of voltage‐gated ClC Cl− channel family, encodes Cl− channels in vascular smooth muscle cells that are volume regulated (ICl,vol) and can be activated by cell swelling (ICl,swell) induced by exposure to hypotonic extracellular solutions or possibly membrane stretch. ICl,b is a basally activated ClC‐3 Cl− current. α‐helices of ClC‐3 are shown as a‐r. ClC‐3 proteins are expressed on both sarcolemmal membrane and intracellular organelles including mitochondria (mito) and endosomes. The proposed model of endosome ion flux and function of Nox1 and ClC‐3 in the signaling endosome is adapted from Miller Jr. et al. (120). Binding of IL‐1β or TNF‐α to the cell membrane initiates endocytosis and formation of an early endosome (EEA1 and Rab5), which also contains NADPH oxidase subunits Nox1 and p22phox, in addition to ClC‐3. Nox1 is electrogenic, moving electrons from intracellular NADPH through a redox chain within the enzyme into the endosome to reduce oxygen to superoxide. ClC‐3 functions as a chloride‐proton exchanger, required for charge neutralization of the electron flow generated by Nox1. The ROS generated by Nox1 result in NF‐κB activation. Both ClC‐3 and Nox1 are necessary for generation of endosomal reactive oxygen species (ROS) and subsequent NF‐κB activation by IL‐1β or TNF‐α in VSMCs. Statins block ClC‐3 channels, which causes hyperpolarization of the cell membrane, closure of Ca2+ channels and vasorelaxation, and inhibition of cell proliferation. PKC, protein kinase C; PP, serine‐threonine protein phosphatases; α−AR, α‐adrenergic receptor; Gi, heterodimeric inhibitory G protein. Nox: NADPH oxidase (61). (Copyright Request: Duan, Hypertension, 2010)



Figure 8.

Comparative two‐dimensional (2D) electrophoresis analysis of protein expression patterns in membranes of cardiac cells from Clcn3+/+ and Clcn3−/− mice. (A) representative 2D gel depicts Coomassie‐stained proteins from wild‐type (Clcn3+/+) mouse heart. (B) Representative 2D gel depicts Coomassie‐stained proteins from Clcn3−/− mouse heart. (C) Spot sets created from images of 2D gels of both wild‐type and Clcn3−/− mouse heart run under the same conditions as the gels in A and B and compared using Bio‐Rad PDQuest version 7.1.1 software. Three gels were run for each mouse heart type; two hearts were pooled to provide proteins for each gel. The filled symbols indicate changes in protein patterns in Clcn3−/− compared to wild type. A total of 35 proteins consistently changed (minimum criteria: more than twofold change) in membranes from Clcn3−/− mouse heart in all 3 experiments (6 missing proteins, 2 new proteins, 9 upregulated proteins, 15 downregulated proteins, and 2 translocated proteins). The open squares (□) in A, B, and C indicate the location (molecular mass 85 kDa and pI 6.9) of the ClC‐3 protein spot (No. 3812) in the 2D gels, which was independently confirmed by Western blotting using a specific anti‐ClC‐3 C670∼687 antibody (180). [Copyright Request: Yamamoto‐Mizuma et al. (180) with permission from Blackwell Publishing].



Figure 9.

Echocardiographic evaluation of cardiac function. (A) Representative M‐mode echocardiography from wild‐type (Clcn3+/+; left) and heart‐specific ClC‐3 knockout (hsClcn3−/−; right) mice. (B) Echocardiographic measurements in Clcn3+/+ and hsClcn3−/− mice. IVSd, interventricular septum thickness at the end of diastole; LVDd, left ventricular (LV) dimension at the end of diastole; LVPWd, LV posterior wall thickness at the end of diastole; IVSs, interventricular septum thickness at the end of systole; LVDs, LV dimension at the end of systole; LVPWs, LV posterior wall thickness at the end of systole; LVEP, calculated LV ejection fraction; %FS, LV fractional shortening; estimated LV mass, LVM (mg) = 1.05[(IVS + LVID + LVPW)3 − (LVID)3], where 1.05 is the specific gravity of the myocardium. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with Clcn3+/+ mice. C. Single longitudinal section (μm) of hearts to demonstrate all four heart chambers. Longitudinal were stained with hematoxylin and eosin (Bar = 2 mm) (Duan D. et al. unpublished data.)



Figure 10.

Effects of inducible heart‐specific ClC‐3 knockout on cardiac volume‐regulated Cl− current (VRCC) and heart function. (A) Representative current traces in isotonic condition and under hypotonic challenge recorded in freshly isolated atrial myocytes from the inducible heart‐specific ClC‐3 knockout (doxyhsClC‐3−/−) mice with doxycycline (on Doxy) in the diet (panel a), or after withdraw of doxycycline (off Doxy) from the diet for 3 weeks (panel b). (c) Summary of VRCC current densities in isotonic and hypotonic solutions, recorded at +80 mV and −80 mV. (B) Representative M‐mode echocardiography from on Doxy (a) and off Doxy (b) mice. (C) Time‐dependent changes in M‐mode echocardiogram of age matched on Doxy or off Doxy for 1.5 and 3 weeks. *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus off Doxy 0 week; #, P < 0.05; ##, P < 0.01; ###, P < 0.001 versus on Doxy at the same time point. (D) Comparison of hearts isolated from age‐matched (11‐week old) doxyhsClcn3−/− mice on Doxy or off Doxy for 3 weeks. Hearts were cleaned up blood and connective tissues and fixed in 4% paraformaldehyde. [Adapted, with permission, from Xiong et al. (174).]



Figure 11.

Effects of inducible heart‐specific ClC‐3 knockout on cardiac volume‐regulated Cl− current (VRCC) and heart function. (A) Representative current traces in isotonic condition and under hypotonic challenge recorded in freshly isolated atrial myocytes from the inducible heart‐specific ClC‐3 knockout (doxyhsClC‐3−/−) mice with doxycycline (on Doxy) in the diet (panel a), or after withdraw of doxycycline (off Doxy) from the diet for 3 weeks (panel b). (c) Summary of VRCC current densities in isotonic and hypotonic solutions, recorded at +80 mV and −80 mV. (B) Representative M‐mode echocardiography from on Doxy (a) and off Doxy (b) mice. (C) Time‐dependent changes in M‐mode echocardiogram of age matched on Doxy or off Doxy for 1.5 and 3 weeks. *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus off Doxy 0 week; #, P < 0.05; ##, P < 0.01; ###, P < 0.001 versus on Doxy at the same time point. (D) Comparison of hearts isolated from age‐matched (11‐week old) doxyhsClcn3−/− mice on Doxy or off Doxy for 3 weeks. Hearts were cleaned up blood and connective tissues and fixed in 4% paraformaldehyde. [Adapted, with permission, from Xiong et al. (174).]



Figure 12.

Modulation of cardiac electrical activity by activation of ClC‐2 channels in cardiac pacemaker cells and myocytes. Changes in action potentials (top panels) and membrane currents (bottom panels) of cardiac pacemaker cells (A) or atrial and ventricular myocytes (B) due to activation of ClC‐2 channels are depicted. ICl,ir is activated by hyperpolarization, cell swelling, and acidosis. Top panels: numbers illustrate conventional phases of a prototype ventricular action potential under control conditions (black) and after activation of ICl (red). Range of estimates for normal physiological values for Cl− equilibrium potential (ECl) is indicated in blue. Bottom panels: range of zero‐current values corresponding to ECl is shown in grey. (A) Activation of ICl,ir in pacemaker cells during hyperpolarization causes acceleration of phase 4 depolarization and automaticity, shortening of action potential duration, and decrease in cycle length and action potential amplitude (dashed red line in top panel). (B) Activation of ICl,ir in atrial and ventricular myocytes during hyperpolarization causes depolarization of resting membrane potential and induction of phase 4 auto depolarization and abnormal electrical impulse (trigger activity) and automaticity (dotted red line in top panel). [Adapted, with permission, from Duan (50)]. (Copyright Request: Duan D. J Physiol 587: 2163‐2177, 2009)



Figure 13.

Whole‐cell currents recorded from SAN cells of guinea‐pig heart. (A) An example of single SAN cells (arrows) isolated from the SAN region of guinea pig heart by enzymatic dispersion. (B) Whole‐cell currents recorded from SAN cells. When cations (Na+ and K+) were included in the extracellular solutions, inward currents were slowly activated upon hyperpolarization under isotonic (a) conditions. Exposure of the same cell to hypotonic extracellular solution caused cell swelling and an increase in the inward current amplitude (b). The difference current caused by hypotonic cell swelling is shown in panel e. Subsequent replacement of 20 mmol/L of NaCl with CsCl caused a significant inhibition of the inward current (c). The Cs+‐sensitive current is shown in panel f. Subsequent addition of 0.2 mmol/L of Cd2+ to the hypotonic solution caused an inhibition of the inward current (d). The Cd2+‐sensitive currents are shown in panel g (91).



Figure 14.

Molecular expression of ClC‐2 in SAN cells. (A) Localization of ClC‐2 chloride channels in guinea‐pig SAN tissue. (a) Section labeled with anti‐Connexin 43 (red) to illustrate the adjacent atrial (AT) septum was positively labeled while the SAN was negative (dark region), which clearly delineates the SAN region from the AT septum (dashed white line). (b) Section stained with 4′,6‐diamidino‐2‐phenylindole (DAPI) (blue) to compare nuclei density in the SAN region and in AT. The SAN region had a higher DAPI staining density (higher nuclei density) than the adjacent AT. (c) Section stained with anti‐ClC‐2 (green). ClC‐2 immunoreactivity is evident in both SAN and AT regions. (d) Merged images of a, b, and c illustrate that ClC‐2 is expressed in the densely nucleated and Cx43 negative SAN region. (B) Agarose gel depicting real time polymerase chain reaction product of ClC‐2 amplified from mRNA prepared from enzymatically dispersed guinea‐pig SA nodal cells. (C) Images of ClC‐2‐like immunofluorescence in a representative SAN cell visualized using fluorescent microscopy. Phase contrast (a) and fluorescent micrographs (b) of a single SAN cell.



Figure 15.

Effects of Anti‐ClC‐2 Ab on ICl,ir in SAN cells. (A) Representative whole‐cell currents recorded from SAN cells under isotonic (panel a) and hypotonic (panel b) conditions in the presence of anti‐ClC‐2 Ab in the pipette solutions. SAN cells were exposed to isotonic solution for at least 10 min before whole‐cell recordings. Currents shown in panel a were recorded right after successful whole‐cell configuration under isotonic conditions. Currents shown in panel b were recorded after exposure to hypotonic solution for 20 min. Pipette and bath solutions were identical to those described in Figure 1B except the pipette solution contained 3 μg/mL anti‐ClC‐2 Ab. (d) Mean I‐V from 5 SAN cells under the same conditions. (B) SAN cells were exposed to hypotonic solution for 20 min to fully activate ICl,ir before whole‐cell recordings. Bath and pipette solutions were the same as in panel A. Representative current traces recorded by voltage‐clamp (protocol is shown in inset) from the SAN cell immediately after membrane rupture (a) and after 20 min of anti‐ClC‐2 Ab dialysis (b). The anti‐ClC‐2 Ab‐sensitive current (a)‐(b) is shown in (c) (current traces) and (d) (mean I‐V, n = 5). Notice the anti‐ClC‐2 Ab‐sensitive current (c) was similar to ICl,ir shown in Figure 1 and the typical ICa and If (b) were not affected by anti‐ClC‐2 Ab.



Figure 16.

Effects of Anti‐ClC‐2 Ab on pacemaker action potential in SAN cells. (A) Representative spontaneous action potentials recorded from an SAN cell by current‐clamp (no current injection) with pipette solution containing no anti‐ClC‐2 Ab under isotonic (a) and hypotonic (b) conditions. SAN cells were exposed to isotonic solution for at least 10 min before action potential recordings. Action potentials shown in panel a were recorded right after successful whole‐cell configuration under isotonic conditions. Action potentials shown in panel b were recorded after exposure to hypotonic solution for 20 min. For comparison, the action potentials recorded under these conditions were superimposed with an expanded time scale in panel c. The dotted lines indicate zero voltage. (B) Spontaneous action potentials recorded from a SAN cell by current clamp using a pipette solution containing pre‐absorbed anti‐ClC‐2 Ab (control) and cell was exposed to isotonic solutions for 10 min (a) and hypotonic solutions for 20 min (b). For comparison, the action potentials recorded under these conditions were superimposed with an expanded time scale in panel c. (C) SAN cells were perfused with isotonic solutions for 20 min before whole‐cell recordings. Action potentials were recorded immediately after membrane rupture (a) and after dialysis of anti‐ClC‐2 Ab for 20 min (b) under the same isotonic conditions. Panel c shows the expanded and superimposed action potentials as shown in panel a and panel b. Note that after 20 min dialysis of anti‐ClC‐2 Ab in to the cell the spontaneous action potential rate was not significantly altered. (D) SAN cells were exposed to hypotonic solution for 20 min to fully activate ICl,ir before whole‐cell recordings. Action potentials were recorded immediately after membrane rupture (a) and after dialysis of anti‐ClC‐2 Ab for 20 min (b). Panel c shows the expanded and superimposed action potentials as shown in panel a and panel b. Note that the spontaneous action potential rate significantly decreased after 20 min dialysis of anti‐ClC‐2 Ab in to the cell, which corresponds with the decrease in inward



Figure 17.

Telemetry electrocardiogram (ECG) recordings in Clcn2−/− mice and their Clcn2+/+ and Clcn2+/− littermates during treadmill exercises. (A) Representative ECG (Lead II) recordings in Clcn2+/+, Clcn2+/−, and Clcn2−/− mice while they were subjected to treadmill exercise at (a) rest period: acclimation at 0 m/min, incline 0o for 5 min; (b) walk period: walking at 5m/min, incline 0o for 5 min; (c) run period: running at 15 m/min, uphill incline 8o for 5 min. (B) Mean heart rate during the last minute of each treadmill exercise segment for the Clcn2+/+ (n = 6), Clcn2+/‐ (n = 5), and Clcn2−/− (n = 7) mice. (C) Mean heart rate of the Clcn2+/+ (n = 5) and Clcn2−/− (n = 4) mice before (Control, Cont) and after the intraperitoneal injection of atropine (Atro), propranolol (Prop), or atropine plus propranolol (Atro + Prop) during the last minute of each treadmill exercise segment (rest, walk, and run). *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus Clcn2+/+; #, P < 0.05; ##, P < 0.01; ###, P < 0.001 versus Clcn2+/−; $, P < 0.05; $$, P < 0.01; $$$, P < 0.001 versus control (Cont); d, P < 0.05 versus rest (91).



Figure 18.

Modulation of cardiac electrical activity by activation of Ca2+‐activated Cl− channels in heart. Changes in action potentials (top) and membrane currents (bottom) due to activation of Ca2+‐activated Cl− channels are depicted. Top panel: numbers illustrate conventional phases of a prototype ventricular action potential under control conditions (black) and after activation of ICl (red). Range of estimates for normal physiological values for ECl is indicated in blue. Bottom panel: Range of zero‐current values corresponding to ECl is shown in grey. Activation of ICl,Ca during [Ca2+]i overload results in oscillatory transient inward current (ITI) and induction of delayed afterdepolarization (DAD) (dotted red lines). [Adapted, with permission, from Duan (50)]. (Copyright Request: Duan D. J Physiol 587: 2163‐2177, 2009.)

References
 1. Bahinski A, Nairn AC, Greengard P, Gadsby DC. Chloride conductance regulated by cyclic AMP‐dependent protein kinase in cardiac myocytes. Nature 340: 718‐721, 1989.
 2. Baker MA, Lane DJ, Ly JD, De Pinto V, Lawen A. VDAC1 is a transplasma membrane NADH‐ferricyanide reductase. J Biol Chem 279: 4811‐4819, 2004.
 3. Batthish M, Diaz RJ, Zeng HP, Backx PH, Wilson GJ. Pharmacological preconditioning in rabbit myocardium is blocked by chloride channel inhibition. Cardiovasc Res 55: 660‐671, 2002.
 4. Baumgarten CM, Clemo HF. Swelling‐activated chloride channels in cardiac physiology and pathophysiology. Prog Biophys Mol Biol 82: 25‐42, 2003.
 5. Baumgarten CM, Fozzard HA. Intracellular chloride activity in mammalian ventricular muscle. Am J Physiol 241: C121‐C129, 1981.
 6. Berthonneche C, Peter B, Schupfer F, Hayoz P, Kutalik Z, Abriel H, Pedrazzini T, Beckmann JS, Bergmann S, Maurer F. Cardiovascular response to beta‐adrenergic blockade or activation in 23 inbred mouse strains. PLoS ONE 4: e6610, 2009.
 7. Bilder RM, Sabb FW, Cannon TD, London ED, Jentsch JD, Parker DS, Poldrack RA, Evans C, Freimer NB. Phenomics: The systematic study of phenotypes on a genome‐wide scale. Neuroscience 164: 30‐42, 2009.
 8. Blume AJ, Beasley J, Goldstein NI. The use of peptides in Diogenesis: A novel approach to drug discovery and phenomics. Biopolymers 55: 347‐356, 2000.
 9. Bogue MA, Grubb SC. The Mouse Phenome Project. Genetica 122: 71‐74, 2004.
 10. Borsani G, Rugarli EI, Taglialatela M, Wong C, Ballabio A. Characterization of a human and murine gene (CLCN3) sharing similarities to voltage‐gated chloride channels and to a yeast integral membrane protein. Genomics 27: 131‐141, 1995.
 11. Boujaoude LC, Bradshaw‐Wilder C, Mao C, Cohn J, Ogretmen B, Hannun YA, Obeid LM. Cystic fibrosis transmembrane regulator regulates uptake of sphingoid base phosphates and lysophosphatidic acid: Modulation of cellular activity of sphingosine 1‐phosphate. J Biol Chem 276: 35258‐35264, 2001.
 12. Bozeat N, Dwyer L, Ye L, Yao T, Duan D. The role of ClC‐3 chloride channels in early and late ischemic preconditioning in mouse heart. FASEB J 19(4): A694‐A695, 2005.
 13. Bozeat N, Dwyer L, Ye L, Yao TY, Hatton WJ, Duan D. VSOACs play an important cardioprotective role in late ischemic preconditioning in mouse heart. Circulation 114: 272‐273 (1425), 2006.
 14. Britton FC, Hatton WJ, Rossow CF, Duan D, Hume JR, Horowitz B. Molecular distribution of volume‐regulated chloride channels (ClC‐2 and ClC‐3) in cardiac tissues. Am J Physiol Heart Circ Physiol 279: H2225‐H2233, 2000.
 15. Britton FC, Ohya S, Horowitz B, Greenwood IA. Comparison of the properties of CLCA1 generated currents and I(Cl(Ca)) in murine portal vein smooth muscle cells. J Physiol 539: 107‐117, 2002.
 16. Britton FC, Wang GL, Huang ZM, Ye L, Horowitz B, Hume JR, Duan D. Functional characterization of novel alternatively spliced ClC‐2 chloride channel variants in the heart. J Biol Chem 280: 25871‐25880, 2005.
 17. Browe DM, Baumgarten CM. Stretch of beta 1 integrin activates an outwardly rectifying chloride current via FAK and Src in rabbit ventricular myocytes. J Gen Physiol 122: 689‐702, 2003.
 18. Browe DM, Baumgarten CM. Angiotensin II (AT1) receptors and NADPH oxidase regulate Cl‐ current elicited by beta1 integrin stretch in rabbit ventricular myocytes. J Gen Physiol 124: 273‐287, 2004.
 19. Burd L, Klug MG, Martsolf JT, Kerbeshian J. Fetal alcohol syndrome: Neuropsychiatric phenomics. Neurotoxicol Teratol 25: 697‐705, 2003.
 20. Caille JP, Ruiz‐Ceretti E, Schanne OF. Intracellular chloride activity in rabbit papillary muscle: Effect of ouabain. Am J Physiol 240: C183‐C188, 1981.
 21. Caputo A, Caci E, Ferrera L, Pedemonte N, Barsanti C, Sondo E, Pfeffer U, Ravazzolo R, Zegarra‐Moran O, Galietta LJ. TMEM16A, a membrane protein associated with calcium‐dependent chloride channel activity. Science 322: 590‐594, 2008.
 22. Carmeliet E. Cardiac ionic currents and acute ischemia: From channels to arrhythmias. Physiol Rev 79: 917‐1017, 1999.
 23. Chen H, Liu LL, Ye LL, McGuckin C, Tamowski S, Scowen P, Tian H, Murray K, Hatton WJ, Duan D. Targeted inactivation of cystic fibrosis transmembrane conductance regulator chloride channel gene prevents ischemic preconditioning in isolated mouse heart. Circulation 110: 700‐704, 2004.
 24. Chen Z, Chua CC, Ho YS, Hamdy RC, Chua BH. Overexpression of Bcl‐2 attenuates apoptosis and protects against myocardial I/R injury in transgenic mice. Am J Physiol Heart Circ Physiol 280: H2313‐H2320, 2001.
 25. Choi BR, Hatton WJ, Hume JR, Liu T, Salama G. Low osmolarity transforms ventricular fibrillation from complex to highly organized, with a dominant high‐frequency source. Heart Rhythm 3: 1210‐1220, 2006.
 26. Cid LP, Montrose‐Rafizadeh C, Smith DI, Guggino WB, Cutting GR. Cloning of a putative human voltage‐gated chloride channel (CIC‐2) cDNA widely expressed in human tissues. Hum Mol Genet 4: 407‐413, 1995.
 27. Cid LP, Niemeyer MI, Ramirez A, Sepulveda FV. Splice variants of a ClC‐2 chloride channel with differing functional characteristics. Am J Physiol Cell Physiol 279: C1198‐C1210, 2000.
 28. Cid LP, Ramirez A, Niemeyer MI, Sepulveda FV. Characterisation of ClC‐2 chloride channel splice variants. J Physiol (Lond) 523P: 4P, 2000.
 29. Clemo HF, Baumgarten CM. Protein kinase C activation blocks ICl(swell) and causes myocyte swelling in a rabbit congestive heart failure model. Circulation 98: I‐695, 1998.
 30. Clemo HF, Danetz JS, Baumgarten CM. Does ClC‐3 modulate cardiac cell volume? Biophys J 76: A203, 1999.
 31. Clemo HF, Stambler BS, Baumgarten CM. Swelling‐activated chloride current is persistently activated in ventricular myocytes from dogs with tachycardia‐induced congestive heart failure. Circ Res 84: 157‐165, 1999.
 32. Collardeau‐Frachon S, Bouvier R, Le GC, Rivet C, Cabet F, Bellon G, Lachaux A, Scoazec JY. Unexpected diagnosis of cystic fibrosis at liver biopsy: A report of four pediatric cases. Virchows Arch 451: 57‐64, 2007.
 33. Collier ML, Hume JR. Unitary chloride channels activated by protein kinase C in guinea pig ventricular myocytes. Circ Res 76: 317‐324, 1995.
 34. Collier ML, Levesque PC, Kenyon JL, Hume JR. Unitary Cl‐ channels activated by cytoplasmic Ca2+ in canine ventricular myocytes. Circ Res 78: 936‐944, 1996.
 35. Conrad M, Rizki MM. The artificial worlds approach to emergent evolution. Biosystems 23: 247‐258, 1989.
 36. Cuppoletti J, Malinowska DH, Tewari KP, Li QJ, Sherry AM, Patchen ML, Ueno R. SPI‐0211 activates T84 cell chloride transport and recombinant human ClC‐2 chloride currents. Am J Physiol Cell Physiol 287: C1173‐C1183, 2004.
 37. Cuppoletti J, Tewari KP, Sherry AM, Ferrante CJ, Malinowska DH. Sites of protein kinase A activation of the human ClC‐2 Cl(‐) channel. J Biol Chem 279: 21849‐21856, 2004.
 38. da Silva GA, Holt JG. Numerical taxonomy of certain coryneform bacteria. J Bacteriol 90: 921‐927, 1965.
 39. Davies WL, Vandenberg JI, Sayeed RA, Trezise AE. Post‐transcriptional regulation of the cystic fibrosis gene in cardiac development and hypertrophy. Biochem Biophys Res Commun 319: 410‐418, 2004.
 40. De Mello WC. Heart failure: How important is cellular sequestration? The role of the renin‐angiotensin‐aldosterone system. J Mol Cell Cardiol 37: 431‐438, 2004.
 41. Deng W, Baki L, Baumgarten CM. Endothelin signalling regulates volume‐sensitive Cl‐ current via NADPH oxidase and mitochondrial reactive oxygen species. Cardiovasc Res 88: 93‐100, 2010.
 42. Diaz RJ, Batthish M, Backx PH, Wilson GJ. Chloride channel inhibition does block the protection of ischemic preconditioning in myocardium. J Mol Cell Cardiol 33: 1887‐1889, 2001.
 43. Diaz RJ, Losito VA, Mao GD, Ford MK, Backx PH, Wilson GJ. Chloride channel inhibition blocks the protection of ischemic preconditioning and hypo‐osmotic stress in rabbit ventricular myocardium. Circ Res 84: 763‐775, 1999.
 44. DiFrancesco D. Properties of the cardiac pacemaker (if) current. Boll Soc Ital Biol Sper 60(Suppl 4): 29‐33, 1984.
 45. DiFrancesco D. Funny channels in the control of cardiac rhythm and mode of action of selective blockers. Pharmacol Res 53: 399‐406, 2006.
 46. Dobrzynski H, Boyett MR, Anderson RH. New insights into pacemaker activity: Promoting understanding of sick sinus syndrome. Circulation 115: 1921‐1932, 2007.
 47. Du XY, Sorota S. Cardiac swelling‐induced chloride current depolarizes canine atrial myocytes. Am J Physiol 272: H1904‐H1916, 1997.
 48. Duan D. Phenomics of cardiac chloride channels: The systematic study of chloride channel function in the heart. J Physiol 587: 2163‐2177, 2009.
 49. Duan D, Cowley S, Horowitz B, Hume JR. A serine residue in ClC‐3 links phosphorylation‐dephosphorylation to chloride channel regulation by cell volume. J Gen Physiol 113: 57‐70, 1999.
 50. Duan D, Fermini B, Nattel S. Sustained outward current observed after I(to1) inactivation in rabbit atrial myocytes is a novel Cl‐ current. Am J Physiol 263: H1967‐H1971, 1992.
 51. Duan D, Fermini B, Nattel S. Alpha‐adrenergic control of volume‐regulated Cl‐ currents in rabbit atrial myocytes. Characterization of a novel ionic regulatory mechanism. Circ Res 77: 379‐393, 1995.
 52. Duan D, Hume JR, Nattel S. Evidence that outwardly rectifying Cl‐ channels underlie volume‐ regulated Cl‐ currents in heart. Circ Res 80: 103‐113, 1997.
 53. Duan D, Liu L, Wang GL, Ye L, Tian H, Yao Y, Chen A, Duan M, Hatton W. Cell volume‐regulated ion channels and ionic remodeling in hypertrophied mouse heart. J Cardiac Failure 10: S72, 2004.
 54. Duan D, Nattel S. Properties of single outwardly rectifying Cl‐ channels in heart. Circ Res 75: 789‐795, 1994.
 55. Duan D, Winter C, Cowley S, Hume JR, Horowitz B. Molecular identification of a volume‐regulated chloride channel. Nature 390: 417‐421, 1997.
 56. Duan D, Ye L, Britton F, Horowitz B, Hume JR. UltraRapid communications : A novel anionic inward rectifier in native cardiac myocytes. Circ Res 86: 485, 2000.
 57. Duan D, Ye L, Britton F, Miller LJ, Yamazaki J, Horowitz B, Hume JR. Purinoceptor‐coupled Cl‐ channels in mouse heart: A novel, alternative pathway for CFTR regulation. J Physiol 521(Pt 1): 43‐56, 1999.
 58. Duan D, Zhong J, Hermoso M, Satterwhite CM, Rossow CF, Hatton WJ, Yamboliev I, Horowitz B, Hume JR. Functional inhibition of native volume‐sensitive outwardly rectifying anion channels in muscle cells and Xenopus oocytes by anti‐ClC‐3 antibody. J Physiol 531: 437‐444, 2001.
 59. Duan DD. Volume matters: Novel roles of the volume‐regulated CLC‐3 channels in hypertension‐induced cerebrovascular remodeling. Hypertension 56: 346‐348, 2010.
 60. Duan DY, Liu LL, Bozeat N, Huang ZM, Xiang SY, Wang GL, Ye L, Hume JR. Functional role of anion channels in cardiac diseases. Acta Pharmacol Sin 26: 265‐278, 2005.
 61. Dutzler R, Campbell EB, Cadene M, Chait BT, MacKinnon R. X‐ray structure of a ClC chloride channel at 3.0 A reveals the molecular basis of anion selectivity. Nature 415: 287‐294, 2002.
 62. Elble RC, Pauli BU. Tumor suppression by a proapoptotic calcium‐activated chloride channel in mammary epithelium. J Biol Chem 276: 40510‐40517, 2001.
 63. Estevez R, Boettger T, Stein V, Birkenhager R, Otto E, Hildebrandt F, Jentsch TJ. Barttin is a Cl‐ channel beta‐subunit crucial for renal Cl‐ reabsorption and inner ear K +secretion. Nature 414: 558‐561, 2001.
 64. Frace AM, Maruoka F, Noma A. Control of the hyperpolarization‐activated cation current by external anions in rabbit sino‐atrial node cells. J Physiol (Lond) 453: 307‐318, 1992.
 65. Freimer N, Sabatti C. The human phenome project. Nat Genet 34: 15‐21, 2003.
 66. Fritsch J, Edelman A. Modulation of the hyperpolarization‐activated Cl‐ current in human intestinal T84 epithelial cells by phosphorylation. J Physiol 490(Pt 1): 115‐128, 1996.
 67. Furukawa T, Horikawa S, Terai T, Ogura T, Katayama Y, Hiraoka M. Molecular cloning and characterization of a novel truncated from (ClC‐2 beta) of ClC‐2 alpha (ClC‐2G) in rabbit heart. FEBS Lett 375: 56‐62, 1995.
 68. Furukawa T, Ogura T, Katayama Y, Hiraoka M. Characteristics of rabbit ClC‐2 current expressed in Xenopus oocytes and its contribution to volume regulation. Am J Physiol 274: C500‐C512, 1998.
 69. Furukawa T, Ogura T, Zheng YJ, Tsuchiya H, Nakaya H, Katayama Y, Inagaki N. Phosphorylation and functional regulation of ClC‐2 chloride channels expressed in Xenopus oocytes by M cyclin‐dependent protein kinase. J Physiol 540: 883‐893, 2002.
 70. Gadsby DC, Nairn AC. Control of CFTR channel gating by phosphorylation and nucleotide hydrolysis. Physiol Rev 79: S77‐S107, 1999.
 71. Gao L, Kim KJ, Yankaskas JR, Forman HJ. Abnormal glutathione transport in cystic fibrosis airway epithelia. Am J Physiol 277: L113‐L118, 1999.
 72. Gerlai R. Phenomics: Fiction or the future? Trends Neurosci 25: 506‐509, 2002.
 73. Giles WR, Imaizumi Y. Comparison of potassium currents in rabbit atrial and ventricular cells. J Physiol 405: 123‐145, 1988.
 74. Guan YY, Wang GL, Zhou JG. The ClC‐3 Cl‐ channel in cell volume regulation, proliferation and apoptosis in vascular smooth muscle cells. Trends Pharmacol Sci 27: 290‐296, 2006.
 75. Guggino SE. Can we generate new hypotheses about Dent disease from gene analysis? Exp Physiol 94: 191‐196, 2009.
 76. Guggino WB, Banks‐Schlegel SP. Macromolecular interactions and ion transport in cystic fibrosis. Am J Respir Crit Care Med 170: 815‐820, 2004.
 77. Guggino WB, Stanton BA. New insights into cystic fibrosis: Molecular switches that regulate CFTR. Nat Rev Mol Cell Biol 7: 426‐436, 2006.
 78. Hagiwara N, Masuda H, Shoda M, Irisawa H. Stretch‐activated anion currents of rabbit cardiac myocytes. J Physiol (Lond) 456: 285‐302, 1992.
 79. Hartzell C, Putzier I, Arreola J. Calcium‐activated chloride channels. Annu Rev Physiol 67: 719‐758, 2005.
 80. Harvey RD. Cardiac chloride currents. News Physiol Sci 11: 175‐181, 1996.
 81. Harvey RD, Hume JR. Autonomic regulation of a chloride current in heart. Science 244: 983‐985, 1989.
 82. Harvey RD, Hume JR. Histamine activates the chloride current in cardiac ventricular myocytes. J Cardiovas Electrophysiol 1: 309‐317, 1990.
 83. Hermoso M, Satterwhite CM, Andrade YN, Hidalgo J, Wilson SM, Horowitz B, Hume JR. ClC‐3 is a fundamental molecular component of volume‐sensitive outwardly rectifying Cl‐ channels and volume regulation in HeLa cells and Xenopus laevis oocytes. J Biol Chem 277: 40066‐40074, 2002.
 84. Heusch G, Liu GS, Rose J, Cohen MV, Downey JM. No confirmation for a causal role of volume‐regulated chloride channels in ischemic preconditioning in rabbits. J Mol Cell Cardiol 32: 2279‐2285, 2000.
 85. Hiraoka M, Kawano S, Hirano Y, Furukawa T. Role of cardiac chloride currents in changes in action potential characteristics and arrhythmias. Cardiovasc Res 40: 23‐33, 1998.
 86. Houser SR, Piacentino V, III, Weisser J. Abnormalities of calcium cycling in the hypertrophied and failing heart. J Mol Cell Cardiol 32: 1595‐1607, 2000.
 87. Huang X, Godfrey TE, Gooding WE, McCarty KS, Jr, Gollin SM. Comprehensive genome and transcriptome analysis of the 11q13 amplicon in human oral cancer and synteny to the 7F5 amplicon in murine oral carcinoma. Genes Chromosomes Cancer 45: 1058‐1069, 2006.
 88. Huang ZM, Britton FC, An C, Yuan C, Ye L, Hatton WJ, Duan D. Characterization of ClC‐2 channel/PKA interaction in mouse heart. FASEB J 22, 721.5. 2008.
 89. Huang ZM, Prasad C, Britton FC, Ye LL, Hatton WJ, Duan D. Functional role of CLC‐2 chloride inward rectifier channels in cardiac sinoatrial nodal pacemaker cells. J Mol Cell Cardiol 47: 121‐132, 2009.
 90. Hume JR, Duan D, Collier ML, Yamazaki J, Horowitz B. Anion transport in heart. Physiol Rev 80: 31‐81, 2000.
 91. Hume JR, Wang GX, Yamazaki J, Ng LC, Duan D. CLC‐3 chloride channels in the pulmonary vasculature. Adv Exp Med Biol 661: 237‐247, 2010.
 92. Intengan HD, Schiffrin EL. Vascular remodeling in hypertension: Roles of apoptosis, inflammation, and fibrosis. Hypertension 38: 581‐587, 2001.
 93. January CT, Fozzard HA. Delayed afterdepolarizations in heart muscle: Mechanisms and relevance. Pharmacol Rev 40: 219‐227, 1988.
 94. Jentsch TJ, Stein V, Weinreich F, Zdebik AA. Molecular structure and physiological function of chloride channels. Physiol Rev 82: 503‐568, 2002.
 95. Jordt SE, Jentsch TJ. Molecular dissection of gating in the ClC‐2 chloride channel. EMBO J 16: 1582‐1592, 1997.
 96. Kajita H, Omori K, Matsuda H. The chloride channel ClC‐2 contributes to the inwardly rectifying Cl‐ conductance in cultured porcine choroid plexus epithelial cells. J Physiol 523(Pt 2): 313‐324, 2000.
 97. Kajita H, Whitwell C, Brown PD. Properties of the inward‐rectifying Cl‐ channel in rat choroid plexus: Regulation by intracellular messengers and inhibition by divalent cations. Pflugers Arch 440: 933‐940, 2000.
 98. Kajita H, Whitwell C, Brown PD. Properties of the inward‐rectifying Cl‐ channel in rat choroid plexus: Regulation by intracellular messengers and inhibition by divalent cations. Pflugers Arch 440: 933‐940, 2000.
 99. Kogan I, Ramjeesingh M, Li C, Kidd JF, Wang Y, Leslie EM, Cole SP, Bear CE. CFTR directly mediates nucleotide‐regulated glutathione flux. EMBO J 22: 1981‐1989, 2003.
 100. Komukai K, Brette F, Orchard CH. Electrophysiological response of rat atrial myocytes to acidosis. Am J Physiol Heart Circ Physiol 283: H715‐H724, 2002.
 101. Komukai K, Brette F, Pascarel C, Orchard CH. Electrophysiological response of rat ventricular myocytes to acidosis. Am J Physiol Heart Circ Physiol 283: H412‐H422, 2002.
 102. Krieg RE, Lockhart WR. Classification of enterobacteria based on overall similarity. J Bacteriol 92: 1275‐1280, 1966.
 103. Kunzelmann K, Schreiber R, Boucherot A. Mechanisms of the inhibition of epithelial Na(+) channels by CFTR and purinergic stimulation. Kidney Int 60: 455‐461, 2001.
 104. Kunzelmann K, Schreiber R, Nitschke R, Mall M. Control of epithelial Na+ conductance by the cystic fibrosis transmembrane conductance regulator. Pflugers Arch 440: 193‐201, 2000.
 105. Lang F, Busch GL, Ritter M, Volkl H, Waldegger S, Gulbins E, Haussinger D. Functional significance of cell volume regulatory mechanisms. Physiol Rev 78: 247‐306, 1998.
 106. Lemonnier L, Shuba Y, Crepin A, Roudbaraki M, Slomianny C, Mauroy B, Nilius B, Prevarskaya N, Skryma R. Bcl‐2‐dependent modulation of swelling‐activated Cl‐ current and ClC‐3 expression in human prostate cancer epithelial cells. Cancer Res 64: 4841‐4848, 2004.
 107. Levesque PC, Hume JR. ATPo but not cAMPi activates a chloride conductance in mouse ventricular myocytes. Cardiovasc Res 29: 336‐343, 1995.
 108. Li Y, Trush MA. Diphenyleneiodonium, an NAD(P)H oxidase inhibitor, also potently inhibits mitochondrial reactive oxygen species production. Biochem Biophys Res Commun 253: 295‐299, 1998.
 109. Linsdell P, Hanrahan JW. Glutathione permeability of CFTR. Am J Physiol 275: C323‐C326, 1998.
 110. Liu J, Noble PJ, Xiao G, Abdelrahman M, Dobrzynski H, Boyett MR, Lei M, Noble D. Role of pacemaking current in cardiac nodes: Insights from a comparative study of sinoatrial node and atrioventricular node. Prog Biophys Mol Biol 96: 294‐304, 2007.
 111. Liu L, Ye L, McGuckin C, Hatton WJ, Duan D. Disruption of Clcn3 gene in mice facilitates heart failure during pressure overload. J Gen Physiol 122: 76, 2003.
 112. Liu YJ, Wang XG, Tang YB, Chen JF, Lv XF, Zhou JG, Guan YY. Simvastatin ameliorates rat cerebrovascular remodeling during hypertension via inhibition of volume‐regulated chloride channel. Hypertension (in press), 2010.
 113. Loewen ME, MacDonald DW, Gaspar KJ, Forsyth GW. Isoform‐specific exon skipping in a variant form of ClC‐2. Biochim Biophys Acta 1493: 284‐288, 2000.
 114. Malinowska DH, Kupert EY, Bahinski A, Sherry AM, Cuppoletti J. Cloning, functional expression, and characterization of a PKA‐activated gastric Cl‐ channel. Am J Physiol 268: C191‐C200, 1995.
 115. Miller FJ, Jr., Filali M, Huss GJ, Stanic B, Chamseddine A, Barna TJ, Lamb FS. Cytokine activation of nuclear factor kappa B in vascular smooth muscle cells requires signaling endosomes containing Nox1 and ClC‐3. Circ Res 101: 663‐671, 2007.
 116. Minagawa N, Nagata J, Shibao K, Masyuk AI, Gomes DA, Rodrigues MA, LeSage G, Akiba Y, Kaunitz JD, Ehrlich BE, LaRusso NF, Nathanson MH. Cyclic AMP regulates bicarbonate secretion in cholangiocytes through release of ATP into bile. Gastroenterology 133: 1592‐1602, 2007.
 117. Mizoguchi K, Maeta H, Yamamoto A, Oe M, Kosaka H. Amelioration of myocardial global ischemia/reperfusion injury with volume‐regulatory chloride channel inhibitors in vivo. Transplantation 73: 1185‐1193, 2002.
 118. Moore RC, Lee IY, Silverman GL, Harrison PM, Strome R, Heinrich C, Karunaratne A, Pasternak SH, Chishti MA, Liang Y, Mastrangelo P, Wang K, Smit AF, Katamine S, Carlson GA, Cohen FE, Prusiner SB, Melton DW, Tremblay P, Hood LE, Westaway D. Ataxia in prion protein (PrP)‐deficient mice is associated with upregulation of the novel PrP‐like protein doppel. J Mol Biol 292: 797‐817, 1999.
 119. Moss WW, Webster WA. Phenetics and numerical taxonomy applied to systematic nematology. J Nematol 2: 16‐25, 1970.
 120. Nagel G, Hwang TC, Nastiuk KL, Nairn AC, Gadsby DC. The protein kinase A‐regulated cardiac Cl‐ channel resembles the cystic fibrosis transmembrane conductance regulator. Nature 360: 81‐84, 1992.
 121. Nakajima T, Sugimoto T, Kurachi Y. Effects of anions on the G protein‐mediated activation of the muscarinic K+ channel in the cardiac atrial cell membrane. Intracellular chloride inhibition of the GTPase activity of GK. J Gen Physiol 99: 665‐682, 1992.
 122. Naren AP, Kirk KL. CFTR chloride channels: binding partners and regulatory networks. News Physiol Sci 15: 57‐61, 2000.
 123. O'Driscoll KE, Hatton WJ, Burkin HR, Leblanc N, Britton FC. Expression, localization and functional properties of Bestrophin 3 channel isolated from mouse heart. Am J Physiol Cell Physiol 295: C1610‐C1624, 2008.
 124. O'Driscoll KE, Leblanc N, Britton FC. Molecular and functional characterization of murine Bestrophin 1 cloned from Heart. FASEB J 22: 1201.25, 2008.
 125. Ohba M. Effects of tonicity on the pacemaker activity of guinea‐pig sino‐atrial node. Jpn J Physiol 36: 1027‐1038, 1986.
 126. Okada Y, Shimizu T, Maeno E, Tanabe S, Wang X, Takahashi N. Volume‐sensitive chloride channels involved in apoptotic volume decrease and cell death. J Membr Biol 209: 21‐29, 2006.
 127. Paigen K, Eppig JT. A mouse phenome project. Mamm Genome 11: 715‐717, 2000.
 128. Park K, Begenisich T, Melvin JE. Protein kinase A activation phosphorylates the rat ClC‐2 Cl‐ channel but does not change activity. J Membr Biol 182: 31‐37, 2001.
 129. Patel DG, Higgins RS, Baumgarten CM. Swelling‐activated cl current, ICl,swell, is chronically activated in diseased human atrial myocytes. Biophys J 84: 233, 2003.
 130. Rees SA, Vandenberg JI, Wright AR, Yoshida A, Powell T. Cell swelling has differential effects on the rapid and slow components of delayed rectifier potassium current in guinea pig cardiac myocytes. J Gen Physiol 106: 1151‐1170, 1995.
 131. Reisin IL, Prat AG, Abraham EH, Amara JF, Gregory RJ, Ausiello DA, Cantiello HF. The cystic fibrosis transmembrane conductance regulator is a dual ATP and chloride channel. J Biol Chem 269: 20584‐20591, 1994.
 132. Ren Z, Raucci FJ, Jr., Browe DM, Baumgarten CM. Regulation of swelling‐activated Cl(‐) current by angiotensin II signalling and NADPH oxidase in rabbit ventricle. Cardiovasc Res 77: 73‐80, 2008.
 133. Ruiz PE, Ponce ZA, Schanne OF. Early action potential shortening in hypoxic hearts: Role of chloride current(s) mediated by catecholamine release. J Mol Cell Cardiol 28: 279‐290, 1996.
 134. Sabirov RZ, Okada Y. ATP release via anion channels. Purinergic Signal 1: 311‐328, 2005.
 135. Sachar DB. Genomics and phenomics in Crohn's disease. Gastroenterology 122: 1161‐1162, 2002.
 136. Schilling CH, Edwards JS, Palsson BO. Toward metabolic phenomics: Analysis of genomic data using flux balances. Biotechnol Prog 15: 288‐295, 1999.
 137. Schork NJ. Genetics of complex disease: Approaches, problems, and solutions. Am J Respir Crit Care Med 156: S103‐S109, 1997.
 138. Schroeder BC, Cheng T, Jan YN, Jan LY. Expression cloning of TMEM16A as a calcium‐activated chloride channel subunit. Cell 134: 1019‐1029, 2008.
 139. Sherry AM, Stroffekova K, Knapp LM, Kupert EY, Cuppoletti J, Malinowska DH. Characterization of the human pH‐ and PKA‐activated ClC‐2G(2 alpha) Cl‐ channel. Am J Physiol 273: C384‐C393, 1997.
 140. Skach WR. CFTR: New members join the fold. Cell 127: 673‐675, 2006.
 141. Solbach TF, Paulus B, Weyand M, Eschenhagen T, Zolk O, Fromm MF. ATP‐binding cassette transporters in human heart failure. Naunyn Schmiedebergs Arch Pharmacol 377: 231‐243, 2008.
 142. Sorota S. Swelling‐induced chloride‐sensitive current in canine atrial cells revealed by whole‐cell patch‐clamp method. Circ Res 70: 679‐687, 1992.
 143. Soule M, Baker B. Phenetics of natural populations. IV. The population asymmetry parameter in the butterfly Coenonympha tullia. Heredity 23: 611‐614, 1968.
 144. Spitzer KW, Walker JL. Intracellular chloride activity in quiescent cat papillary muscle. Am J Physiol 238: H487‐H493, 1980.
 145. Staley K, Smith R, Schaack J, Wilcox C, Jentsch TJ. Alteration of GABAA receptor function following gene transfer of the CLC‐2 chloride channel. Neuron 17: 543‐551, 1996.
 146. Sugita M, Yue Y, Foskett JK. CFTR Cl‐ channel and CFTR‐associated ATP channel: Distinct pores regulated by common gates. EMBO J 17: 898‐908, 1998.
 147. Thiemann A, Grunder S, Pusch M, Jentsch TJ. A chloride channel widely expressed in epithelial and non‐epithelial cells. Nature 356: 57‐60, 1992.
 148. Tomaselli GF, Marban E. Electrophysiological remodeling in hypertrophy and heart failure. Cardiovasc Res 42: 270‐283, 1999.
 149. van Borren MM, Verkerk AO, Vanharanta SK, Baartscheer A, Coronel R, Ravesloot JH. Reduced swelling‐activated Cl(‐) current densities in hypertrophied ventricular myocytes of rabbits with heart failure. Cardiovasc Res 53: 869‐878, 2002.
 150. Vandenberg JI, Bett GC, Powell T. Contribution of a swelling‐activated chloride current to changes in the cardiac action potential. Am J Physiol 273: C541‐C547, 1997.
 151. Vaughan‐Jones RD. Non‐passive chloride distribution in mammalian heart muscle: Micro‐electrode measurement of the intracellular chloride activity. J Physiol (Lond) 295: 83‐109, 1979.
 152. Vaughan‐Jones RD. Chloride activity and its control in skeletal and cardiac muscle. Philos Trans R Soc Lond B Biol Sci 299: 537‐548, 1982.
 153. Verkerk AO, Tan HL, Ravesloot JH. Ca2+‐activated Cl‐ current reduces transmural electrical heterogeneity within the rabbit left ventricle. Acta Physiol Scand 180: 239‐247, 2004.
 154. Verkerk AO, Veldkamp MW, Baartscheer A, Schumacher CA, Klopping C, van Ginneken AC, Ravesloot JH. Ionic mechanism of delayed afterdepolarizations in ventricular cells isolated from human end‐stage failing hearts. Circulation 104: 2728‐2733, 2001.
 155. Verkerk AO, Veldkamp MW, Bouman LN, van Ginneken AC. Calcium‐activated Cl(‐) current contributes to delayed afterdepolarizations in single Purkinje and ventricular myocytes. Circulation 101: 2639‐2644, 2000.
 156. Verkerk AO, Wilders R, Coronel R, Ravesloot JH, Verheijck EE. Ionic remodeling of sinoatrial node cells by heart failure. Circulation 108: 760‐766, 2003.
 157. Verkerk AO, Wilders R, Zegers JG, van Borren MM, Ravesloot JH, Verheijck EE. Ca(2+)‐activated Cl(‐) current in rabbit sinoatrial node cells. J Physiol 540: 105‐117, 2002.
 158. Vilela RM, Lands LC, Meehan B, Kubow S. Inhibition of IL‐8 release from CFTR‐deficient lung epithelial cells following pre‐treatment with fenretinide. Int Immunopharmacol 6: 1651‐1664, 2006.
 159. Volk AP, Heise CK, Hougen JL, Artman CM, Volk KA, Wessels D, Soll DR, Nauseef WM, Lamb FS, Moreland JG. CLC‐3 and ICLswell are required for normal neutrophil chemotaxis and shape change. J Biol Chem 283: 34315‐34326, 2008.
 160. Walsh KB, Long KJ. Properties of a protein kinase C‐activated chloride current in guinea pig ventricular myocytes. Circ Res 74: 121‐129, 1994.
 161. Wang GX, Hatton WJ, Wang GL, Zhong J, Yamboliev I, Duan D, Hume JR. Functional effects of novel anti‐ClC‐3 antibodies on native volume‐sensitive osmolyte and anion channels in cardiac and smooth muscle cells. Am J Physiol Heart Circ Physiol 285: H1453‐H1463, 2003.
 162. Wang X, Venable J, LaPointe P, Hutt DM, Koulov AV, Coppinger J, Gurkan C, Kellner W, Matteson J, Plutner H, Riordan JR, Kelly JW, Yates JR, III, Balch WE. Hsp90 cochaperone Aha1 downregulation rescues misfolding of CFTR in cystic fibrosis. Cell 127: 803‐815, 2006.
 163. Wei L, Xiao AY, Jin C, Yang A, Lu ZY, Yu SP. Effects of chloride and potassium channel blockers on apoptotic cell shrinkage and apoptosis in cortical neurons. Pflugers Arch 448: 325‐334, 2004.
 164. Wong KR, Trezise AE, Crozatier B, Vandenberg JI. Loss of the normal epicardial to endocardial gradient of cftr mRNA expression in the hypertrophied rabbit left ventricle. Biochem Biophys Res Commun 278: 144‐149, 2000.
 165. Wright J, Morales MM, Sousa‐Menzes J, Ornellas D, Sipes J, Cui Y, Cui I, Hulamm P, Cebotaru V, Cebotaru L, Guggino WB, Guggino SE. Transcriptional adaptation to Clcn5 knockout in proximal tubules of mouse kidney. Physiol Genomics 33: 341‐354, 2008.
 166. Xiang SY, Schegg K, Ye LL, Hatton WJ, Duan D. VDAC‐1 may interact with CFTR to impart important cellular function in mouse heart. FASEB J 21(726.3): A799, 2007.
 167. Xiang SY, Ye LL, Hatton WJ, Duan D. ATPo‐activated chloride channels play a key role in postconditioning‐induced cardioprotection in mouse heart. FASEB J 22: 1130.10, 2008.
 168. Xiong D, Heyman NS, Airey J, Zhang M, Singer CA, Rawat S, Ye L, Evans R, Burkin DJ, Tian H, McCloskey DT, Valencik M, Britton FC, Duan D, Hume JR. Cardiac‐specific, inducible ClC‐3 gene deletion eliminates native volume‐sensitive chloride channels and produces myocardial hypertrophy in adult mice. J Mol Cell Cardiol 48: 211‐219, 2010.
 169. Xiong D, Wang GX, Burkin DJ, Yamboliev IA, Singer CA, Rawat S, Scowen P, Evans R, Ye L, Hatton WJ, Tian H, Keller PS, McCloskey DT, Duan D, Hume JR. Cardiac‐specific overexpression of the human short CLC‐3 chloride channel isoform in mice. Clin Exp Pharmacol Physiol 36: 386‐393, 2009.
 170. Xiong D, Ye L, Neveux I, Burkin DJ, Scowen P, Evans R, Valencik M, Duan D, Hume JR. Cardiac specific inactivation of ClC‐3 gene reveals cardiac hypertrophy and compromised heart function. FASEB J 22: 970.25, 2008.
 171. Xu Y, Dong PH, Zhang Z, Ahmmed GU, Chiamvimonvat N. Presence of a calcium‐activated chloride current in mouse ventricular myocytes. Am J Physiol Heart Circ Physiol 283: H302‐H314, 2002.
 172. Yamamoto S, Ehara T. Acidic extracellular pH‐activated outwardly rectifying chloride current in mammalian cardiac myocytes. Am J Physiol Heart Circ Physiol 290: H1905‐H1914, 2006.
 173. Yamamoto‐Mizuma S, Wang GX, Hume JR. P2Y purinergic receptor regulation of CFTR chloride channels in mouse cardiac myocytes. J Physiol 556: 727‐737, 2004.
 174. Yamamoto‐Mizuma S, Wang GX, Liu LL, Schegg K, Hatton WJ, Duan D, Horowitz TL, Lamb FS, Hume JR. Altered properties of volume‐sensitive osmolyte and anion channels (VSOACs) and membrane protein expression in cardiac and smooth muscle myocytes from Clcn3‐/‐ mice. J Physiol 557: 439‐456, 2004.
 175. Yang YD, Cho H, Koo JY, Tak MH, Cho Y, Shim WS, Park SP, Lee J, Lee B, Kim BM, Raouf R, Shin YK, Oh U. TMEM16A confers receptor‐activated calcium‐dependent chloride conductance. Nature 455: 1210‐1215, 2008.
 176. Ye L, Dwyer L, Duan D. In vivo study of the role of cystic fibrosis transmembrane conductance regulator Cl− channels in early and late ischemic preconditioning. Heart Disease 4(362): 91, 2005.
 177. Yin Z, Tong Y, Zhu H, Watsky MA. ClC‐3 is required for LPA‐activated Cl‐ current activity and fibroblast‐to‐myofibroblast differentiation. Am J Physiol Cell Physiol 294: C535‐C542, 2008.
 178. Zhang Z, Xu Y, Song H, Rodriguez J, Tuteja D, Namkung Y, Shin HS, Chiamvimonvat N. Functional roles of Cav1.3 ({alpha}1D) calcium channel in sinoatrial nodes: Insight gained using gene‐targeted null mutant mice. Circ Res 90: 981‐987, 2002.
 179. Zygmunt AC. Intracellular calcium activates a chloride current in canine ventricular myocytes. Am J Physiol 267: H1984‐H1995, 1994.
 180. Zygmunt AC, Gibbons WR. Calcium‐activated chloride current in rabbit ventricular myocytes. Circ Res 68: 424‐437, 1991.
 181. Zygmunt AC, Gibbons WR. Properties of the calcium‐activated chloride current in heart. J Gen Physiol 99: 391‐414, 1992.

Contact Editor

Submit a note to the editor about this article by filling in the form below.

* Required Field

Article Title: Phenomics of Cardiac Chloride Channels
 

How to Cite

Dayue Darrel Duan. Phenomics of Cardiac Chloride Channels. Compr Physiol 2013, 3: 667-692. doi: 10.1002/cphy.c110014