Research and progress on ClC‑2 (Review).

Chloride channel 2 (ClC-2) is one of the nine mammalian members of the ClC family. The present review discusses the molecular properties of ClC‑2, including CLCN2, ClC‑2 promoter and the structural properties of ClC‑2 protein; physiological properties; functional properties, including the regulation of cell volume. The effects of ClC‑2 on the digestive, respiratory, circulatory, nervous and optical systems are also discussed, in addition to the mechanisms involved in the regulation of ClC‑2. The review then discusses the diseases associated with ClC‑2, including degeneration of the retina, Sjögren's syndrome, age‑related cataracts, degeneration of the testes, azoospermia, lung cancer, constipation, repair of impaired intestinal mucosa barrier, leukemia, cystic fibrosis, leukoencephalopathy, epilepsy and diabetes mellitus. It was concluded that future investigations of ClC‑2 are likely to be focused on developing specific drugs, activators and inhibitors regulating the expression of ClC‑2 to treat diseases associated with ClC‑2. The determination of CLCN2 is required to prevent and treat several diseases associated with ClC‑2.

Introduction

Chloride channels (ClCs) are a type of permeable channel protein for chloride ions or other anions on the cell membrane, and ClC proteins are encoded by genes of the ClC family. ClCs have nine family members, which are classified into three distinct subfamilies: ClC-1, ClC-2, ClC-Ka/K1 and ClC-Kb/K2; ClC-3, ClC-4 and ClC-5; and ClC-6 and ClC-7 (1). ClC-2 is one of the nine mammalian members of the ClC family, and was initially isolated from the rat heart and brain (2,3) and then from the rabbit heart (4,5). ClC-2 is a two-pore homodimeric, voltage-gated Cl− channel (5–8). ClC-2 can be activated by hyperpolarization (3,9), cell swelling (2,9), extracelluar hypotonicity (2) and extracellular acidification (9,10). ClC-2 is almost ubiquitously expressed (3,11), including in ureteric bud cells (12), intestine (13–17), gastric parietal cells (18,19), the liver (20), lung (11,21–26), rat retina (27), parotid acinar cells (28), guinea pig cardiac muscle (29), neuronal cells (30), rat and human airways (17), bovine trabecular meshwork (31), human trabecular meshwork (32,33) and rat trabecular meshwork (34). In addition, ClC-2 can regulate cell volume (2,4,35), control response to swelling (11,36–39), and regulates post-synaptic responses to GABA and glycine (11,40,41).

Although ClC-2 has a wide variety of properties and functions, repots of CLC-3 prior to 1994 are limited, with only six published between 1953 and 1994. Between 1994 and 2015, ClC-2 attracted increasing attention as ClC-2 was understood to possess several molecular, functional and physiological properties, and be associated with several diseases, including degeneration of the retina, Sjögren's syndrome, age-related cataracts, degeneration of the testes, azoospermia, lung cancer, constipation, repair of impaired intestinal mucosa barrier, leukemia, cystic fibrosis, leukoencephalopathy, epilepsy and diabetes mellitus. However, reviews on ClC-2 are limited. The present review aimed to discuss the molecular, functional and physiological properties of ClC-2, in addition to mechanisms involved in the regulation of ClC-2 and diseases associated with ClC-2.

Molecular properties of ClC-2

CLCN2 and ClC-2 promoter

The ClC-2 protein is encoded by the CLCN2 gene, which is composed of 898 amino acids and is located in chromosome 3q27.1. The major transcription start site of the ClC-2 gene has been identified, and is localized 100 bp upstream of the putative translation initiation codon (21). A previous study (42) on the possible evolution of ClC-2 gene suggested that ClC-2 may have evolved by gene duplication, mutation and DNA rearrangement (27,43).

The ClC-2 promoter belongs to a GC-rich and TATA-less class (44). Within the 1,930-bp region, one of three CAAT boxes is close to the ClC-2 coding sequence, and the other two are at the middle (44). Within a 391-bp region upstream, the first three of four GC boxes are conserved in human ClC-2, as reported by Cid et al (21,44).

Structural properties of ClC-2 protein

Despite wide functional diversity, ClC family members share a conserved protein structure, including a transmembrane region, which is involved in chloride anion transport, and two intracellular copies of the cystathionine-β-synthase (CBS) domain (CBS1 and CBS2) (5–8,45,46) and an N-terminus. Therefore, the protein structure of ClC-2 is the same in the other ClC family members (45,46), as shown in Fig. 1.

Figure 1.

Basic structure of ClC-2 as a two-pore homodimeric channel. ClC-2 is a double-barreled channel with two identical, predominantly independent pores. (A) 18 α-helices are labeled A-R, and the two similar halves within the transmembrane domain (α-helices B-I and J-Q), which are oriented in opposite directions to the membrane, and are shown in green and cyan. The sequence regions, which contribute to the Cl− selectivity filter, are indicated by orange arrows, and the respective conserved sequences are shown; CBS1 is colored red and CBS2 is colored blue. (B) Structure viewed from the extracellular side. The two subunits of the homodimeric protein are shown in red and blue, and bound anions are shown in green. ClC-2, chloride channel 2; CBS, cystathionine-β-synthase.

Several other studies on the structural properties of ClC-2 have reported that the ClC-2 dimer is the minimum functional structure (47), protons act independently from the possible effects of the N-terminus on gating (48) and the hetero-dimerization of ClC-2 can modify the unitary conductance of protopores (49).

Biophysical properties of ClC-2

In 1992, it was reported that ClC-2 expressed in Xenopus oocytes generates Cl− currents, which activate slowly upon hyperpolarization and show a linear instantaneous current-voltage association (3). Further studies on the biophysical properties on ClC-2 have focused predominantly on Cl− currents, including ClC-2-like inwardly rectifying Cl− currents (3,50–54) and ClC-2 outwardly rectifying Cl− currents (36,37,52,55–59).

ClC-2-like inwardly rectifying Cl− currents have been detected in the human T84 cell line (50), drosophila ClC-2 variants in HEK-293 cells (51) and rat type IV spiral ligament fibrocytes (52). The conductivity sequence of the inwardly rectifying currents is Cl− ≥ Br− > I− (3). ClC-2 inwardly rectifying Cl− currents are inhibited by 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS) (3) and Cd2+ (50), and regulated by cell swelling (53), extracellular pH, Cl− and Ca2+ (52). However, the results of another study on ClC-2 inwardly rectifying Cl−currents differed, reporting that ClC-2 did not significantly contribute to inward-rectifying anion conductance in the mouse choroid plexus (54).

ClC-2 outwardly rectifying Cl− currents have been found in the T84 human adenocarcinoma cell line (36,37,55), human neurons (56), human parotid acinar cells (57) and rat type IV spiral ligament fibrocytes (52). The outwardly rectifying currents are characterized by a time-dependent decay at depolarizing voltages, and the anion permeability sequence I−>Br >Cl−>F−, and the currents show sensitivity to 1,9-dideoxyforskolin, DIDS (58) and tamoxifen (55). ClC-2 outwardly rectifying Cl− currents are regulated by extracellular pH, Cl− and Ca2+ (52). Another study showed that ClC-2 may contribute to cell volume regulation following hyposmotic stress produced by outwardly rectifying Cl−currents, however, cell volume regulation in T84 cells is independent of ClC-2 activity (59).

ClC-2 inwardly/outwardly rectifying Cl− currents are simultaneously found in rat type IV spiral ligament fibrocytes (52), and they are modulated by extracellular pH, Cl− and Ca2+. According to previous studies, at least two chloride channels are involved in modulating membrane anion conductance (52). Another study on Cl− currents in rabbit heart cells suggested that rabClC-2α/2β may provide two homologous protein kinase A-activated chloride anion channels, with or without extracellular hypotonicity (60).

Functional properties of ClC-2

Previous studies have shown that ClC-2 has several functional properties, including effects on cell volume (2,4,20,35,61), the digestive system (11,16,18,19,62–64), the eye (65–67), respiratory system (11,24,26,68–70), circulatory system (35,71–75) and nervous system (76). In addition, several studies have reported the functions of the NH2 terminus (77) and carboxy terminus (78) in ClC-2, and the similar functional properties between chloride-selective ion channels, including ClC-2, in protozoa (79–82).

Regulation of cell volume

The activation of ClC-2 in response to cell swelling and low extracellular pH is involved in the dissociation of an NH2-terminal region of ClC-2 (10). The dephosphorylation of serine/threonine residues in the channel or an associated protein appears to be necessary for current activation following cell swelling in T84 cells (53) and for a ClC-2-like current in ascidian embryo cells during the cell cycle (83). Hypotonic cell swelling activates an outwardly rectifying anionic conductance, with different characteristics from those of ClC-2 previously reported (84). The endogenous currents suggest that protein tyrosine phosphorylation is involved in the signal transduction pathway in certain cell types (85–87), and that epidermal growth factor enhances the hypotonicity-induced anionic efflux (85). However, tyrosine kinase p56lck activates these swelling-sensitive chloride channels without cell swelling, even during cell shrinkage, in lymphocytes (88).

Effect of ClC-2 on the digestive system

Nehrke et al (62) detected the regulatory volume decrease following cell swelling, which showed that ClC-2 can control cell volume in mouse parotid acinar cells. Others functions of ClC-2 have been reported, including HCl secretion across the parietal cell secretory membrane in the rabbit gastric mucosa (19), mediating the basolateral membrane exit of Cl− in the distal colon of guinea pigs (16), regulating gastric acid secretion (11,18,63), and affecting gastric glands and its cell layers (63). However, one study suggested that ClC-2 may not be a Cl−-transporting protein for gastric acid secretion in parietal cells (64).

Effect of ClC-2 on the eye

Previous studies have shown that CIC-2 has a protective effect on trabecular cells under pressure stress (65), can regulate the inward rectification in Drosophila retinal photoreceptors (66), and can modulate cellular volume, intracellular Cl− and other cellular functions in trabecular meshwork cells (67).

Effect of ClC-2 on the respiratory, circulatory and nervous systems

ClC-2 is gestationally regulated and is predominantly expressed in the fetal lung (26,68). Cystic fibrosis transmembrane conductance regulator (CFTR)-independent fluid accumulation is induced by keratinocyte growth factor in fetal lung explants (69), and keratinocyte growth factor is necessary for ClC-2-like pH-sensitive Cl− secretion in the fetal airway epithelia (24). It has been shown that ClC-2 affects fetal lung fluid production and lung cyst morphology (70).

It has been reported that a novel Cl− inward rectifier channel is found in the cardiac atrial and ventricular myocytes of several species, including the guinea pig (71–73), mouse (71,73) and rat (72–75). The Cl− inward rectifier channels have properties of activation by hypotonic cell swelling and extracellular acidification, inward rectification, time-dependent slow activation and Cd2+-sensitive inhibition (71–75). Experiments have shown that ClC-2 affects the positive chronotropic effect of acute exercise stress (73) and modulates cardiac pacemaker activity (73), but cannot alter intrinsic heart rate (73).

Another study (76) investigating the effect of ClC-2 on inhibitory interneurons showed that membrane voltage-/intracellular chloride-dependent ClC-2 can selectively regulate GABAA receptor (GABAA R)-mediated synaptic inputs from basket cells.

Mechanisms of ClC-2 regulation by regulators

Cell swelling can increase ClC-2 activity (2,9), and ClC-2 can be activated by hyperpolarization (3,9), extracelluar hypotonicity (2) and extracellular acidification (9,10). Previous studies have shown that ClC-2 is also regulated by hormones (89–91), drugs (92–100), proteins (101–107), kinases (108–114), transcription factors (115,116), inhibitors (117–120), scorpion venom (121), α1-adrenoceptor (122), Plasmodium berghei (123), adenosine triphosphate (124,125), ClH3 (40,126–137), permeant anions (138,139), membrane cholesterol (140) and the tyrosine endocytosis motif (141).

Regulation of ClC-2 by hormones

Thyroid hormones (T3 and T4) can regulate renal function (142,143), Na+/K+ ATPase (144), Na+/H+ exchanger (145), and affect kidney growth and development (146). Previous studies have demonstrated that hyperthyroidism and hypothyroidism are associated with chloride (147,148), and the reversal of glomerular filtration by T4 is observed in patients with hypothyroidism (149). In addition, a previous study (89) on T4 suggested that ClC-2 is involved in chloride transport regulated by thyroid hormones in rat renal proximal tubules.

Estrogen is associated with a decrease in the urinary excretion of sodium and chloride (150–153). A study by Nascimento et al (90) showed that ClC-2 may be involved in estrogen-induced Cl− transport in the rat kidney. Another study (91) showed that ClC-2 is also involved in the rat renal tubule transcellular chloride transport regulated by arginine vasopressin, a neurohypophysial hormone.

Regulation of ClC-2 by drugs

Lubiprostone can induce intestinal fluid secretion (154), treat constipation (155–157) and activate ClC-2 Cl− currents in a concentration-dependent manner (94), and ClC-2 can recover mucosal barrier function in the ischemia-injured intestine (92). Studies have shown that the activation of ClC-2 by lubiprostone can stimulate the recovery of intestinal barrier function in the ischemia-injured porcine ileum and colon (93), and may have a protective and therapeutic effect on murine models of colitis (99). The prostaglandin E2 receptor 4 [EP (4)] receptor (94–97) can stimulate ClC-2 and CFTR chloride channels, and it has been shown (98) that lubiprostone can activate CFTR by the EP (4) receptor in oocytes. The Chinese medicinal compounds Guanxinkang and diltiazem hydrochloride can decrease the mRNA and protein expression of ClC-2 in rats with myocardial ischemia reperfusion injury (100).

Regulation of ClC-2 by proteins

The functional expression and activation of ClC-2 is reduced by dynein (a protein complex) (101) and p34cdc2/cyclin B (102), however, the inrerferon-γ glycoprotein can activate ClC-2 in lung epithelial cells through mRNA stabilization, and increase ClC-2 transcripts in Calu-3 cells (107). ClC-2 is regulated by protein phosphatase 1 (102), interactions between the actin cytoskeleton (a filamentous protein structure) and the N-terminus of ClC-2 (104). M phase-specific p34cdc2/cyclin B can phosphorylate the ubiquitination of ClC-2 (103). Heat shock protein 90 can increase ClC-2 current amplitude and the intracellular Cl− concentration, and enhance channel sensitivity to intracellular Cl− (105). ClC-2 is also associated with the transmembrane glycoprotein, GlialCAM, in the brain, and GlialCAM can involve ClC-2 in the homeostasis of myelin, which is defective in leukodystrophy (106).

Regulation of ClC-2 by kinases

ClC-2 membrane abundance is increased by serum and glucocorticoid inducible kinases (SGKs) and decreased by NEDD4-2, an enzyme of the NEDD4 family (108). In rats, protein kinase A can directly phosphorylate ClC-2, whereas protein kinase C and Ca2+/calmodulin-dependent protein kinase II cannot (113). PIKfyve is a FYVE finger-containing phosphoinositide kinase (158) and is a potent stimulator of ClC-2-activity (112), which can contribute to the SGK1-dependent regulation of ClC-2 (112).

Janus kinase (JAK) 2 and JAK3 (tyrosine kinase) can downregulate the activation of ClC-2 and offset Cl− exit (109,111), however, their functions and regulatory mechanism are different. JAK2 is involved in the signaling of leptin (159), growth hormones (160), erythropoietin, thrombopoietin, granulocyte colony-stimulating factor (161) and a variety of cytokines (161,162). JAK2 inhibitors can treat myeloproliferative disorders (163–170), and JAK2 can be activated by hyperosmotic shock (171,172). By contrast, JAK3 can promote proliferation and act against the apoptosis of lymphocytes and tumor cells (173–177). It is also present in acute megakaryoplastic leukemia (178,179). Therefore, cell proliferation and apoptosis is associated with Cl− channel activity (110). In addition, the substitution of lysine by alanine can inactivate JAK3 (180). In addition to JAK2 and JAK3, SPS1-related proline/alanine-rich kinase and oxidative stress-responsive kinase 1 can also downregulate ClC-2 (114).

Regulation of ClC-2 by transcription factors

The Sp1 and Sp3 transcription factors can control the rate of transcription of genetic information from DNA to messenger RNA (181,182). Reducing interactions between Sp1 or Sp3 and the ClC-2 promoter can lead to a postnatal decrease in the expression of ClC-2 in lung epithelia (116). In addition, the glycosylation of SP1 produces the 105-kD isoform of SP1 and is involved in regulating the expression of ClC-2 (115).

Regulation of ClC-2 by inhibitors

Previous studies have shown that ClC-2 is inhibited by methadone (117), gating modifier of anion channels 2 (GaTx2) (118), DIDS (119) and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) (120). However, DASU-02, as an inhibitor of common chloride channels, cannot inhibit ClC-2 (117). The inhibitory effects on ClC-2 by these inhibitors are entirely different. Methadone can inhibit the activities of ClC-2, which shows that ClC-2 may be involved in chloride anion secretion (117). GaTx2, as a peptide toxin inhibitor of ClC-2, can inhibit channel activation gating, but not other chloride channels or voltage-gated potassium channels (118). DIDS can significantly reduce the increased mRNA and protein levels of ClC-2 following ischemia-hypoxia damage (119). NPPB, as a ClC inhibitor, can affect the phagocytosis of human trabecular meshwork cells, which shows that ClC-2 may be involved in the regulation of its phagocytic process (120).

Other factors in the functional regulation of ClC-2

According to previous studies, in addition to the above-mentioned regulatory mechanisms, scorpion venom (121), α1-adrenoceptor (122), Plasmodium berghei (123), adenosine triphosphate (124,125), ClH3 (137), GABAAR (40,126–128), permeant anions (138,139), membrane cholesterol (140) and β-cyclodextrin sensitive clusters (141) can also regulate or affect ClC-2. A peptide of scorpion venom can induce slower activation kinetics of ClC-2 (121). α1-adrenoceptor activation can significantly reduce protein levels of ClC-2 in the villus and crypt epithelial cells from the acutely denervated jejunum, but not the innervated jejunum (122). The activation of ClC-2 is involved in the altered permeability caused by Plasmodium berghei infection in Plasmodium berghei-infected mouse CLCN2 (123). ATP not only decelerates ClC-2 common gating for sufficient electrical stability of neurons (124), it also alters its surface expression (125). Intra-/extra-cellular permeant anions can affect the Vm-dependence of ClC-2 (138). Specially, Cl− as a permeant anion can increase pore occupancy, thus obstructing the closure of the protopore gate of ClC-2 (139). In addition, alterations in the (Cl−)i between 10 and 200 mM can decelerate ClC-2 channel closing at a positive Vm (139). Membrane cholesterol can regulate the activation of ClC-2, and the increased activation of ClC-2 is involved in the relocalization of ClC-2 to detergent-soluble microdomains (140). β-cyclodextrin sensitive clusters with other molecules can mantain ClC-2 activity (141).

Previous studies have reported that ClH3 encodes the Caenorhabditis elegans homologs of ClC-2 (129–131). ClH3-dependent regulation not only alters the voltage dependence of ClC-2 channels and inhibits hereditary sensory neuropathy (HSN) excitability (137), but may also affect chloride influx (137) in Caenorhabditis elegans. However, the chloride efflux pathway of ClC-2 is associated with synaptic inhibition regulated by the GABAAR (40,126–128). In addition, the HSNs can release acetylcholine, serotonin and multiple neuropeptides (133–136), which excite the VC motor neurons (132). These functions of HSNs may be associated with clh-3-dependent regulation and the voltage dependence of ClC-2 channels.

Diseases associated with ClC-2

Previous studies have shown that disruption and abnormity of ClC-2 can cause a number of diseases, including ophthalmological disease (11,183–185), otorhinolaryngological disease (186), disease of the reproductive system (11,187), disease of the respiratory system (188), disease of the digestive system (189–194), disease of the hematological system (195), genetic diseases (23,25,26,42,44,74,76,196–198), diseases of the nervous system (30,40,41,168,199–212) and metabolic diseases (213). The majority of studies have focused on diseases of the nervous system.

Ophthalmological and otorhinolaryngological disease associated with ClC-2

Severe degeneration of the retina occurs in ClC-2-knockout mice, although no other notable eye abnormalities are present (11). Possible reasons for degeneration of the retina caused by ClC-2-knockout include the following (11): i) ClC-2 disruption causes the death of photoreceptor cells; ii) depletion of ClC-2 may impair the transport and alter the ionic environment of photoreceptors. Other studies on ophthalmological diseases associated with ClC-2 include Sjögren's syndrome (183,184,214–218) and age-related cataracts (185).

Sjögren's syndrome is a chronic autoimmune disease, and previous studies have shown that eye diseases caused by Sjögren's syndrome are primarily through the destruction of lacrimal glands (LGs) (214), keratoconjunctivitis sicca (215). In addition, LG pathologic properties in Sjögren's syndrome appear in rabbits induced by autoimmune dacryoadenitis (216–218). Acini and interlobar ducts have the lowest mRNA abundance of ClC2γ, and the intralobar duct has the highest (183,184), and alterations in ClC2γ may alter lacrimal secretion, particularly Cl− transport (184). The causes of age-related cataracts include aging, inheritance, local nutritional disorders, immune and metabolic abnormalities, trauma, poisoning and radiation. According to a study by Ouyang (185), the expression level of ClC-2 is associated with the development of age-related cataracts.

Reports on otorhinolaryngological diseases associated with ClC-2 are limited, however, Li et al reported on the pathogenesis of nasal polyps. The study (186) showed that ClC-2 was not expressed in normal nasal mucosa, however, ClC-2 proteins were expressed in epithelial cells and sub-epithelial mucous glands in patients affected with nasal polyps. This suggested that ClC-2 is involved in the pathogenesis of nasal polyps.

Diseases of the reproductive system associated with ClC-2

In previous studies, a number of diseases of the reproductive system have been associated with ClC-2, including degeneration of the testes (11) and azoospermia (187,219,220).

It was shown that severe degeneration of the testes appears in ClC-2-knockout mice, however, no other reproductive abnormalities are observed (11). A study by Bösl et al suggested that ClC-2 disruption causes the death of germ cells, impaired transport and alterations to the ionic environment of germ cells, and that germ cells rely on the transepithelial transport mediated by Sertoli cells (11).

Azoospermia is a medical condition in which males do not have a measurable level of sperm in their semen, and is associated with low levels of fertility or infertility. Its pathogenesis involves CFTR disruption in CF, affecting male fertility (219). In addition, the male sterility resulting from reduced fluid volume is associated with disruption of Cl− channels from other ClCN genes (220). Another study (187) by Edwards et al reported that ClCN2nmf240 homozygotes have azoospermic symptoms severe degradation of spermatogenesis, and shortages of spermatocytes, spermoblasts and sperm.

Diseases of the respiratory system associated with ClC-2

Reports on diseases of the respiratory system associated with ClC-2 are limited. A previous study (188) reported that ClC-2 may be important in the invasion, development and occurrence of lung cancer, and that ClC-2 may be a novel molecular target for clinical therapy in non-small cell lung cancer.

Diseases of the digestive system associated with ClC-2

There have been several reports on diseases of digestive system associated with ClC-2, including constipation (18,26,189–193,221) and repair of impaired intestinal mucosa barrier (194).

CFTR is major chloride channel regulating chloride secretion in the small intestine (221), however, ClC-2 can also regulate chloride secretion (18,26). Hypotonicity can also activate chloride currents in the rat ileum (189). Several studies have reported that ClC-2 is expressed in the intestinal epithelium, in T84 human intestinal cells, rat intestinal tissue (3) and the murine duodenum (190). In addition, previous studies have shown that ClC-2 can regulate chloride secretion in rodent neonatal airways (26), and regulate gastric chloride secretion (18). However, another study (191) described contradictory findings, reporting that ClC-2 was not involved in Cl− secretion, but was involved in Cl− absorption in the distal colon. According to these studies, ClC-2 inhibitors may be used to treat constipation by decreasing NaCl and water absorption in the colon (191).

Irritable bowel syndrome (IBS) is characterized by chronic abdominal pain, discomfort and bloating associated with altered bowel habits, including diarrhea and/or constipation. Current therapy for constipation caused by IBS has side effects, including deterioration of the condition or electrolyte disturbances (192). Previous studies have shown that, in addition to the above-mentioned side effects, lubiprostone can also increase gut motility and frequency of stool passage, relieve abdominal pain and discomfort (192), stimulate chloride secretion and improve bowel function (193).

A previous study (194) by Chen et al reported that the activation of ClC-2 can activate tight junction proteins and repair impaired intestinal mucosa barrier. In addition, ClC-2 and tight junction proteins are involved in maintenance of the intestinal mucosal barrier, and acute biliary obstruction-induced destruction of the intestinal mucosa barrier is associated with ClC-2 in enterocytes (194).

Diseases of the hematological system associated with ClC-2

There have been few studies on diseases of the hematological system associated with ClC-2. A previous study reported the expression of swelling-and/or pH-regulated ClC-2 chloride channels in human leukemia (195). In addition, it was suggested that the molecular identification of chloride channels may provide a novel approach for the treatment of leukemia (195).

Genetic diseases associated with ClC-2

According to previous reports, there have been several studies on the association between CF and ClC-2 (3,11,23,25,26,68,70,196–198,222,223). Several studies have reported that ClC-2 may be an alternative pathway for chloride anion secretion in CF (3,11,23), and that ClC-2 is a potential target for therapy in CF (25,197). CF is a genetic disease, which primarily affects the lungs, in addition to the pancreas, liver, kidneys and intestine (222). CF is caused by mutations in the gene encoding CFTR, the only member of the ABC transporter family known to be a cAMP-activated chloride channel (223). ClC-2 is a candidate alternative chloride channel in respiratory epithelia (196). ClC-2 is also involved in lung morphogenesis (26,68), and can conduct chloride in mature respiratory epithelia (23,25,197). ClC-2 mRNA and protein are expressed in unaffected tissues in CF, which may make up for defects in the expression of CFTR (68).

However, another previous study (198) reported that ClC-2 is unlikely to be a candidate rescue channel in CF, as disruption of ClC-2 and CFTR channel genes did not cause morphological alterations in the intestine, lung or pancreas affected by CF; neither disrupted ClC-2 or CFTR reduced Cl− secretion (198). In addition, two studies (70,196) refer to the importance of detecting modifications of CF by ClC-2, and underlined the importance of examining potential polymorphisms in subjects affected with CF and potential mutations in the coding region of ClC-2.

Diseases of the nervous system associated with ClC-2

There have been more studies on diseases of the nervous system associated with ClC-2, compared with other diseases associated with ClC-2. Studies on diseases of the nervous system associated with ClC-2 have predominantly focused on leukoencephalopathy (199–202,224) and epilepsy (30,40,203–212,225).

Leukoencephalopathy can refer specifically to any of the following diseases: Progressive multifocal leukoencephalopathy, toxic leukoencephalopathy, leukoencephalopathy with vanishing white matter, leukoencephalopathy with neuroaxonal spheroids, reversible posterior leukoencephalopathy syndrome, megalencephalic leukoencephalopathy with subcortical cysts (MLC) and hypertensive leukoencephalopathy. Among these, MLC is a rare type of leukodystrophy, which is characterized by macrocephaly emerging in the first years of life (224).

The disruption of ClC-2 can cause fluid accumulation resulting in myelin vacuolation in mice, similar to that observed in humans affected with MLC from mutations in MLC1 or GlialCAM. GlialCAM is a ClC-2 binding partner and the first auxiliary subunit of ClC-2 (199). According to a study by Jeworutzki et al (199), neither the stimulation of GlialCAM on ClC-2 currents, nor mislocalization of this Cl− channel were found to damage glial Cl− transport. Mutated GlialCAM in MLC can target ClC-2 to cell contacts in glia and activate its currents (200). A study by Jeworutzki et al (200) showed that GlialCAM can target the common gate deficient ClC-2 mutant, E211V/H816A, to cell contacts without altering its function. Another study suggested that ClC-2 is not important for MLC1 or GlialCAM localization in the brain (201), but that it is involved in the pathogenesis of MLC (201).

However, a separate study (202) reported different conclusions, as there was no evidence that the CLCN2 gene is associated with MLC. Despite not referring to the association between the CLCN2 gene and MLC, this study demonstrated that mice lacking the ClC-2 protein had white matter abnormalities with vacuole formation in myelin sheaths, similar to the intramyelinic vacuoles in MLC.

Epilepsy comprises a set of neurological diseases characterized by epileptic seizures, and a heterogeneous disorder characterized by recurrent unprovoked seizures, which affect ~1–3% of the population during their lifetimes (225). ClC-2 mRNA and protein are found in neurons and astrocytes (30,40,203). In addition, ClC-2 protein is present at the end feet of astrocytes contacting blood vessels and neurons close to inhibitory synapses (30). Inwardly rectifying hyperpolarization-activated ClC-2-like currents are found in hippocampal pyramidal cells (40,127) and in astrocytes (204–207). In hippocampal pyramidal cells and astrocytes (208), ClC-2 may have different effects. In neurons, ClC-2 can prevent the accumulation of chloride anions above equilibrium due to the activation of ClC-2 by intracellular Cl− (41,127). Under these conditions, GABAA R activity may become excitatory (208). A previous study (208) reported that loss of function mutations of ClC-2 lead to increased excitability in certain neurons, and that hyperpolarization-activated chloride currents are detected in cortical astrocytes, but absent in tissues from ClC-2-null mice (207). Another study (211) showed that several ClC-2 sequence abnormalities previously found in patients affected with epilepsy are likely to represent innocuous polymorphisms, detected by sequencing of a large collection of human DNA and electrophysiological analysis.

Several other studies have reported that a susceptibility locus for idiopathic generalized epilepsy (IGE) is on chromosome 3q26 (the location of the CLCN2 gene) (209), and three mutations on ClC-2 cosegregated with IGE with autosomal dominant inheritance (210) have been identified.

In addition to the above-mentioned studies on the association between epilepsy and ClC-2, an association between temporal lobe epilepsy (TLE) and ClC-2 has been reported. TLE with spontaneous recurrent attacks, and learning and memory disabilities, is associated with neurodegeneration, abnormal reorganization of the circuitry and loss of functional suppression in hippocampus (212). A study by Ge et al reported that ClC-2 contributes towards tonic inhibition, modulated by α5 subunit-containing GABAA Rs in the CA1 area (212).

Metabolic diseases associated with ClC-2

Few studies on metabolic diseases associated with ClC-2 have been performed, however, a study on diabetes mellitus was found. Diabetes mellitus is a metabolic disease, which generally causes chronic delayed wound healing. In a study by Pan et al (213), a high glucose concentration inhibited keratinocyte migration by downregulating ClC-2, suggesting ClC-2 may be important during delayed wound healing processes. In addition, the study reported that ClC-2 is an important modulator of cell migration in keratinocytes, although it did not discuss how ClC-2 is involved in keratinocyte migration.

Conclusion

With the continuous progress in experimental and clinical studies on the ClC family, it is clear that the activation of ClC-2 occurs via hyperpolarization (3,9), cell swelling (2,9), extracelluar hypotonicity (2) and extracellular acidification (9,10), and that the expression of ClC-2 is ubiquitous (3,11) in ureteric bud cells (12), the intestine (13–17), gastric parietal cells (18,19), the liver (20), the lungs (21–25), rat retina (27), parotid acinar cells (28), guinea pig cardiac muscle (29), neuronal cells (30), rat and human airways (17), bovine trabecular meshwork (31), human trabecular meshwork (32,33) and rat trabecular meshwork (34). Although there has been progress in understanding the CLCN2 gene, the molecular structure of the ClC-2 protein, the structure of ClC-2 chloride channels, and the functional properties and mechanisms regulating ClC-2, they remain to be fully elucidated and there have been contradictions in previous studies (226). According to previous studies, it is known that the disruption of ClC-2 can lead to several diseases. Although ClC-2 is associated with the pathogenesis of several diseases, the association between pathogenesis and ClC-2 remains to be fully elucidated.

At present, >6,500 types of genetic disease have been identified, among which ~3,000 are caused by a single gene defect. In this review, the mechanisms underlying the association between the CLCN2 gene and diseases, including azoospermia and IGE, were discussed. In the future, investigations focused on the determination of the CLCN2 gene may identify novel methods to treat and prevent several diseases associated with ClC-2. In addition, as the overexpression and underexpression of ClC-2 can cause diseases, the development of specific ClC-2 activators and inhibitors, and understanding the mechanism of action between the functional properties of ClC-2 and these activators and inhibitors, is required in future investigations to regulate the expression of ClC-2.

As described above, ClC-2 can be regulated by hormones, drugs and scorpion venom. Certain hormones in humans are produced in small quantities, however, they have a substantial effect on health, with underproduction or overproduction leading to a variety of diseases. In addition, scorpion venom can be used to treat cancer and lower blood pressure, and is applied for hemostasis, anticoagulation, as an analgesic and a nerve growth factor. Therefore, future investigations focused on the regulatory mechanisms of hormones, drugs and scorpion venom may assist in developing specific drugs for treating diseases associated with ClC-2.

In conclusion, ClC-2 is important in several diseases and, in order to fully elucidate the structure and function of ClC-2, and the mechanisms regulating ClC-2 associated with disease treatment, examining the associations between ClC-2 and regulators, including hormones, proteins, kinases, transcription factors, scorpion venom, adenosine triphosphate, clh-3, permeant anions, membrane cholesterol, tyrosine endocytosis motif and α1-adrenoceptor is required to develop novel treatment strategies. Therefore, further investigations are required in the future.