A volume-regulated chloride current (ICl.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 ICl.vol has yet to be determined. Although three putative chloride channel proteins expressed from cloned genes (P-glycoprotein, pICln and ClC-2 ) have been proposed to be the molecular equivalent of ICl.vol, neither P-glycoprotein nor pICln is thought to be a chloride channel or part thereof, and the properties of expressed ClC-2 channels differ from native ICl.vol. 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 ICl.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 ICl.vol in many native mammalian cells.

Anion transport proteins in mammalian cells participate in a wide variety of cell and intracellular organelle functions, including regulation of electrical activity, pH, volume, and the transport of osmolites and metabolites, and may even play a role in the control of immunological responses, cell migration, cell proliferation, and differentiation. Although significant progress over the past decade has been achieved in understanding electrogenic and electroneutral anion transport proteins in sarcolemmal and intracellular membranes, information on the molecular nature and physiological significance of many of these proteins, especially in the heart, is incomplete. Functional and molecular studies presently suggest that four primary types of sarcolemmal anion channels are expressed in cardiac cells: channels regulated by protein kinase A (PKA), protein kinase C, and purinergic receptors (I(Cl.PKA)); channels regulated by changes in cell volume (I(Cl.vol)); channels activated by intracellular Ca(2+) (I(Cl.Ca)); and inwardly rectifying anion channels (I(Cl.ir)). In most animal species, I(Cl.PKA) is due to expression of a cardiac isoform of the epithelial cystic fibrosis transmembrane conductance regulator Cl(-) channel. New molecular candidates responsible for I(Cl.vol), I(Cl.Ca), and I(Cl.ir) (ClC-3, CLCA1, and ClC-2, respectively) have recently been identified and are presently being evaluated. Two isoforms of the band 3 anion exchange protein, originally characterized in erythrocytes, are responsible for Cl(-)/HCO(3)(-) exchange, and at least two members of a large vertebrate family of electroneutral cotransporters (ENCC1 and ENCC3) are responsible for Na(+)-dependent Cl(-) cotransport in heart. A 223-amino acid protein in the outer mitochondrial membrane of most eukaryotic cells comprises a voltage-dependent anion channel. The molecular entities responsible for other types of electroneutral anion exchange or Cl(-) conductances in intracellular membranes of the sarcoplasmic reticulum or nucleus are unknown. Evidence of cardiac expression of up to five additional members of the ClC gene family suggest a rich new variety of molecular candidates that may underlie existing or novel Cl(-) channel subtypes in sarcolemmal and intracellular membranes. The application of modern molecular biological and genetic approaches to the study of anion transport proteins during the next decade holds exciting promise for eventually revealing the actual physiological, pathophysiological, and clinical significance of these unique transport processes in cardiac and other mammalian cells.

In many mammalian cells, ClC-3 volume-regulated chloride channels maintain a variety of normal cellular functions during osmotic perturbation. The molecular mechanisms of channel regulation by cell volume, however, are unknown. Since a number of recent studies point to the involvement of protein phosphorylation/dephosphorylation in the control of volume-regulated ionic transport systems, we studied the relationship between channel phosphorylation and volume regulation of ClC-3 channels using site-directed mutagenesis and patch-clamp techniques. In native cardiac cells and when overexpressed in NIH/3T3 cells, ClC-3 channels were opened by cell swelling or inhibition of endogenous PKC, but closed by PKC activation, phosphatase inhibition, or elevation of intracellular Ca2+. Site-specific mutational studies indicate that a serine residue (serine51) within a consensus PKC-phosphorylation site in the intracellular amino terminus of the ClC-3 channel protein represents an important volume sensor of the channel. These results provide direct molecular and pharmacological evidence indicating that channel phosphorylation/dephosphorylation plays a crucial role in the regulation of volume sensitivity of recombinant ClC-3 channels and their native counterpart, ICl.vol.

Background Adult mammalian hearts have a limited ability to generate new cardiomyocytes. Proliferation of existing adult cardiomyocytes (ACM) is a potential source of new cardiomyocytes. Understanding the fundamental biology of ACM proliferation could be of great clinical significance for treating myocardial infarction (MI). We aim to understand the process and regulation of ACM proliferation and its role in new cardiomyocyte formation of post-MI mouse hearts. Methods β-actin-GFP transgenic mice and fate-mapping Myh6-MerCreMer-tdTomato/lacZ mice were used to trace the fate of ACMs. In a co-culture system with neonatal rat ventricular myocytes (NRVMs), ACM proliferation was documented with clear evidence of cytokinesis observed with time-lapse imaging. Cardiomyocyte proliferation in the adult mouse post-MI heart was detected by cell cycle markers and EdU incorporation analysis. Echocardiography was used to measure cardiac function and histology was performed to determine infarction size. Results In-vitro, mononucleated and bi/multi-nucleated ACMs were able to proliferate at a similar rate (7.0%) in the co-culture. Dedifferentiation proceeded ACM proliferation, which was followed by redifferentiation. Redifferentiation was essential to endow the daughter cells with cardiomyocyte contractile function. Intercellular propagation of Ca2+ from contracting NRVMs into ACM daughter cells was required to activate the Ca2+ dependent calcineurin-nuclear factor of activated T cells signaling pathway to induce ACM redifferentiation. The properties of NRVM Ca2+ transients influenced the rate of ACM redifferentiation. Hypoxia impaired the function of gap junctions by dephosphorylating its component protein connexin 43, the major mediator of intercellular Ca2+ propagation between cardiomyocytes, thereby impairing ACM redifferentiation. In-vivo, ACM proliferation was found primarily in the MI border zone. An ischemia resistant connexin 43 mutant enhanced the redifferentiation of ACM-derived new cardiomyocytes after MI and improved cardiac function. Conclusions Mature ACMs can reenter the cell cycle and form new cardiomyocytes through a three-step process, dedifferentiation, proliferation and redifferentiation. Intercellular Ca2+ signal from neighboring functioning cardiomyocytes through gap junctions induces the redifferentiation process. This novel mechanism contributes to new cardiomyocyte formation in post-MI hearts in mammals.

N. Increased TMEM16A-encoded calcium-activated chloride channel activity is associated with pulmonary hypertension. Pulmonary artery smooth muscle cells (PASMCs) are more depolarized and display higher Ca 2ϩ levels in pulmonary hypertension (PH). Whether the functional properties and expression of Ca 2ϩ -activated ClϪ channels (Cl Ca), an important excitatory mechanism in PASMCs, are altered in PH is unknown. The potential role of Cl Ca channels in PH was investigated using the monocrotaline (MCT)-induced PH model in the rat. Three weeks postinjection with a single dose of MCT (50 mg/kg ip), the animals developed right ventricular hypertrophy (heart weight measurements) and changes in pulmonary arterial flow (pulse-waved Doppler imaging) that were consistent with increased pulmonary arterial pressure and PH. Whole cell patch experiments revealed an increase in niflumic acid (NFA)-sensitive Ca 2ϩ -activated Cl Ϫ current [ICl(Ca)] density in PASMCs from large conduit and small intralobar pulmonary arteries of MCT-treated rats vs. aged-matched saline-injected controls. Quantitative RT-PCR and Western blot analysis revealed that the alterations in I Cl(Ca) were accompanied by parallel changes in the expression of TMEM16A, a gene recently shown to encode for Cl Ca channels. The contraction to serotonin of conduit and intralobar pulmonary arteries from MCT-treated rats exhibited greater sensitivity to nifedipine (1 M), an L-type Ca 2ϩ channel blocker, and NFA (30 or 100 M, with or without 10 M indomethacin to inhibit cyclooxygenases) or T16A Inh-A01 (10 M), TMEM16A/Cl Ca channel inhibitors, than that of control animals. In conclusion, augmented Cl Ca/TMEM16A channel activity is a major contributor to the changes in electromechanical coupling of PA in this model of PH. TMEM16A-encoded channels may therefore represent a novel therapeutic target in this disease. pulmonary arterial tone; TMEM16A; anoctamin-1; Ca 2ϩ -activated Cl Ϫ channel; patch-clamp technique PULMONARY HYPERTENSION (PH) is defined as a sustained high blood pressure (Ͼ25 mmHg at rest and Ͼ30 mmHg during exercise) in the main pulmonary artery (PA) that ultimately leads to failure of the right hand side of the heart and death (4). Characteristic pathophysiological manifestations of PH are enhanced vasoconstriction, thickening of the arterial muscle wall, and a propensity for thrombosis, as a result of changes in all layers of the blood vessel, but little is known about the molecular mechanisms that drive these pathological responses. It is well established that pulmonary arterial smooth muscle cells (PASMCs) from animal models of PH and human PH patients are more depolarized and exhibit a higher intracellular calcium concentration ([Ca 2ϩ ] i ) than cells from healthy individuals and several ionic conductances are altered in PASMCs from animal models of PH and PH patients (4,13,29,43,68,70). Except for one recent study carried out using the chronic hypoxic model of PH in the rat (58), there is little information regarding the potential role of Ca 2ϩ -activa...

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. 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. 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. 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.

Abstract-Although the cationic inward rectifiers (Kir and hyperpolarization-activated I f channels) have been well characterized in cardiac myocytes, the expression and physiological role of anionic inward rectifiers in heart are unknown. In the present study, we report the functional and molecular identification of a novel chloride (Cl Ϫ ) inward rectifier (Cl.ir) in mammalian heart. Under conditions in which cationic inward rectifier channels were blocked, membrane hyperpolarization (Ϫ40 to Ϫ140 mV) activated an inwardly rectifying whole-cell current in mouse atrial and ventricular myocytes. Under isotonic conditions, the current activated slowly with a biexponential time course (time constants averaging 179.7Ϯ23.4 [meanϮSEM] and 2073.6Ϯ287.6 ms at Ϫ120 mV). Hypotonic cell swelling accelerated the activation and increased the current amplitude whereas hypertonic cell shrinkage inhibited the current. The inwardly rectifying current was carried by Cl Ϫ (I Cl.ir ) and had an anion permeability sequence of Cl Ϫ ϾI Ϫ Ͼ Ͼaspartate. I Cl.ir was blocked by 9-anthracene-carboxylic acid and cadmium but not by stilbene disulfonates and tamoxifen. A similar I Cl.ir was also observed in guinea pig cardiac myocytes. The properties of I Cl.ir are consistent with currents generated by expression of ClC-2 Cl Ϫ channels. Reverse transcription polymerase chain reaction and Northern blot analysis confirmed transcriptional expression of ClC-2 in both atrial and ventricular tissues and isolated myocytes of mouse and guinea pig hearts. These results indicate that a novel I Cl.ir is present in mammalian heart and support a potentially important role of ClC-2 channels in the regulation of cardiac electrical activity and cell volume under physiological and pathological conditions. The full text of this article is available at http://www.circresaha.org. (Circ Res. 2000;86:e63-e71.)

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.