Physiological and Pathological Significance of Chloride Channels

Abstract

Cl⁻ influx and efflux through Cl⁻ channels play a role in regulating the homeostasis of biological functions. Therefore, the hyperfunction or dysfunction of Cl⁻ channels elicits pathological mechanisms. The Cl⁻ channel superfamily includes voltage-gated Cl⁻ (ClC) channels, Ca²⁺-activated Cl⁻ channels (Cl_Ca; TMEM16A/TMEM16B), cystic fibrosis transmembrane conductance regulator channels, and ligand-gated Cl⁻ channels. These channels are ubiquitously expressed to regulate ion homeostasis, muscle tonus, membrane excitability, cell volume, survival, neurotransmission, and transepithelial transport. The activation or inhibition of Cl⁻ channels changes the membrane potential, thereby affecting cytosolic Ca²⁺ signals. An elevation in cytosolic [Ca²⁺] triggers physiological and pathological responses in most cells. However, the roles of Cl⁻ channels have not yet been examined as extensively as cation (Na⁺, Ca²⁺, and K⁺) channels. We recently reported the functional expression of: (i) TMEM16A/Cl_Ca channels in portal vein and pulmonary arterial smooth muscle cells (PASMC), pinealocytes, and brain capillary endothelial cells; (ii) TMEM16B/Cl_Ca channels in pinealocytes; (iii) ClC-3 channels in PASMC and chondrocytes; and (iv) ClC-7 channels in chondrocytes. We also showed that the down-regulation of TMEM16A and ClC-7 channel expression was associated with cirrhotic portal hypertension and osteoarthritis, respectively, whereas the enhanced expression of TMEM16A and ClC-3 channels was involved in the pathogenesis of cerebral ischemia and pulmonary arterial hypertension, respectively. Further investigations on the physiological/pathological functions of Cl⁻ channels will provide insights into biological functions and contribute to the screening of novel target(s) of drug discovery for associated diseases.

1. INTRODUCTION

Chloride ion (Cl⁻) efflux/influx through Cl⁻ channels regulate the homeostasis of biological functions. The activation or inhibition changes the membrane potential and, thus, affects the cytosolic concentration of Ca²⁺ ([Ca²⁺]_cyt). Changes in [Ca²⁺]_cyt regulates physiological and pathological events in most cells.¹⁾ [Ca²⁺]_cyt is mainly regulated by Ca²⁺ influx/extrusion through plasmalemmal proteins and Ca²⁺ release/uptake via intracellular Ca²⁺ store sites.^2–4) A rise in [Ca²⁺]_cyt is elicited by two pathways: Ca²⁺ influx via plasmalemmal Ca²⁺-permeable ion channels (voltage-gated Ca²⁺ channels (VDCC), store-operated Ca²⁺ (SOC) channels, and receptor-operated Ca²⁺ (ROC) channels) and Ca²⁺ release via ryanodine and inositol 1,4,5-trisphophate (IP₃) receptors on the sarco/endoplasmic reticulum (SR/ER). Increased [Ca²⁺]_cyt is reversed to the resting level by Ca²⁺ extrusion through Ca²⁺ pump (Ca²⁺-ATPase) and Na⁺/Ca²⁺ exchangers on the plasma membrane, and Ca²⁺ uptake into cytosolic Ca²⁺ store sites via SR/ER Ca²⁺-ATPase and mitochondrial Ca²⁺ uniporters.

The Cl⁻ channel family includes voltage-gated Cl⁻ channels (ClC), Ca²⁺-activated Cl⁻ channels (Cl_Ca; TMEM16A and TMEM16B), cystic fibrosis transmembrane conductance regulator (CFTR) channels, and ligand-gated Cl⁻ channels (glycine and γ-amino-butyric acid type A (GABA_A) receptors).^5,6) Fewer physiological/pathological/pharmacological analyses have been conducted on Cl⁻ channels in many cell types than on cation (Na⁺, Ca²⁺, and K⁺) channels. This difference may be attributed to the molecular entity of Cl⁻ channels remaining unknown and few specific modulators of Cl⁻ channels being identified.

We recently demonstrated the functional expression of four Cl⁻ channels: (i) TMEM16A/Cl_Ca channels in portal vein smooth muscle cells (PVSMC),^7–10) pulmonary arterial SMC (PASMC),¹¹⁾ brain capillary endothelial cells (BCEC),^12,13) and pinealocytes¹⁴⁾; (ii) TMEM16B Cl_Ca channels in pinealocytes¹⁴⁾; (iii) voltage-gated and swelling-activated ClC-3 channels in PASMC¹⁵⁾ and chondrocytes¹⁶⁾; and (iv) voltage-gated ClC-7 channels in chondrocytes.^17,18) In addition, changes in the expression changes of TMEM16A, ClC-3, and ClC-7 channels have been related with the pathological mechanisms of cirrhotic portal hypertension,⁹⁾ pulmonary arterial hypertension (PAH),¹⁵⁾ cerebral ischemia,¹³⁾ and osteoarthritis (OA).^16,18,19) This review discusses the physiological and pathological significance of Cl⁻ channels in several cell types.

2. CL⁻ CHANNEL SUPERFAMILY

Cl⁻ channels mediate the various physiological mechanisms underlying Cl⁻ balance, muscle contraction, membrane excitability, neuronal transmission, volume regulation, cell fate, and transepithelial transport. The Cl⁻ channel gene family is classified into at least four groups: ClC channels, CFTR channels, Cl_Ca channels (TMEM16A and TMEM16B), and ligand-gated Cl⁻ channels (GABA_A and glycine receptors).^5,6)

Voltage-gated ClC channels are widely expressed for the regulation of physiological functions, including stabilization of the membrane potential, the organelle acidification, volume regulation, cell survival, and transepithelial transport.²⁰⁾ To date, nine ClC proteins (ClC-1–7, Ka, and Kb) have been identified in mammals. Among ClC family, ClC-1/2/Ka/Kb proteins are responsible for plasma membrane Cl⁻ channels. On the other hand, ClC-3/4/5/6/7 proteins are considered to work as intracellular Cl⁻/H⁺ transporters rather than classical Cl⁻ channels.^6,20) These ClC channels have a characteristic electrophysiological profile.^5,6,20) ClC-1/4/5/7 currents are evoked by membrane depolarization, whereas ClC-2 channels are slowly activated by membrane hyperpolarization. ClC-3 currents are evoked by depolarizing/hyperpolarizing stimulations. ClC-Ka/Kb channels generate weak currents dependent on membrane potentials. These ClC channels also exhibit distinct pharmacological sensitivity.^5,6,20) Niflumic acid broadly blocks ClC channels. 4,4′-Diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) inhibits ClC-3/4/7 (and weakly ClC-2/5) channels. 9-Anthracenecarboxylic acid (9-AC) weakly suppresses ClC-1/2 channels, but not ClC-3/5 channels. Acidification elicits ClC-2/7 currents and blocks ClC-4/5 currents, while hypoosmotic stress activates ClC-2/3 channels.

CFTR channels have twelve transmembrane domains, nucleotide-binding domains, and a regulatory domain.^5,6) CFTR are predominantly localized in the apical plasma membrane of epithelial tissues, such as the lung, intestine, sweat duct, pancreas, and testis. Their channel activation following cAMP-dependent phosphorylation and ATP binding to nucleotide-binding domains is associated with normal fluid transport across epithelia. The CFTR was originally identified as the pathogenesis of CF. A deletion mutation in phenylalanine at the 508 position (ΔF508) is the most frequent cause of CF and disrupts the functional CFTR channels. They are voltage-independent channels that are enhanced by cAMP, protein kinase C, and protein kinase A. They are blocked by CFTR_inh-172, but less potently by conventional blockers for Cl⁻ channels.

Cl_Ca channels are widely expressed and play various roles in the regulation of transepithelial transport, sensory transduction, smooth muscle contraction, nociception, and neuronal excitation. TMEM16A/B belonging to the TMEM16 gene family that consists of 10 genes (TMEM16A to TMEM16K, except for TMEM16I) in mammals, are responsible for native Cl_Ca channel conductance.^6,21) TMEM16A and TMEM16B channels include ten putative transmembrane domains, a Ca²⁺-binding pocket, and calmodulin-binding sites.^22,23) TMEM16A channels are mainly expressed in vascular SMC (VSMC), interstitial cells of Cajal, airway epithelial cells, nociceptive neurons, and cancers. TMEM16B channels localize to retinal photoreceptor, olfactory, and hippocampus neurons.²¹⁾ Although both channels generate Cl_Ca currents, their biophysical characteristics significantly differ.²¹⁾ The half maximal [Ca²⁺]_cyt required for the TMEM16A activation (0.4 to 0.6 µM) is lower than that of TMEM16B channels (1 to 3 µM). Furthermore, the time constants of the TMEM16A activation/deactivation are more than 10-fold slower than those of TMEM16B. The single TMEM16A channel conductance (3.5 to 8.3 pS) is also slightly higher than that of TMEM16B channels (0.8 to 3.9 pS). However, both channels share some pharmacological profiles.^6,21,23) They are blocked by conventional blockers for Cl⁻ channels (e.g., DIDS, 9-AC, and niflumic acid). Moreover, T16A_inh-A01 and Ani9 inhibit the activity of TMEM16A channels.

The ligand-gated ion channel gene family contains ionotropic GABA_A and glycine receptors, which are mainly involved in inhibitory neurotransmission in the central nervous system.^5,6) GABA and glycine are used in the brain and spinal cord, respectively. GABA_A and glycine receptors are pentameric channels that are selectively permeable to Cl⁻. They have a cysteine loop at the large extracellular N-terminal region, four transmembrane domains, and an extracellular C-terminal region. GABA_A receptors are enhanced by GABA and classical benzodiazepines (alprazolam and diazepam) and are antagonized by picrotoxin and bicuculline. Glycine receptors are enhanced by β-alanine, taurine, and glycine and are blocked by strychnine and picrotoxin.

3. TMEM16A CL_CA CHANNELS IN THE PORTAL VEIN

The portal vein exhibits spontaneous contractions that are important for blood flow from mesenteric beds to the liver. Since they are mediated by the activity of Cl_Ca channels,²⁴⁾ their molecular identification is essential for understanding the regulatory mechanisms underlying gastro-liver circulation. TMEM16A proteins have been reported to produce Cl_Ca channel conductance in VSMC.^21,24) In PVSMC, TMEM16A proteins and alternative spliced variants are largely responsible for the activity of Cl_Ca channels⁷⁾ (Fig. 1). Single-molecule photobleaching analyses using a total interference reflection fluorescent (TIRF) microscope²⁵⁾ enable the formation of functional Cl_Ca channels by dimeric TMEM16A proteins to be visualized⁷⁾ (Fig. 2). TMEM16A channels have a dimerization domain at the N-terminal.²¹⁾ In VSMC, the activation of Cl_Ca channels shifts the resting membrane potential (RMP) to a depolarizing direction and enhances Ca²⁺ influx via VDCC, resulting in enhanced myogenic tone.^24,26,27) Therefore, TMEM16A channel blockers attenuate spontaneous contractions in PVSM.⁹⁾

Fig. 1. TMEM16A Cl_Ca Channels in Mouse PVSMC

(A) Expression of TMEM16A proteins at the plasma membrane of mouse PVSMC. Left and right panels show transmitted and immunofluorescent images. (B) Whole-cell Cl_Ca currents in PVSMC, which were stimulated from a holding potential of −60 mV to the test potentials (−80 to +120 mV) for 1 s. The dashed line indicates the zero current level. (C) Current density-voltage relationship measured at the end of the test pulses in the absence and presence of a Cl_Ca channel blocker, niflumic acid. (D) Putative topology of mouse full-length TMEM16A at that time.²²⁾ Four square boxes (a, b, c, and d) indicate alternatively spliced segments. (E) Percentage of TMEM16A isoforms. Data are shown as means ± standard error (S.E.). [Modified from⁷⁾].

Fig. 2. Subunit Stoichiometry of TMEM16A Cl_Ca Channels

(A) Conceptual diagram of the GFP photobleaching at the single molecular level. In GFP-expressing cells, the photobleaching of GFP fluorescence in a whole-cell area gradually occurs with time. On the other hand, the photobleaching of a single GFP molecule involves a discrete process in an all-or-none manner. Therefore, the bleaching steps of GFP signals under a TIRF microscope indicate the number of these subunits within a single fluorescent particle. (B to D) Changes in the fluorescent intensity of the BK_Caα-GFP (B), VDCCα1C-GFP (C), or TMEM16A-GFP (D) subunit in human embryonic kidney 293 (HEK293) cells after the photobleaching stimulation (upper). Arrows and solid lines indicate bleaching step(s). The dotted line indicates the complete bleaching (basal) level. The number of bleaching steps and percentage of HEK293 cells expressing these subunits (middle) and the subunit stoichiometry estimated by these results (bottom). Note that BK_Caα and VDCCα1C channels are expressed as a tetramer and monomer, respectively, whereas TMEM16A channels form a dimer. [Modified from^7,25,31,32)].

The ion channel activity is occasionally modulated by interactions with auxiliary subunits, the cytoskeleton, and scaffold proteins.²²⁾ TMEM16A channels have been reported to interact with the scaffold proteins, the ezrin-radixin-moesin complex in salivary gland epithelial cells.²⁸⁾ TMEM16A channels is also modulated by interactions with the actin in PVSMC.⁸⁾ The amino acid sequence of mouse TMEM16A channels (GenBank accession number, NM_178642) suggests two candidates for the actin-binding domain, ¹⁹²LLEAGL and ⁷⁸⁶IIEIRL, based on the conserved actin-binding motif (L/I-X-D/E-X-X-L/I).²⁹⁾ In addition, caveolin 1 deficiency has been shown to up-regulate TMEM16A in PVSMC.¹⁰⁾ Caveolin 1 is a scaffold protein that assembles several molecules as a complex in caveolae, which are raft structures on the surface of plasma membrane.³⁰⁾ Caveolin 1 interacts with VDCC,³¹⁾ large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels,^31,32) and Ca²⁺/calmodulin-dependent kinases^33,34) and their complexes accumulate in the caveolae of VSMC. These functional couplings may be recognized as a specific domain of efficient and effective Ca²⁺ signaling in VSMC. These regulatory mechanisms are informative for elucidating the physiological functions of VSMC Cl_Ca channels.

Portal hypertension induces as a complication in hepatic diseases, including cirrhosis. Cirrhosis-induced fibrosis causes an increase in hepatic vascular tone and vascular remodeling, leading to portal hypertension. The hepatic venous pressure gradient normally ranges between 1 and 5 mmHg. Portal hypertension is clinically defined as an increase in hepatic venous pressure gradient to 10 mmHg and higher.³⁵⁾ In PVSMC from cirrhotic portal hypertensive mice, Cl_Ca currents and spontaneous contractions were found to be inhibited by the down-regulation of TMEM16A expression⁹⁾ (Figs. 3A–E). This down-regulation was mimicked by a treatment with angiotensin II⁹⁾ (Fig. 3F), which is elevated in the blood of cirrhosis patients.³⁶⁾ Similarly, angiotensin II has been shown to down-regulate TMEM16A expression in basilar³⁷⁾ and aortic³⁸⁾ SMC. In contrast, angiotensin II has been reported to up-regulate its expression in aortic SMC.³⁹⁾

Fig. 3. Down-Regulation of TMEM16A Cl_Ca Channels in Cirrhotic Portal Hypertension

(A) Down-regulation of the TMEM16A gene in PVSM from bile duct ligation (BDL) mice, which are widely used as an experimental model of cirrhotic portal hypertension. (B) Decreased TMEM16A proteins in BDL-PVSMC. α-SMA, α-smooth muscle actin. (C) Reduced Cl_Ca currents in BDL-PVSMC, which were stimulated from a holding potential of −60 mV to the test potentials (−100 to +100 mV) for 1 s and then repolarized to −80 mV for 500 ms. (D) Current density-voltage relationship of outward Cl_Ca currents in sham- and BDL-PVSMC. The peak amplitude of outward currents at +100 mV (inset). (E) Spontaneous contractions in the absence and presence of a TMEM16A channel blocker (T16A_inh-A01) in sham- and BDL-PVSM. (F) The down-regulation of TMEM16A expression in angiotensin II-treated PVSM. Data are shown as means ± S.E. * p < 0.05, ** p < 0.01 versus sham or control. [Modified from⁹⁾].

In VSMC, decreased Cl_Ca conductance shifts the RMP to a hyperpolarizing direction and inhibits Ca²⁺ influx via VDCC, which negatively regulates the formation of Ca²⁺-dependent action potentials and spontaneous contractions.^24,26,27) Therefore, the down-regulation of TMEM16A channels may protect against portal pressure increase and associated symptoms. Vasopressin analogues, non-selective adrenergic β receptor blockers, and somatostatin analogues are now used to prevent portal hypertension in cirrhosis. Although novel drug target(s) for portal hypertension have been discussed, these strategies are far from satisfactory. Further studies are necessary for elucidating the pathological mechanism(s) in cirrhotic portal hypertension.

4. TMEM16A CL_CA CHANNELS IN THE PULMONARY ARTERY

Pulmonary circulation is essential for oxygen absorption and carbon dioxide excretion. The tone of PASM is regulated by RMP and [Ca²⁺]_cyt. A rise in [Ca²⁺]_cyt stimulates pulmonary vascular remodeling and vasoconstriction.⁴⁰⁾ In PASMC, Cl_Ca channels are formed by TMEM16A proteins (Fig. 4) and regulate Ca²⁺ signaling through ROC and SOC channels.¹¹⁾ ROC channels are activated following receptor stimulation by growth factors and vasoconstrictors and elicit an increase in [Ca²⁺]_cyt.⁴⁰⁾ SOC channels are enhanced by Ca²⁺ depletion in the SR and induce an elevation in [Ca²⁺]_cyt to refill Ca²⁺ in the depleted SR.⁴⁰⁾

Fig. 4. TMEM16A Cl_Ca Channels in Human PASMC

(A) Whole-cell Cl_Ca currents in human PASMC, which were stimulated from a holding potential of −60 mV to the test potentials (−80 to +100 mV) for 500 ms. Black and gray arrowheads indicate outward peak currents and inward tail currents, respectively. (B) Current density-voltage relationship at the peak amplitude during depolarization (black arrowhead in (A)). (C) Expression analysis of TMEM16A proteins in PASMC using two specific primary antibodies (ab53212 and ab53213; Abcam, Cambridge, U.K.). Alexa Fluor 488 (green) and DAPI (blue) were used as a secondary antibody and nuclear marker, respectively. Data are shown as means ± S.E. [Modified from¹¹⁾].

The cytosolic concentration of Cl⁻ ([Cl⁻]_cyt) in PASMC is high at approximately 50 mM²⁶⁾; therefore, the reversal potential of Cl⁻ is less negative (approximately −25 mV) than that for K⁺ (−80 mV). Since the RMP in PASMC is approximately −65 to −50 mV,⁴¹⁾ an increase in Cl⁻ conductance (due to the up-regulation of Cl⁻ channels) generates inward currents (Cl⁻ efflux) and causes depolarization, resulting in Ca²⁺ influx via VDCC and contraction. The up-regulated expression of TMEM16A has been reported in and systemic hypertension³⁹⁾ and pulmonary hypertension.⁴²⁾

5. SWELLING-ACTIVATED CLC-3 CHANNELS IN THE PULMONARY ARTERY

The diagnostic criteria for pulmonary hypertension were recently revised to a pulmonary arterial pressure (PAP) of > 20 mmHg at rest,⁴³⁾ while the normal PAP is 14 ± 3 mmHg.⁴⁴⁾ PAH is a fatal, progressive, and rare disease. It is characterized by pulmonary vascular remodeling and vasoconstriction, which leads to the narrowing of distal pulmonary arteries and, thus, progressive increases in PAP. The elevation of PAP causes right ventricular hypertrophy/failure.⁴³⁾

Pulmonary vascular remodeling is mainly attributed to the excessive proliferation of PASMC. Excessive cell proliferation needs a sustained elevation in [Ca²⁺]_cyt through increases in the expression of Ca²⁺-permeable channels.⁴¹⁾ Previous studies reported the functional expression in PASMC and their up-/down-regulation in PASMC from experimental PAH animals and PAH patients⁴¹⁾: e.g., VDCC,⁴⁵⁾ transient receptor potential (TRP) channels,^46–49) Orai/stromal interaction molecule (STIM) channels,⁴⁸⁾ voltage-gated K⁺ (K_V) channels,⁵⁰⁾ and BK_Ca channels,⁵⁰⁾ in addition to Ca²⁺-sensing receptors.^51,52)

In addition, the up-regulated expression of voltage-gated ClC-3 channels in PASMC from PAH patients contributes to swelling-activated Cl⁻ currents under hypoosmotic conditions and excessive cell proliferation¹⁵⁾ (Fig. 5). Similar up-regulation has been observed in the PASMC of PAH rats⁵³⁾ and the aortic SMC of neointimal hyperplasia mice⁵⁴⁾ and diabetic rats.⁵⁵⁾ Since proliferation is accompanied by swelling of cell volume, the mechanism(s) of swelling-activated Cl⁻ channels is highlighted in vascular remodeling. In VSMC, ClC-3 channels are involved in proliferation as well as myogenic contractions and volume regulation.^27,56) Endothelin receptor antagonists, phosphodiesterase 5 inhibitors, and prostacyclin analogues are used for PAH patients. A soluble guanylate cyclase activator and prostaglandin I₂ receptor activator have been approved for PAH patients.^43,44) However, the five-year survival rate is 65–70% in the U.S.A. and Europe.⁴⁴⁾ There remains an unmet medical need and novel drugs are required. Up-regulated ClC-3 channels, in addition to Ca²⁺-permeable ion channels, may be a novel molecular target for specific PAH drug(s).

Fig. 5. Up-Regulated Expression of ClC-3 Channels in PASMC from PAH Patients

(A) Up-regulated ClC-3 proteins in PASMC from normal subjects and idiopathic PAH (IPAH) patients. (B, C) Swelling-activated and DIDS-sensitive Cl⁻ currents in normal- (B) and IPAH- (C) PASMC treated with control or ClC-3 siRNA. PASMC were stimulated from a holding potential of 0 mV to the test potentials (−80 to +80 mV) for 200 ms. (D) The reduced proliferation of normal- and IPAH-PASMC treated with ClC-3 siRNA. Data are shown as means ± S.E. * p < 0.05, ** p < 0.01 vs. normal or control siRNA. [Modified from¹⁵⁾].

6. TMEM16A CL_CA CHANNELS IN BRAIN CAPILLARIES

The blood–brain barrier (BBB) is consisted of BCEC, which regulate brain homeostasis. It restricts the substance movement between the brain and circulation and protects neurons against peripheral noxious substances. The barrier function is maintained by the proliferation and death of BCEC.⁵⁷⁾ The survival and death of vascular EC require an elevation in [Ca²⁺]_cyt, which are modulated by ion channels.⁵⁸⁾ For example, small-conductance Ca²⁺-activated K⁺ channels,⁵⁹⁾ inward-rectifier K⁺ channels,⁵⁹⁾ and Orai/STIM channels⁶⁰⁾ are expressed to facilitate BCEC proliferation and death. In addition, TMEM16A channels generate Cl_Ca conductance and affect both the RMP and [Ca²⁺]_cyt, which are positive regulators of the proliferation, migration, and trans-endothelial permeability of BCEC¹²⁾ (Fig. 6).

Fig. 6. TMEM16A Cl_Ca Channels in Bovine BCEC

(A) The abundant expression of the TMEM16A gene in bovine BCEC (t-BBEC117). (B) The protein expression of TMEM16A channels in BCEC. (C) Whole-cell Cl_Ca currents in BCEC treated with control or TMEM16A siRNA. BCEC were stimulated from a holding potential of −40 mV to the test potentials (−80 to +100 mV) for 500 ms and subsequently repolarized to −80 mV for 250 ms. (D) Inhibitory effects of BCEC proliferation by TMEM16A siRNA. (E) Inhibition of BCEC migration by TMEM16A siRNA. (F) Effects of Cl_Ca channel blockers (niflumic acid and T16A_inh-A01) on trans-endothelial permeability (as trans-endothelial electrical resistance (TEER)). Data are shown as means ± standard deviation (S.D.). * p < 0.05, ** p < 0.01 vs. control or control siRNA [Modified from¹²⁾].

Since [Cl⁻]_cyt in endothelial tissues is estimated to be approximately 40 mM,²⁶⁾ the equilibrium potential of Cl⁻ is around −30 mV. The RMP of BCEC is approximately −40 mV⁶¹⁾; therefore, the blockade of Cl_Ca channels leads to membrane hyperpolarization. Hyperpolarization due to K⁺ channel activation promotes Ca²⁺ influx via non-selective cation channels in BCEC.^59,60) Hyperpolarization due to Cl_Ca channel inhibition facilitates Ca²⁺ influx via the same signaling pathway. Breakdown of the BBB disrupts homeostasis in the brain and, thus, causes neurodegenerative disorders, such as Parkinson’s disease, Alzheimer’s disease, and multiple sclerosis.⁵⁷⁾ TMEM16A may be a novel drug target for patients with BBB dysfunctions.

BCEC are exposed to hypoxic environments during cerebral ischemia, leading to BBB disruption. TMEM16A are up-regulated by hypoxia, which increases the proliferation and permeability of BCEC.¹³⁾ Similar hypoxia-induced up-regulation was observed in the cardiac vascular EC of neonatal mice.⁶²⁾ Hypoxia-inducible factor-1α (HIF-1α) is an important regulator for changing expression during hypoxia.⁶³⁾ The expression of HIF-1α is maintained at a low level under normoxia and is immediately up-regulated by hypoxia in order to induce the up-/down-regulation of target proteins. In BCEC, HIF-1α is constitutively expressed even under normoxia.⁶⁴⁾ Thus, the working hypothesis that up-regulated HIF-1α by hypoxia alter the expression of targeted channels may not fit. The hypoxia-induced up-regulation of TMEM16A channels and facilitated BCEC proliferation may induce BBB dysfunction during cerebral ischemia.

7. VOLTAGE-GATED CLC-7 CHANNELS IN CHONDROCYTES

In diarthrodial joints, the bone surface is covered by articular cartilage, which contains 1 to 10% chondrocytes.⁶⁵⁾ Articular chondrocytes are responsible for the synthesis and secretion of the collagens, extracellular matrix, and proteoglycans, which cover articular cartilage to protect against biochemical and mechanical stress. In the process of responding to various stimuli, the activity of ion channels regulates chondrocyte functions though changes in the RMP and [Ca²⁺]_cyt. The RMP is regulated by the balance between K⁺⁶⁶⁾ and Cl⁻¹⁹⁾ conductance in articular chondrocytes. Therefore, the genetic knockdown and pharmacological blockade of Cl⁻ channels induce the hyperpolarization.^17,18) In non-excitable cells, including chondrocytes, membrane hyperpolarization facilitates Ca²⁺ influx via non-selective cation channels.¹⁾ [Ca²⁺]_cyt increase facilitates the synthesis/secretion of the extracellular matrix of cartilage.^19,65) DNA microarray data showed the expression of human ClC-3/7 and rodent ClC-3/4/6 genes in chondrocytes.⁶⁷⁾ In addition, voltage-gated ClC-7 channels are predominantly and functionally expressed in chondrocytes and regulate the RMP and [Ca²⁺]_cyt¹⁸⁾ (Figs. 7A–D).

Fig. 7. Voltage-Gated ClC-7 Channels in Human Chondrocytes

(A) The high expression of the ClC-7 gene in human chondrocytes (OUMS-27). (B) Expression analyses of ClC-7 proteins in the plasma membrane of chondrocytes by Western blotting (membrane fraction; left) and immunocytochemical staining (right). (C) Acidic pH- and DIDS-sensitive Cl⁻ currents in chondrocytes treated with control or ClC-7 siRNA. Chondrocytes were stimulated from a holding potential of −40 mV to the test potentials (−100 to +100 mV) for 500 ms. Dotted lines indicate the zero current level. (D) Reduced DIDS-sensitive currents in acidic pH solution in ClC-7 siRNA-treated chondrocytes. (E) Expression changes in ClC-7 transcripts due to hypoosmotic stress in chondrocytes. (F) Decreased DIDS-sensitive currents in acidic pH solution in hypoosmotic medium. Data are shown as means ± S.E. * p < 0.05, * p < 0.01 vs. 350 mOsm. [Modified from¹⁸⁾].

OA is caused by the degradation of articular cartilage and causes pain and inflammation in the joints. The pathological roles of ion channels in OA are of interest. The osmolality of synovial fluid is lower in OA (249 to 277 mOsm) than in healthy subjects (295 to 340 mOsm).⁶⁸⁾ When chondrocytes are exposed to a hypoosmotic stress, they swell and regain their original cell volume due to the efflux of osmolytes. This is referred to as regulatory volume decrease.⁶⁹⁾ The expression and activity of ClC-7 channels are down-regulated by hypoosmotic stress, which mimics an extracellular OA environment¹⁸⁾ (Figs. 7E, F). This down-regulation induces membrane hyperpolarization followed by [Ca²⁺]_cyt increase, resulting in the death of chondrocytes. Previous studies demonstrated that the expression of BK_Ca channels and intermediate-conductance Ca²⁺-activated K⁺ channels was up-regulated in the chondrocytes of OA patients, whereas that of epithelial Na⁺ channels (ENaCα), two-pore-domain K⁺ channels (TASK-2), Na⁺-activated K⁺ channels (K_Ca4.2), and TRP vanilloid 4 (TRPV4) channels was down-regulated.^67,70) Conflicting findings have been obtained on expression changes in TMEM16A channels in OA chondrocytes.^67,70) Therefore, further studies on the pathophysiological roles of chondrocyte Cl⁻ channels will lead to an understanding of their functions and facilitate drug development for OA.

8. SWELLING-ACTIVATED CLC-3 CHANNELS IN CHONDROCYTES

The cell volume regulation during osmotic stress requires the maintenance of cell homeostasis. When the cell is exposed to a hypoosmotic environment in the extracellular compartment, it elicits the passive uptake of water and, thus, the cell swells. Cell swelling activates ion efflux via K⁺ and Cl⁻ channels and the extrusion of the increased water volume in the regulatory volume decrease process. The cell volume eventually recovers to the normal level.⁶⁹⁾ In chondrocytes, Cl⁻ channels enhanced by swelling play a role in the regulatory volume decrease process,⁷¹⁾ which leads to apoptosis.⁶⁹⁾ Apoptosis in articular chondrocytes has been suggested to play a role in the development and progression of OA. It is also evoked by a number of proinflammatory cytokines, such as tumor necrosis factor-α, interleukin (IL)-1β, and IL-6.⁷²⁾ The degradation of cartilage is facilitated in the pathogenesis of OA by the up-regulation of matrix metalloproteinases.

The ion channel activity regulates the RMP and [Ca²⁺]_cyt in chondrocytes^19,65,66): e.g., BK_Ca channels,^73,74) Orai/STIM channels,⁷⁵⁾ and ClC-7 channels.¹⁸⁾ In addition, ClC-3 proteins are responsible for swelling-activated Cl⁻ currents and regulatory volume decrease under hypoosmotic stress and are involved in the pathogenesis of OA via the release of prostaglandin E₂ (PGE₂)¹⁶⁾ (Fig. 8). This release of PGE₂, a well-known lipid mediator that contributes to inflammatory pain, may be mediated by [Ca²⁺]_cyt increase, which enhances phospholipase A₂ and produces PGE₂ through the arachidonic cascade. Since OA is a degenerative and inflammatory joint disease with the degradation and loss of articular cartilage,⁷²⁾ ClC-3 may be a new drug target for inflammatory diseases, including OA.

Fig. 8. Swelling-Activated ClC-3 Channels in Human Chondrocytes

(A) Swelling-activated and DIDS-sensitive Cl⁻ currents induced by hypoosmotic solution in human chondrocytes (OUMS-27), which were hyperpolarized/depolarized from a holding potential of 0 mV to the test potentials (−100 and +100 mV) for 200 ms. (B) Current density-voltage relationships for swelling-activated Cl⁻ currents under isosmotic and hypoosmotic conditions in the absence and presence of DIDS. (C) Expression of ClC-3 proteins in chondrocytes. (D) Decrease in swelling-activated Cl⁻ currents in ClC-7 siRNA-treated chondrocytes. (E) The amount of PGE₂ released from control and ClC-3 siRNA-treated chondrocytes following isosmotic or hypoosmotic stimulations. * p < 0.05 vs. control siRNA or 350 mOsm/control siRNA; ^# p < 0.05 vs. 180 mOsm/control siRNA. [Modified from¹⁶⁾].

9. TMEM16A AND TMEM16B CL_CA CHANNELS IN PINEAL GLANDS (PG)

The PG regulate the circadian (sleep/wake) rhythm via the synthesis/secretion of melatonin. This is positively regulated by the sympathetic innervation.⁷⁶⁾ Noradrenaline binds to β₁- adrenergic receptors and stimulates cAMP production. cAMP promotes arylalkylamine-N-acetyltransferase (AANAT; the melatonin-synthesizing enzyme) and facilitates the synthesis of melatonin in pinealocytes. Noradrenaline also binds to α₁-adrenergic receptors and induces IP₃-induced Ca²⁺ release, which is considered to enhance β₁-adrenergic receptor signaling. On the other hand, melatonin production is negatively modulated by parasympathetic innervation.⁷⁶⁾ Acetylcholine binds to nicotinic acetylcholine receptors and induces depolarization, which results in Ca²⁺ influx via VDCC in pinealocytes. The resulting increase in [Ca²⁺]_cyt causes the exocytosis of glutamate. This glutamate binds to metabotropic glutamate receptor-3 and inhibits cAMP production, which results in reduced AANAT activity and melatonin production.

Pinealocytes, which are mainly present in the mammalian PG,⁷⁶⁾ express several channels, including VDCC,^77,78) K_V channels,^77,79) Ca²⁺-activated K⁺ channels,^80–82) and SOC channels.⁸⁰⁾ In addition, the TMEM16A and TMEM16B proteins are the predominant Cl_Ca channel subtypes, and their conductance of Cl⁻ is involved in the secretion of melatonin from PG¹⁴⁾ (Figs. 9A–E). Although both proteins are expressed in the olfactory epithelium,⁸³⁾ dorsal root ganglia,⁸⁴⁾ and uterine smooth muscles,⁸⁵⁾ heteromeric formation had not yet been demonstrated. On the other hand, pineal Cl_Ca channels are composed of heterodimeric TMEM16A and TMEM16B complexes, in addition to homodimers¹⁴⁾ (Figs. 9F–H). These findings provide new information on the underlying mechanism(s) of the circadian (sleep/wake) rhythm through melatonin production in PG.

Fig. 9. TMEM16A and TMEM16B Cl_Ca Channels in Rat PG

(A) Inhibitory effects of Cl_Ca channel blockers (niflumic acid and T16A_inh-A01) on melatonin secretion induced by noradrenaline (norepinephrine) in rat PG. (B) The abundant expression of the TMEM16A and TMEM16B genes in PG. (C) The protein expression of TMEM16A and TMEM16B in PG. (D) The expression of TMEM16A and TMEM16B proteins in the plasma membrane of rat pinealocytes. (E) Whole-cell Cl_Ca currents in pinealocytes transfected with control, TMEM16A, TMEM16B, or TMEM16A + TMEM16B siRNA. Pinealocytes were stimulated from a holding potential of −40 mV to the test potentials (−80 to +100 mV) for 500 ms and subsequently repolarized to −80 mV for 250 ms. (F) Bimolecular fluorescence complementation assay for detecting heteromeric TMEM16A/TMEM16B channels in HEK293 cells co-transfected with TMEM16A-VN/TMEM16B-VC or TMEM16A-VC/TMEM16B-VN. (G) Efficiency of fluorescence resonance energy transfer (E_FRET) of TMEM16B-CFP alone (as a negative control), TMEM16B-CFP/YFP-TMEM16B (as a positive control), or TMEM16B-CFP/YFP-TMEM16A HEK293 cells. (H) Co-immunoprecipitation to detect the TMEM16A/TMEM16B heteromer in PG. N indicates a negative control lane. * p < 0.05, ** p < 0.01. [Modified from¹⁴⁾].

10. CONCLUSION

As described above, plasmalemmal Cl⁻ channels exert a number of physiological functions in most cells, and their expression and functions change under pathological conditions (Fig. 10). Therefore, Cl⁻ channels associated with the pathogenesis of diseases are considered to be suitable targets for ion channel drug discovery. Few selective and specific activators/inhibitors for Cl⁻ channels have been identified. Lubiprostone, an activator of voltage-gated ClC-2 channels, was recently launched as the first Cl⁻ channel modulator to treat chronic constipation.⁸⁶⁾ We are optimistic for the future development of Cl⁻ channel modulators as drugs for various diseases.

Fig. 10. Physiological and Pathological Significance of Cl_Ca (TMEM16A and TMEM16B) and ClC (ClC-3 and ClC-7) Channels

TMEM16A channels in PVSMC are involved in the regulation of spontaneous contractions and are down-regulated in cirrhotic portal hypertension. TMEM16A and ClC-3 channels in PASMC regulate contractions, proliferation, and cell volume, and their up-regulation is involved in the pathogenesis of PAH. TMEM16A channels in BCEC function in cell proliferation, migration, and permeability, and up-regulated by hypoxia stress, including cerebral ischemia. Chondrocyte ClC-7 channels contribute to cell survival and the maintenance of RMP and increase by hypotonic stress, such as OA. Chondrocyte ClC-3 channels are responsible for swelling-activated Cl⁻ channels and inflammatory PGE₂ release. TMEM16A and TMEM16B proteins form heterodimeric Cl_Ca channels, in addition to homodimeric Cl_Ca channels, in pinealocytes, which regulate the RMP and melatonin production. Further research on the physiological and pathological functions of Cl⁻ channels will provide a comprehensive understanding of biological functions and contribute to the identification of novel targets of drug discovery for associated diseases.

Acknowledgments

The author would like to express his deepest gratitude to his supervisors: Emeritus Prof. Yuji Imaizumi (Nagoya City University, Japan), the late Emeritus Prof. Minoru Watanabe (Nagoya City University, Japan), Prof. Shoichi Shimada (Osaka University), and Prof. Jason Yuan (University of California, San Diego, U.S.A.). The author also deeply thanks all the collaborators and laboratory members in the Department of Molecular/Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University. The present study was supported by Grants-in-Aid for Scientific Research on Innovative Areas (17H05537), Scientific Research (C) (25460104, 16K08278, and 19K07125), and Scientific Research (B) (22H02787) from the Japan Society for the Promotion of Science. This investigation was also supported by Grants-in-Aid from the Japan Research Foundation for Clinical Pharmacology (2012A19 and 2018A19), Shorai Foundation for Science and Technology, Suzuken Memorial Foundation (18-100), and Institute of Drug Discovery Science at Nagoya City University (2018-04 and NCU-IDDS-A202105), and Grants-in-Aid for Research in Nagoya City University (18-16, 1943007, and 2150019).

Conflict of Interest

The author declares no conflict of interest.

Notes

This review of the author’s work was written by the author upon receiving the 2023 Pharmaceutical Society of Japan Award for Divisional Scientific Promotion.

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