GaTx2

CLC-2 is a positive modulator of oligodendrocyte precursor cell differentiation and myelination.[Pubmed:29344669]

Mol Med Rep. 2018 Mar;17(3):4515-4523.

Oligodendrocytes (OLs) are myelin-forming cells that are present within the central nervous system. Impaired oligodendrocyte precursor cell (OPC) differentiation into mature OLs is a major cause of demyelination diseases. Therefore, identifying the underlying molecular mechanisms of OPC differentiation is crucial to understand the processes of myelination and demyelination. It has been acknowledged that various extrinsic and intrinsic factors are involved in the control of OPC differentiation; however, the function of ion channels, particularly the voltagegated chloride channel (CLC), in OPC differentiation and myelination are not fully understood. The present study demonstrated that CLC2 may be a positive modulator of OPC differentiation and myelination. Western blotting results revealed that CLC2 was expressed in both OPCs and OLs. Furthermore, CLC2 currents (ICLC2) were recorded in both types of cells. The inhibition of ICLC2 by GaTx2, a blocker of CLC2, was demonstrated to be higher in OPCs compared with OLs, indicating that CLC2 may serve a role in OL differentiation. The results of western blotting and immunofluorescence staining also demonstrated that the expression levels of myelin basic protein were reduced following GaTx2 treatment, indicating that the differentiation of OPCs into OLs was inhibited following CLC2 inhibition. In addition, following western blot analysis, it was also demonstrated that the protein expression of the myelin proteins yin yang 1, myelin regulatory factor, Smadinteracting protein 1 and sexdetermining region Ybox 10 were regulated by CLC2 inhibition. Taken together, the results of the present study indicate that CLC2 may be a positive regulator of OPC differentiation and able to contribute to myelin formation and repair in myelinassociated diseases by controlling the number and open state of CLC-2 channels.

Chloride channel ClC- 2 enhances intestinal epithelial tight junction barrier function via regulation of caveolin-1 and caveolar trafficking of occludin.[Pubmed:28161538]

Exp Cell Res. 2017 Mar 1;352(1):113-122.

Previous studies have demonstrated that the chloride channel ClC-2 plays a critical role in intestinal epithelial tight junction (TJ) barrier function via intracellular trafficking of TJ protein occludin. To study the mechanism of ClC-2-mediated TJ barrier function and intracellular trafficking of occludin, we established ClC-2 over-expressing Caco-2 cell line (Caco-2(CLCN2)) by full length ClC-2 ORF transfection. ClC-2 over-expression (Caco-2(CLCN2)) significantly enhanced TJ barrier (increased TER by >/=2 times and reduced inulin flux by 50%) compared to control Caco-2(pEZ) cells. ClC-2 over-expression (Caco-2(CLCN2)) increased occludin protein level compared to control Caco-2(pEZ) cells. Surface biotinylation assay revealed reduced steady state endocytosis of occludin in Caco-2(CLCN2) cells. Furthermore, ClC-2 over-expression led to reduction in caveolin-1 protein level and diminishment of caveolae assembly. Caveolae disruption increased TJ permeability in control but not ClC-2 over-expressing Caco-2(CLCN2) cells. Selective ClC-2 channel blocker GaTx2 caused an increase in caveolin-1 protein level and reduced occludin level. Delivery of cell permeable caveolin-1 scaffolding domain reduced the occludin protein level. Over all, these results suggest that ClC- 2 enhances TJ barrier function in intestinal epithelial cells via regulation of caveolin-1 and caveolae-mediated trafficking of occludin.

Dual activation of CFTR and CLCN2 by lubiprostone in murine nasal epithelia.[Pubmed:23316067]

Am J Physiol Lung Cell Mol Physiol. 2013 Mar 1;304(5):L324-31.

Multiple sodium and chloride channels on the apical surface of nasal epithelial cells contribute to periciliary fluid homeostasis, a function that is disrupted in patients with cystic fibrosis (CF). Among these channels is the chloride channel CLCN2, which has been studied as a potential alternative chloride efflux pathway in the absence of CFTR. The object of the present study was to use the nasal potential difference test (NPD) to quantify CLCN2 function in an epithelial-directed TetOn CLCN2 transgenic mouse model (TGN-K18rtTA-hCLCN2) by using the putative CLCN2 pharmacological agonist lubiprostone and peptide inhibitor GaTx2. Lubiprostone significantly increased chloride transport in the CLCN2-overexpressing mice following activation of the transgene by doxycycline. This response to lubiprostone was significantly inhibited by GaTx2 after CLCN2 activation in TGN-CLCN2 mice. Cftr(-/-) and Clc2(-/-) mice showed hyperpolarization indicative of chloride efflux in response to lubiprostone, which was fully inhibited by GaTx2 and CFTR inhibitor 172 + GlyH-101, respectively. Our study reveals lubiprostone as a pharmacological activator of both CFTR and CLCN2. Overexpression and activation of CLCN2 leads to improved mouse NPD readings, suggesting it is available as an alternative pathway for epithelial chloride secretion in murine airways. The utilization of CLCN2 as an alternative chloride efflux channel could provide clinical benefit to patients with CF, especially if the pharmacological activator is administered as an aerosol.

Isolation and characterization of a high affinity peptide inhibitor of ClC-2 chloride channels.[Pubmed:19574231]

J Biol Chem. 2009 Sep 18;284(38):26051-62.

The ClC protein family includes voltage-gated chloride channels and chloride/proton exchangers. In eukaryotes, ClC proteins regulate membrane potential of excitable cells, contribute to epithelial transport, and aid in lysosomal acidification. Although structure/function studies of ClC proteins have been aided greatly by the available crystal structures of a bacterial ClC chloride/proton exchanger, the availability of useful pharmacological tools, such as peptide toxin inhibitors, has lagged far behind that of their cation channel counterparts. Here we report the isolation, from Leiurus quinquestriatus hebraeus venom, of a peptide toxin inhibitor of the ClC-2 chloride channel. This toxin, GaTx2, inhibits ClC-2 channels with a voltage-dependent apparent K(D) of approximately 20 pm, making it the highest affinity inhibitor of any chloride channel. GaTx2 slows ClC-2 activation by increasing the latency to first opening by nearly 8-fold but is unable to inhibit open channels, suggesting that this toxin inhibits channel activation gating. Finally, GaTx2 specifically inhibits ClC-2 channels, showing no inhibitory effect on a battery of other major classes of chloride channels and voltage-gated potassium channels. GaTx2 is the first peptide toxin inhibitor of any ClC protein. The high affinity and specificity displayed by this toxin will make it a very powerful pharmacological tool to probe ClC-2 structure/function.