Dayue Duan

Molecular identification of a volume-regulated chloride channel

A volume-regulated chloride current ( I Cl.vol) is ubiquitously present in mammalian cells, and is required for the regulation of electrical activity, cell volume, intracellular pH, immunological responses, cell proliferation and differentiation. However, the molecule responsible for I Cl.vol has yet to be determined. Although three putative chloride channel proteins expressed from cloned genes (P-glycoprotein, p I Cln (ref. 5) and ClC-2 (ref. 6)) have been proposed to be the molecular equivalent of I Cl.vol, neither P-glycoprotein nor p I Cln is thought to be a chloride channel or part thereof,, and the properties of expressed ClC-2 channels differ from native I Cl.vol (refs. 3, 6). Here we report that functional expression in NIH/3T3 cells of a cardiac clone of another member of the ClC family, ClC-3, results in a large basally active chloride conductance, which is strongly modulated by cell volume and exhibits many properties identical to those of I Cl.vol in native cells,. A mutation of asparagine to lysine at position 579 at the end of the transmembrane domains of ClC-3 abolishes the outward rectification and changes the anion selectivity from I− > Cl− to Cl− > I− but leaves swelling activation intact. Because ClC-3 is a channel protein belonging to a large gene family of chloride channels,, these results indicate that ClC-3 encodes I Cl.vol in many native mammalian cells.

Functional and molecular expression of volume‐regulated chloride channels in canine vascular smooth muscle cells

1 We examined the possibility of functional and molecular expression of volume‐regulated Cl− channels in vascular smooth muscle using the whole‐cell patch‐clamp technique and quantitative reverse transcriptase‐polymerase chain reaction (RT‐PCR) on cells from canine pulmonary and renal arteries. 2 Decreasing external osmolarity induced cell swelling, which was accompanied by activation of Cl−‐dependent outward‐rectifying membrane currents with an anion permeability sequence of SCN− > I− > Br− > Cl− > aspartate−. These currents were sensitive to block by DIDS, extracellular ATP and the antioestrogen compound tamoxifen. 3 Experiments were performed to determine whether the molecular form of the volume‐regulated chloride channel (ClC‐3) is expressed in pulmonary and renal arteries. Quantitative RT‐PCR confirmed expression of ClC‐3 in both types of smooth muscle. ClC‐3 expression was 76.4 % of β‐actin in renal artery and 48.0 % of β‐actin in pulmonary artery. 4 We conclude that volume‐regulated Cl− channels are expressed in vascular smooth muscle cells and exhibit functional properties similar to those found in other types of cells, presumably contributing to the regulation of cell volume, electrical activity and, possibly, myogenic tone.

Altered properties of volume‐sensitive osmolyte and anion channels (VSOACs) and membrane protein expression in cardiac and smooth muscle myocytes from Clcn3‐/‐ mice

ClC‐3, a member of the large superfamily of ClC voltage‐dependent Cl– channels, has been proposed as a molecular candidate responsible for volume‐sensitive osmolyte and anion channels (VSOACs) in some cells, including heart and vascular smooth muscle. However, the reported presence of native VSOACs in at least two cell types from transgenic ClC‐3 disrupted (Clcn3−/−) mice casts considerable doubt on this proposed role for ClC‐3. We compared several properties of native VSOACs and examined mRNA transcripts and membrane protein expression profiles in cardiac and pulmonary arterial smooth muscle cells from Clcn3+/+ and Clcn3−/− mice to: (1) test the hypothesis that native VSOACs are unaltered in cells from Clcn3−/− mice, and (2) test the possibility that targeted inactivation of the Clcn3 gene using a conventional murine global knock‐out approach may result in compensatory changes in expression of other membrane proteins. Our experiments demonstrate that VSOAC currents in myocytes from Clcn3+/+ and Clcn3−/− mice are remarkably similar in terms of activation and inactivation kinetics, steady‐state current densities, rectification, anion selectivity (I− > Cl−≫ Asp−) and sensitivity to block by glibenclamide, niflumic acid, DIDS and extracellular ATP. However, additional experiments revealed several significant differences in other fundamental properties of native VSOACs recorded from atrial and smooth muscle cells from Clcn3−/− mice, including: differences in regulation by endogenous protein kinase C, differential sensitivity to block by anti‐ClC‐3 antibodies, and differential sensitivities to [ATP]i and free [Mg2+]i. These results suggest that in response to Clcn3 gene deletion, there may be compensatory changes in expression of other proteins that alter VSOAC channel subunit composition or associated regulatory subunits that give rise to VSOACs with different properties. Consistent with this hypothesis, in atria from Clcn3−/− mice compared to Clcn3+/+ mice, quantitative analysis of ClC mRNA expression levels revealed significant increases in transcripts for ClC‐1, ClC‐2, and ClC‐3, and protein expression profiles obtained using two‐dimensional polyacrylamide gel electrophoresis revealed complex changes in at least 35 different unidentified membrane proteins in cells from Clcn3−/− mice. These findings emphasize that caution needs to be exercised in simple attempts to interpret the phenotypic consequences of conventional global Clcn3 gene inactivation.

Functional inhibition of native volume-sensitive outwardly rectifying anion channels in muscle cells and Xenopus oocytes by anti-ClC-3 antibody

1 Intracellular dialysis of NIH/3T3 cells with a commercially available anti‐ClC‐3 polyclonal antibody (Ab) for ≈30 min completely inhibited expressed guinea‐pig ClC‐3 currents (IgpClC‐3), while intracellular dialysis with antigen‐preabsorbed anti‐ClC‐3 Ab failed to affect IgpClC‐3. 2 Anti‐ClC‐3 Ab was used as a selective probe to examine the relationship between endogenous ClC‐3 expression and native volume‐sensitive outwardly rectifying anion channels (VSOACs) in guinea‐pig cardiac cells, canine pulmonary arterial smooth muscle cells (PASMCs) and Xenopus laevis oocytes. Intracellular dialysis or injection of anti‐ClC‐3 Ab abolished native VSOAC function in cardiac cells and PASMCs and significantly reduced VSOACs in oocytes. In contrast, native VSOAC function was unaltered by antigen‐preabsorbed anti‐ClC‐3 Ab. 3 It is suggested that endogenous ClC‐3 represents a major molecular entity responsible for native VSOACs in cardiac and smooth muscle cells and Xenopus oocytes. Anti‐ClC‐3 Ab should be a useful experimental tool to directly test the relationship between endogenous ClC‐3 expression and native VSOAC function, and help resolve existing controversies related to the regulation and physiological role of native VSOACs in a wide variety of different cells.

Phenomics of cardiac chloride channels: the systematic study of chloride channel function in the heart

Recent studies have identified several chloride (Cl−) channel genes in the heart, including CFTR, ClC‐2, ClC‐3, CLCA, Bestrophin, and TMEM16A. Gene targeting and transgenic techniques have been used to delineate the functional role of cardiac Cl− channels in the context of health and disease. It has been shown that Cl− channels may contribute to cardiac arrhythmogenesis, myocardial hypertrophy and heart failure, and cardioprotection against ischaemia–reperfusion. The study of physiological or pathophysiological phenotypes of cardiac Cl− channels, however, may be 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 multi‐protein complexes of the Cl− channel subproteome and similar phenotypes can be attained from alternative protein pathways within cellular networks, which are influenced by genetic and environmental factors. Therefore, 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 (such as gene knockouts or knockins) under the scope of genome/proteome/phenome, may provide a more complete understanding of the integrated function of each cardiac Cl− channel in the context of health and disease.

Evidence That Outwardly Rectifying Cl− Channels Underlie Volume-Regulated Cl− Currents in Heart

Swelling-induced Cl- current (ICl.swell) is present in most cardiac tissues, but the unitary channel underlying ICls.well is unknown. We used the cell-attached patch-clamp technique to assess the properties of single channels underlying ICls.well and the basally active Cl- current (ICl.b) in rabbit atrial myocytes. Under isotonic conditions, single outwardly rectifying Cl- channels (ORCCs) with a slope conductance of 28 +/- 1 pS at the reversal potential were observed in 21 (5.7%) of 367 patches. Unconditional kinetic analysis revealed at least three open and four closed-channel states. Hypotonic superfusion-induced swelling resulted in the appearance of active channels in 41 (15.5%) of 265 patches without channel activity under isotonic conditions and caused a second active channel to appear in 3 of 14 patches showing a single channel under isotonic conditions. Overall, channels were seen in 54 of 336 patches under hypotonic conditions (16.1%, P < .001 versus isotonic conditions). The current-voltage relations, reversal potential-[Cl-]o relations, open probability, and kinetics of swelling-induced channels were indistinguishable from those of ORCCs under isotonic conditions. Unitary ORCCs, ICl.b, and ICl.swell were strongly and similarly inhibited by tamoxifen. Swelling-induced increases in macroscopic Cl- current were attributable to an increase in the number of active ORCCs with no significant effects on single-channel amplitude or open probability. Estimated macroscopic currents based on cell surface area, patch dimensions, single-channel ORCC current amplitude, open probability, and density were consistent with measured values of ICl.b and ICl.swell. We conclude that ORCCs underlie volume-regulated basal and swelling-induced Cl- currents in isolated rabbit atrial myocytes.

Functional role of anion channels in cardiac diseases.

AbstractIn comparison to cation (K+, Na+ and Ca2+) channels, much less is currently known about the functional role of anion (Cl−) channels in cardiovascular physiology and pathophysiology. Over the past 15 years, various types of Cl− currents have been recorded in cardiac cells from different species including humans. All cardiac Cl− channels described to date may be encoded by five different Cl− channel genes: the PKA- and PKC-activated cystic fibrosis tansmembrane conductanceregulator (CFTR), the volume-regulated CIC-2 und CIC-3, and the Ca2+ - activated CLCA or Bestrophin. Recent studies using multiple approaches to examine the functional role of Cl− channels in the context of health and disease have demonstrated that Cl− channels might contribute to: 1) arrhythmogenesis in mycocardial injury; 2) Cardiac ischemic preconditioning; and 3) the adaptive remodeling of the heart during myocardial hypertrophy and heart failure. Therefore anion channels represent very attractive novel targets for therapeutic approaches to the treatment of heart diseases. Recent evidence suggests that Cl− channels, like cation channels, might function as a multiprotein complex or functional module. In the post-genome era, the emergence of functional proteomics has necessitated a new paradigm shift to the structural and functional assessment of integrated Cl− channel multiprotein complexes in the heart, which could provide new insight into our understanding of the underlying mechanisms responsible for heart disease and protection.

Targeted Inactivation of Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Gene Prevents Ischemic Preconditioning in Isolated Mouse Heart

Background—Recent evidence suggests that chloride channels may be involved in ischemic preconditioning (IPC). In this study, we tested whether the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels, which are expressed in the heart and activated by protein kinase A and protein kinase C, are important for IPC in isolated heart preparations from wild-type (WT) and CFTR knockout (CFTR−/−) mice. Methods and Results—Hearts were isolated from age-matched WT or CFTR−/− (B6.129P2-Cftrtm1Unc and STOCKCftrtm1Unc-TgN 1Jaw) mice and perfused in the Langendorff or working-heart mode. All hearts were allowed to stabilize for 10 minutes before they were subjected to 30 or 45 minutes of global ischemia followed by 40 minutes of reperfusion (control group) or 3 cycles of 5 minutes of ischemia and reperfusion (IPC group) before 30 or 45 minutes of global ischemia and 40 minutes of reperfusion. Hemodynamic indices were recorded to evaluate cardiac functions. Release of creatine phosphate kinase (CPK) in the samples of coronary effluent and infarct size of the ventricles were used to estimate myocardial tissue injury. In WT adult hearts, IPC protected cardiac function during reperfusion and significantly decreased ischemia-induced CPK release and infarct size. A selective CFTR channel blocker, gemfibrozil, abrogated the protective effect of IPC. Furthermore, targeted inactivation of the CFTR gene in 2 different strains of CFTR−/− mice also prevented IPC’s protection of cardiac function and myocardial injury against sustained ischemia. Conclusions—CFTR Cl− channels may serve as novel and crucial mediators in mouse heart IPC.

Purinoceptor-coupled Cl− channels in mouse heart: a novel, alternative pathway for CFTR regulation

1 P2‐purinoceptors couple extracellular ATP to the activation of a Cl− current (ICl,ATP) in heart. We studied the molecular mechanism and intracellular signalling pathways of ICl,ATP activation in mouse heart. 2 Extracellular adenosine‐5′‐O‐(3‐thiotriphosphate) (ATPγS; 100 μM) activated ICl,ATP in both atrial and ventricular myocytes. A specific PKC inhibitor, bisindolylmaleimide blocked the effect of ATPγS while a PKC activator, phorbol 12,13‐dibutyrate (PDBu) activated a current with identical properties to ICl,ATP. Maximal activation of ICl,ATP by ATPγS or PDBu occluded further modulation by the other agonist, suggesting that they may activate the same population of Cl− channels. 3 Isoprenaline increased ICl,ATP pre‐activated by ATPγS or PDBu, while isoprenaline or forskolin alone failed to activate any Cl− current in these myocytes. Adenosine 3′,5′‐cyclic monophosphothionate, a PKA inhibitor, prevented ATPγS or PDBu activation of ICl,ATP. Thus, ICl,ATP is regulated by dual intracellular phosphorylation pathways involving both PKA and PKC in a synergistic manner similar to cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channels. 4 Glibenclamide (50 μM) significantly blocked ICl,ATP activated by ATPγS or by the CFTR channel activator, levamisole. 5 The slope conductance of the unitary ICl,ATP in cell‐attached patches was 11·8 ± 0·3 pS, resembling the known properties of CFTR Cl− channels in cardiac myocytes. 6 The reverse transcription polymerase chain reaction and Northern blot analysis revealed CFTR mRNA expression in mouse heart. 7 We conclude that ICl,ATP in mouse heart is due to activation of CFTR Cl− channels through a novel intracellular signalling pathway involving purinergic activation of PKC and PKA.

Intracellular cyclic AMP inhibits native and recombinant volume-regulated chloride channels from mammalian heart.

1 ClC‐3 encodes a volume‐regulated Cl− channel (ICl,vol) in heart. We studied the regulation of native and recombinant cardiac ICl,vol by intracellular cyclic AMP (cAMPi). 2 Symmetrical high Cl− concentrations were used to effectively separate outwardly rectifying ICl,vol from other non‐rectifying Cl− currents, such as the cystic fibrosis transmembrane conductance regulator (CFTR) and Ca2+‐activated Cl− currents (ICl,CFTR and ICl,Ca, respectively), which are concomitantly expressed in cardiac myocytes. 3 8‐Bromo‐cyclic AMP (8‐Br‐cAMP) significantly inhibited ICl,vol in most guinea‐pig atrial myocytes. In ≈30 % of the atrial myocytes examined, 8‐Br‐cAMP increased macroscopic Cl− currents. However, the 8‐Br‐cAMP‐stimulated difference currents exhibited a linear current‐voltage (I–V) relation, consistent with activation of ICl,CFTR, not ICl,vol. 4 In canine atrial myocytes, isoprenaline (1 μM) consistently reduced ICl,vol in Ca2+‐free hypotonic bath solutions with strong intracellular Ca2+ (Ca2+i) buffering. In Ca2+‐containing hypotonic bath solutions with weak Ca2+i buffering, however, isoprenaline increased net macroscopic Cl− currents. Isoprenaline‐stimulated difference currents were not outwardly rectifying, consistent with activation of ICl,Ca, not ICl,vol. 5 In NIH/3T3 cells transfected with gpClC‐3 (the gene encoding ICl,vol), 8‐Br‐cAMP consistently inhibited ICl,ClC‐3. These effects were prevented by a protein kinase A (PKA) inhibitor, KT5720, or by mutation of a single consensus protein kinase C (PKC) phosphorylation site (S51A) on the N‐terminus of ClC‐3, which also mediates PKC inhibition of ICl,ClC‐3. 6 We conclude that cAMPi causes inhibition of ICl,vol in mammalian heart due to cross‐phosphorylation of the same PKC consensus site on ClC‐3 by PKA. Our results suggest that contamination of macroscopic ICl,vol by ICl,CFTR and/or ICl,Ca may account for some of the inconsistent and controversial effects of cAMPi on ICl,vol previously reported in native cardiac myocytes.