CLC Channels Research Articles

Presynaptic CLC-3 determines quantal size of inhibitory transmission in the hippocampus

The absence of the chloride channel CLC-3 in Clcn3−/− mice results in hippocampal degeneration with a distinct temporal-spatial sequence reminiscent of neuronal loss in temporal lobe epilepsy. We examined how the loss of CLC-3 might impact GABAergic synaptic transmission in the hippocampus. An electrophysiological study of synaptic function in Clcn3+/+ and Clcn3-−/− mice in hippocampal slices before the onset of neurodegeneration, revealed a significant decrease in the amplitude and frequency of mIPSCs. We found that CLC-3 colocalizes with the vesicular GABA transporter VGAT in the CA1 region of the hippocampus. Cl−-induced acidification of inhibitory synaptic vesicles showed a significant dependence on CLC-3 expression. The decrement in inhibitory transmission in the Clcn3−/− animals suggests a decrease in neurotransmitter loading of synaptic vesicles which we attributed to defective vesicular acidification. Our observations extend the role of Cl− in inhibitory transmission from that of a postsynaptic permeant species to a presynaptic regulatory element.

Anion- and Proton-Dependent Gating of ClC-4 Anion/Proton Transporter under Uncoupling Conditions

ClC-4 is a secondary active transporter that exchanges Cl − ions and H + with a 2:1 stoichiometry. In external SCN −, ClC-4 becomes uncoupled and transports anions with high unitary transport rate. Upon voltage steps, the number of active transporters varies in a time-dependent manner, resembling voltage-dependent gating of ion channels. We here investigated modification of the voltage dependence of uncoupled ClC-4 by protons and anions to quantify association of substrates with the transporter. External acidification shifts voltage dependence of ClC-4 transport to more positive potentials and leads to reduced transport currents. Internal pH changes had less pronounced effects. Uncoupled ClC-4 transport is facilitated by elevated external [SCN −] but impaired by internal Cl − and I −. Block by internal anions indicates the existence of an internal anion-binding site with high affinity that is not present in ClC channels. The voltage dependence of ClC-4 coupled transport is modulated by external protons and internal Cl − in a manner similar to what is observed under uncoupling conditions. Our data illustrate functional differences but also similarities between ClC channels and transporters.

Synergistic Substrate binding in the CLC-Ec1 Exchanger

Secondary active transporters couple the electrochemical gradients of two or more substrates to catalyze their stoichiometric exchange across biological membranes. In canonical models of alternating access exchange binding of different molecules is mutually exclusive or competitive. CLC-ec1 is a structurally known H+/Cl- exchanger of the CLC family and it has served as a guide in understanding the basic functional properties of CLC channels and transporters. Two glutamates (E148, E203) define the external and internal end points of the H+ permeation pathway. However the coupling mechanism of these exchangers is still obscure. We used Isothermal Titration Calorimetry to probe the coupling between H+/Cl- binding and transport by measuring the enthalpy of Cl- binding in different buffers. We found that binding of Cl- and H+ is synergistic: binding of 2 Cl- to CLC-ec1 induces the binding of 1 H+. Thus, the stoichiometries of binding and transport are equal but have opposite sign. Cl- binding induces the uptake of 1 H+ from the buffer in the E203Q mutant despite the fact that this mutant is unable to transport H+. In contrast, Cl- and H+ binding become decoupled in the E148A mutant. Thus, while both glutamates are essential for coupling movement of H+ and Cl- only E148 controls their coupled binding while protonation of E203 takes place in a Cl--independent manner. Decoupling H+ and Cl- fluxes by mutating residues that impair Cl- binding leads to a parallel disruption of their binding synergism, strongly suggesting that this step is critical for the transport process.In conclusion, our results show that during the exchange cycle binding of Cl- at one side of the membrane induces binding of H+ at the other side. This, implies that the transport cycle of CLC-ec1 is drastically different from conventional alternating access mechanisms.

Design, Function and Structure of a Monomeric CLC Transporter

The CLC family of Cl- channels and Cl-/H+ antiporters are homodimeric in structure with each subunit containing a distinct transport pathway. Yet, the role of this conserved dimeric architecture in relation to protein function is not well understood. While certain CLC channels demonstrate dimer-dependent gating through cytosolic domains, the near-normal function of a bacterial CLC transporter strait-jacketed by covalent crosslinks across the dimer interface argues that the transport cycle resides within each subunit and does not require rigid-body rearrangements between subunits. A prediction that follows is that it should be possible to construct a monomeric form of a CLC protein while preserving structural and functional properties. To investigate this, we designed a stable monomeric variant of the E. coli CLC transporter, CLC-ec1, by introducing two tryptophan mutations, I201W and I422W, at the dimer interface. The existence of monomer was confirmed by gel filtration and glutaraldehyde cross-linking in both detergent micelles and PC/PG liposomes. The monomer is functional, exhibiting H+ coupled Cl- transport at rates comparable to the wild-type dimer and with 2:1 stoichiometry. The structure of the monomer was solved to 3.1 A resolution showing a crystal packing arrangement that exposes the previously buried dimer interface. The backbone atoms of this new structure aligns with the original CLC-ec1 crystal structure with 0.4 A RMSD, showing that the intrinsic subunit structure is not affected by these mutations or separation from the dimer state. These results demonstrate that the CLC subunit alone is the basic functional unit for transport and that cross-subunit interaction is not required for Cl-/H+ exchange in CLC transporters.

PH-Sensitive CLC Channel Provides a Novel Charge Compensation Pathway for Phagocyte NADPH Oxidase

pH-Sensitive CLC Channel Provides a Novel Charge Compensation Pathway for Phagocyte NADPH Oxidase

ClC-2 Channels in Erythrocytes

ClC-2 is a ubiquitously expressed plasma membrane Cl - channel that reportedly controls the ionic environment in mouse retina and testis. Beyond that, ClC-2 might sense cellular energy status and cellular stress by its carboxy- terminal cystathionine-beta-synthase (CBS) domains and by its molecular interaction with the heat shock protein Hsp90, respectively. In mature human and mouse erythrocytes, ClC-2 is activated by oxidative stress and by malaria infection. This article describes possible function of erythrocyte ClC-2 channels for the programmed death of oxidatively injured erythrocytes and for the regulatory volume decrease of malaria-infected erythrocytes.

Lubiprostone Activates Cl− Secretion via cAMP Signaling and Increases Membrane CFTR in the Human Colon Carcinoma Cell Line, T84

Lubiprostone, used clinically (b.i.d.) to treat constipation, has been reported to increase transepithelial Cl(-) transport in T84 cells by activating ClC-2 channels. To identify the underlying signaling pathway, we explored the effects of short-term and overnight lubiprostone treatment on second messenger signaling and Cl(-) transport. Cl(-) transport was assessed either as I(sc) across T84 monolayers grown on Transwells and mounted in Ussing chambers or by the iodide efflux assay. [cAMP](i) was measured by enzyme immunoassay, and [Ca(2+)](i) by Fluo-3 fluorescence. Quantitation of apical cell surface CFTR protein levels was assessed by Western blotting and biotinylation with the EZ-Link Sulfo-NHS-LC-LC-Biotin. ClC-2 mRNA level was studied by RT-PCR. Lubiprostone and the cAMP stimulator, forskolin, caused comparable and maximal increases of I(sc) in T84 cells. The I(sc) effects of lubiprostone and forskolin were each suppressed if the tissue had previously been treated with the other agent. These responses were unaltered even if the monolayers were treated with lubiprostone overnight. Lubiprostone-induced increases in iodide efflux were ~80% of those obtained with forskolin. Lubiprostone increased [cAMP](i). H89, bumetanide, or CFTR(inh)-172 greatly attenuated lubiprostone-stimulated Cl(-) secretion, whereas the ClC-2 inhibitor CdCl(2) did not. Compared to controls, FSK-treatment increased membrane-associated CFTR by 1.9 fold, and lubiprostone caused a 2.6-fold increase in apical membrane CFTR as seen by immunoblotting following cell surface biotinylation. Lubiprostone activates Cl(-) secretion in T84 cells via cAMP, protein kinase A, and by increasing apical membrane CFTR protein.

PECAM‐targeted delivery of SOD inhibits endothelial inflammatory response

Elevated generation of reactive oxygen species (ROS) by endothelial enzymes, including NADPH-oxidase, is implicated in vascular oxidative stress and endothelial proinflammatory activation involving exposure of vascular cell adhesion molecule-1 (VCAM-1). Catalase and superoxide dismutase (SOD) conjugated with antibodies to platelet/endothelial cell adhesion molecule 1 (PECAM-1) bind specifically to endothelium and inhibit effects of corresponding ROS, H(2)O(2), and superoxide anion. In this study, anti-PECAM/SOD, but not anti-PECAM/catalase or nontargeted enzymes, including polyethylene glycol (PEG)-SOD, inhibited 2- to 3-fold VCAM expression caused by tumor necrosis factor (TNF), interleukin-1β, and lipopolysaccharide. Anti- PECAM/SOD, but not nontargeted counterparts, accumulated in vascular endothelium after intravenous injection, localized in endothelial endosomes, and inhibited by 70% lipopolysaccharide-caused VCAM-1 expression in mice. Anti-PECAM/SOD colocalized with EEA-1-positive endothelial vesicles and quenched ROS produced in response to TNF. Inhibitors of NADPH oxidase and anion channel ClC3 blocked TNF-induced VCAM expression, affirming that superoxide produced and transported by these proteins, respectively, mediates inflammatory signaling. Anti-PECAM/SOD abolished VCAM expression caused by poly(I:C)-induced activation of toll-like receptor 3 localized in intracellular vesicles. These results directly implicate endosomal influx of superoxide in endothelial inflammatory response and suggest that site-specific interception of this signal attained by targeted delivery of anti-PECAM/SOD into endothelial endosomes may have anti-inflammatory effects.

Permeant anions contribute to voltage dependence of ClC‐2 chloride channel by interacting with the protopore gate

It has been shown that the voltage (V(m)) dependence of ClC Cl(-) channels is conferred by interaction of the protopore gate with H(+) ions. However, in this paper we present evidence which indicates that permeant Cl(-) ions contribute to V(m)-dependent gating of the broadly distributed ClC-2 Cl() channel. The apparent open probability (P(A)) of ClC-2 was enhanced either by changing the [Cl(-)](i) from 10 to 200 mM or by keeping the [Cl(-)](i) low (10 mM) and then raising [Cl(-)](o) from 10 to 140 mM. Additionally, these changes in [Cl(-)] slowed down channel closing at positive V(m) suggesting that high [Cl(-)] increased pore occupancy thus hindering closing of the protopore gate. The identity of the permeant anion was also important since the P(A)(V(m)) curves were nearly identical with Cl(-) or Br(-) but shifted to negative voltages in the presence of SCN() ions. In addition, gating, closing rate and reversal potential displayed anomalous mole fraction behaviour in a SCN(-)/Cl() mixture in agreement with the idea that pore occupancy by different permeant anions modifies the V(m) dependence ClC-2 gating. Based on the ec1-ClC anion pathway, we hypothesized that opening of the protopore gate is facilitated when Cl(-) ions dwell in the central binding site. In contrast, when Cl(-) ions dwell in the external binding site they prevent the gate from closing. Finally, this Cl(-)-dependent gating in ClC-2 channels is of physiological relevance since an increase in [Cl(-)](o) enhances channel opening when the [Cl(-)](i) is in the physiological range.

Chloride transport across kidney epithelia through CLC chloride channels.

This review summarizes recent progress in elucidating the chloride-transporting mechanisms in kidney epithelia, focusing particularly on those which act through the newly identified chloride channels. A family of chloride channel proteins (ClC chloride channels) consisting of at least 9 members (ClC-1, 2, 3, 4, 5, 6, 7, K1 and K2) has recently been identified in mammals. Although all of these ClC channels, except for skeletal muscle-specific ClC-1, are expressed in the kidney, only ClC-K1 and K2 are kidney-specific ClC chloride channels, suggesting that they play an important role in the kidney. The functional properties and intrarenal localization of these chloride channels are summarized, and their involvement in certain tubular dysfunctions and physiological roles are discussed in this report.

A Three-State Multi-Ion Kinetic Model for Conduction Properties of ClC-0 Chloride Channel

A three-state, multiion kinetic model was proposed to enable the conduction properties of the mammalian channel ClC-0 to be well characterized. Using this rate-theory based model, the current-voltage and conductance-concentration relations were obtained. The five parameters needed were determined by fitting the data of conduction experiments of the wild-type ClC-0 and its K519C mutant. The model was then tested against available calculation and simulation data, and the energy differences between distinct chloride-occupancy states computed agreed with an independent calculation on the binding free energies solved by using the Poisson-Boltzmann equation. The average ion number of conduction and the ion passing duration calculated closely resembled the values obtained from Brownian dynamics simulations. According to the model, the decrease of conductance caused by mutating residue K519 to C519 can be attributed to the effect of K519C mutation on translocation rate constants. Our study sets up a theoretical model for ion permeation and conductance in ClC-0. It provides a starting point for experimentalists to test the three-state model, and would help in understanding the conduction mechanism of ClC-0.

Expression patterns of ClC-3 mRNA and protein in aortic smooth muscle, kidney and brain in diabetic rats

ClC-3, a member of the ClC family of voltage-gated chloride channels, regulates cell proliferation of cultured rat aortic vascular smooth muscle cells, pathogenesis of allergic rhinitis and tumor cell migration. However, its role in diabetic animals is still unknown. To address this issue, we investigated the expression patterns of ClC-3 in diabetic rats. Five-week-old Sprague-Dawley rats were divided into two groups, 50 non-diabetic control rats (non-DM) and 50 diabetic model rats (DM). ClC-3 mRNA and protein expression in aortic smooth muscle, kidney and brain tissues were examined by fluorimeter-based quantitive RT-PCR assay and Western blot analysis, respectively. ClC-3 mRNA and protein were endogenously expressed in aortic smooth muscle, kidney (cortex and medulla) and brain tissues of both control and streptozotocin-induced diabetic rats. ClC-3 mRNA and protein expression levels were significantly higher in aortic smooth muscle and brain tissues of diabetic rats, but significantly decreased in kidney medulla tissue, relative to non-DM controls. There were no significant differences in ClC-3 mRNA and protein expression in kidney cortex between non-diabetic control and diabetic rats. Furthermore, the altered ClC-3 expression patterns in diabetic rat aortic smooth muscle, brain, and kidney medulla tissues all correlated with the changes in blood glucose levels (p < 0.05). In conclusion, our data show for the first time that diabetes alters both the gene and protein expression of ClC-3 channels. These changes may contribute to the impaired vascular, brain and kidney functions observed in diabetes.

Molecular Interaction and Functional Regulation of ClC-3 by Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII) in Human Malignant Glioma

Glioblastoma multiforme is the most common and lethal primary brain cancer in adults. Tumor cells diffusely infiltrate the brain making focal surgical and radiation treatment challenging. The invasion of glioma cells into normal brain is facilitated by the activity of ion channels aiding dynamic regulation of cell volume. Recent studies have specifically implicated ClC-3, a voltage-gated chloride channel, in this process. However, the interaction between ClC-3 activity and cell movement is poorly understood. Here, we demonstrate that ClC-3 is highly expressed on the plasma membrane of human glioma cells where its activity is regulated through phosphorylation via Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). Intracellular infusion of autoactivated CaMKII via patch pipette enhanced chloride currents 3-fold, and this regulation was inhibited by autocamtide-2 related inhibitory peptide, a CaMKII-specific inhibitor. CaMKII modulation of chloride currents was also lost upon stable small hairpin RNA knockdown of ClC-3 channels indicating a specific interaction of ClC-3 and CaMKII. In ClC-3-expressing cells, inhibition of CaMKII reduced glioma invasion to the same extent as direct inhibition of ClC-3. The importance of the molecular interaction of ClC-3 and CaMKII is further supported by our finding that CaMKII co-localizes and co-immunoprecipitates with ClC-3. ClC-3 and CaMKII also co-immunoprecipitate in tissue biopsies from patients diagnosed with grade IV glioblastoma. These tumor samples show 10-fold higher ClC-3 protein expression than nonmalignant brain. These data suggest that CaMKII is a molecular link translating intracellular calcium changes, which are intrinsically associated with glioma migration, to changes in ClC-3 conductance required for cell movement.

1α,25(OH) 2-Vitamin D 3 stimulation of secretion via chloride channel activation in Sertoli cells

Sertoli cell secretory activities are highly dependent on ion channel functions and critical to spermatogenesis. The steroid hormone 1α,25(OH) 2-vitamin D 3 (1,25(OH) 2-D 3) stimulates exocytosis in different cell systems by activating a nongenotropic vitamin D receptor (VDR). Here, we described 1,25(OH) 2-D 3 stimulation of secretion via Cl − channel activation in the mouse immature Sertoli cell line TM4. 1,25(OH) 2-D 3 potentiation of chloride currents was dependent on hormone concentration, and correlated with a significant increase in whole-cell capacitance within 20–40 min. In addition, Cl − currents were potentiated by the nongenomic VDR agonist 1α,25(OH) 2 lumisterol D 3 (JN), while 1,25(OH) 2-D 3 potentiation of channels was suppressed by nongenomic VDR antagonist 1β,25(OH) 2-vitamin D 3 (HL). Treatment of TM4 cells with PKC and PKA activators PMA and forskolin respectively, increased Cl − currents significantly, while PKC and PKA inhibitors Go6983 and H-89, respectively, abolished 1,25(OH) 2-D 3 stimulation of Cl − currents, suggesting phosphorylation pathways in 1,25(OH) 2-D 3 mediated channel responses. RT-PCR demonstrated the expression of outwardly rectifying ClC-3 channels in TM4 cells. Taken together, our results demonstrate a PKA/PKC-dependent 1,25(OH) 2-D 3/VDR nongenotropic pathway leading to Cl − channel and exocytosis activation in Sertoli cells. We conclude that 1,25(OH) 2-D 3 appears to be a modulator of male reproductive functions at least in part by stimulating Sertoli cell secretory functions.

Channelopathies and juvenile myoclonic epilepsy

Channelopathies and juvenile myoclonic epilepsy

Role of the Q-R Linker in H+/Cl- Coupling in a CLC Exchanger

Members of the CLC family function as Cl- channels or as H+/Cl- exchangers. Despite this mechanistic divide the two CLC sub-classes share many functional and structural traits. CLC-ec1, a transporter, can be transformed into a Cl--selective pore by simultaneously removing the intra- and extra-cellular gates. This entailed mutating two residues, E148 and Y445, which are conserved in both CLC channels and transporters. While the extracellular gate regulates channel gating, the intracellular one does not.

Involvement of volume‐sensitive Cl− channels in the proliferation of human subcutaneous pre‐adipocytes

1. Obesity is a significant challenge in terms of public health and preventive medicine. Inhibition of pre-adipocyte proliferation is believed to be important in the proposed anti-obesity mechanism. The aim of the present study was to examine the interplay between Cl(-) channels and their possible involvement in the proliferation of undifferentiated human pre-adipocytes. 2. Pre-adipocytes were isolated from human abdominal subcutaneous adipose tissue. Membrane ion currents were recorded using the whole-cell patch-clamp technique. Expression of the Cl(-) channel ClC-3 gene and protein was determined by reverse transcription-polymerase chain reaction (RT-PCR) and western blot, respectively. Cell proliferation was evaluated using the [(3)H]-thymidine incorporation assay. 3. Electrophysiological recordings revealed a volume-sensitive Cl(-) current (I(Cl.vol)) expressed in pre-adipocytes that was activated under hyposmotic conditions (external osmolarity decreased by 80%) and inhibited by the Cl(-) channel blocker tamoxifen. 4. Expression of the ClC-3 channel gene and protein was confirmed by RT-PCR and western blot analysis. Blocking I(Cl.vol) with tamoxifen supressed the proliferation of pre-adipocytes in a concentration-dependent manner. 5. Collectively, the results of the present study indicate that the volume-sensitive Cl(-) channel participates in regulation of the proliferation of human subcutaneous pre-adipocytes.

Basis of substrate binding and conservation of selectivity in the CLC family of channels and transporters

Ion binding to secondary active transporters triggers a cascade of conformational rearrangements resulting in substrate translocation across cellular membranes. Despite the fundamental role of this step, direct measurements of binding to transporters are rare. We investigated ion binding and selectivity in CLC-ec1, a H+/Cl− exchanger of the CLC family of channels and transporters. Cl− affinity depends on the conformation of the protein: it is highest with the extracellular gate removed, and weakens as the transporter adopts the occluded configuration and with the intracellular gate removed. The central ion-binding site determines selectivity in CLC transporters and channels, a serine to proline substitution at this site confers NO3− selectivity upon the Cl− specific CLC-ec1 transporter and CLC-0 channel. We propose that CLC-ec1 operates through an affinity-switch mechanism and that the bases of substrate specificity are conserved in the CLC channels and transporters.

Bestrophin-1 Enables Ca2+-activated Cl− Conductance in Epithelia

Epithelial cells express calcium-activated Cl(-) channels of unknown molecular identity. These Cl(-) channels play a central role in diseases such as secretory diarrhea, polycystic kidney disease, and cystic fibrosis. The family of bestrophins has been suggested to form calcium-activated Cl(-) channels. Here, we demonstrate molecular and functional expression of bestrophin-1 (BEST1) in mouse and human airways, colon, and kidney. Endogenous calcium-activated whole cell Cl(-) currents coincide with endogenous expression of the Vmd2 gene product BEST1 in murine and human epithelial cells, whereas calcium-activated Cl(-) currents are absent in epithelial tissues lacking BEST1 expression. Blocking expression of BEST1 with short interfering RNA or applying an anti-BEST1 antibody to a patch pipette suppressed ATP-induced whole cell Cl(-) currents. Calcium-dependent Cl(-) currents were activated by ATP in HEK293 cells expressing BEST1. Thus, BEST1 may form the Ca2+-activated Cl(-) current, or it may be a component of a Cl(-) channel complex in epithelial tissues.

The Granular Chloride Channel ClC-3 Is Permissive for Insulin Secretion

Insulin secretion from pancreatic beta cells is dependent on maturation and acidification of the secretory granule, processes necessary for prohormone convertase cleavage of proinsulin. Previous studies in isolated beta cells revealed that acidification may be dependent on the granule membrane chloride channel ClC-3, in a step permissive for a regulated secretory response. In this study, immuno-EM of beta cells revealed colocalization of ClC-3 and insulin on secretory granules. Clcn3(-/-) mice as well as isolated islets demonstrate impaired insulin secretion; Clcn3(-/-) beta cells are defective in regulated insulin exocytosis and granular acidification. Increased amounts of proinsulin were found in the majority of secretory granules in the Clcn3(-/-) mice, while in Clcn3(+/+) cells, proinsulin was confined to the immature secretory granules. These results demonstrate that in pancreatic beta cells, chloride channels, specifically ClC-3, are localized on insulin granules and play a role in insulin processing as well as insulin secretion through regulation of granular acidification.