Biophysical characterization of chloride intracellular channel 6 (CLIC6)
2023; Elsevier BV; Volume: 299; Issue: 11 Linguagem: Inglês
10.1016/j.jbc.2023.105349
ISSN1083-351X
AutoresVeronica Loyo-Celis, Devendra Patel, Shridhar Sanghvi, Kamalpreet Kaur, Devasena Ponnalagu, Yang Zheng, Sahej Bindra, Harmeet Rireika Bhachu, Isabelle Deschênes, Shubha Gururaja Rao, Harpreet Singh,
Tópico(s)Cardiac electrophysiology and arrhythmias
ResumoChloride intracellular channels (CLICs) are a family of proteins that exist in soluble and transmembrane forms. The newest discovered member of the family CLIC6 is implicated in breast, ovarian, lung gastric, and pancreatic cancers and is also known to interact with dopamine-(D(2)-like) receptors. The soluble structure of the channel has been resolved, but the exact physiological role of CLIC6, biophysical characterization, and the membrane structure remain unknown. Here, we aimed to characterize the biophysical properties of this channel using a patch-clamp approach. To determine the biophysical properties of CLIC6, we expressed CLIC6 in HEK-293 cells. On ectopic expression, CLIC6 localizes to the plasma membrane of HEK-293 cells. We established the biophysical properties of CLIC6 by using electrophysiological approaches. Using various anions and potassium (K+) solutions, we determined that CLIC6 is more permeable to chloride-(Cl−) as compared to bromide-(Br−), fluoride-(F−), and K+ ions. In the whole-cell configuration, the CLIC6 currents were inhibited after the addition of 10 μM of IAA-94 (CLIC-specific blocker). CLIC6 was also found to be regulated by pH and redox potential. We demonstrate that the histidine residue at 648 (H648) in the C terminus and cysteine residue in the N terminus (C487) are directly involved in the pH-induced conformational change and redox regulation of CLIC6, respectively. Using qRT-PCR, we identified that CLIC6 is most abundant in the lung and brain, and we recorded the CLIC6 current in mouse lung epithelial cells. Overall, we have determined the biophysical properties of CLIC6 and established it as a Cl− channel. Chloride intracellular channels (CLICs) are a family of proteins that exist in soluble and transmembrane forms. The newest discovered member of the family CLIC6 is implicated in breast, ovarian, lung gastric, and pancreatic cancers and is also known to interact with dopamine-(D(2)-like) receptors. The soluble structure of the channel has been resolved, but the exact physiological role of CLIC6, biophysical characterization, and the membrane structure remain unknown. Here, we aimed to characterize the biophysical properties of this channel using a patch-clamp approach. To determine the biophysical properties of CLIC6, we expressed CLIC6 in HEK-293 cells. On ectopic expression, CLIC6 localizes to the plasma membrane of HEK-293 cells. We established the biophysical properties of CLIC6 by using electrophysiological approaches. Using various anions and potassium (K+) solutions, we determined that CLIC6 is more permeable to chloride-(Cl−) as compared to bromide-(Br−), fluoride-(F−), and K+ ions. In the whole-cell configuration, the CLIC6 currents were inhibited after the addition of 10 μM of IAA-94 (CLIC-specific blocker). CLIC6 was also found to be regulated by pH and redox potential. We demonstrate that the histidine residue at 648 (H648) in the C terminus and cysteine residue in the N terminus (C487) are directly involved in the pH-induced conformational change and redox regulation of CLIC6, respectively. Using qRT-PCR, we identified that CLIC6 is most abundant in the lung and brain, and we recorded the CLIC6 current in mouse lung epithelial cells. Overall, we have determined the biophysical properties of CLIC6 and established it as a Cl− channel. The chloride channels are present in all the cellular membranes and are tightly regulated by various stimuli including voltage, pH, volume, ligands, pressure, and intracellular messengers (1Verkman A.S. Galietta L.J.V. Chloride transport modulators as drug candidates.Am. J. Physiol. 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Cell. 2012; 22: 131-145Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar) and as a soluble protein in the cytosol (21Ulmasov B. Bruno J. Woost P.G. Edwards J.C. Tissue and subcellular distribution of CLIC1.BMC Cell Biol. 2007; 8: 8Crossref PubMed Scopus (59) Google Scholar). CLIC6, the most recent CLIC family member was originally identified in rabbit gastric parietal cells and named parchorin (5Gururaja Rao S. Ponnalagu D. Patel N.J. Singh H. Three decades of chloride intracellular channel proteins: from organelle to organ physiology.Curr. Protoc. Pharmacol. 2018; 80: 11.21.11-11.21.17Crossref Scopus (33) Google Scholar, 22Ponnalagu D. Singh H. Anion channels of Mitochondria.Handb. Exp. Pharmacol. 2017; 240: 71-101Crossref PubMed Scopus (61) Google Scholar, 23Friedli M. Guipponi M. Bertrand S. Bertrand D. Neerman-Arbez M. Scott H.S. et al.Identification of a novel member of the CLIC family, CLIC6, mapping to 21q22.12.Gene. 2003; 320: 31-40Crossref PubMed Scopus (30) Google Scholar, 24Griffon N. Jeanneteau F. Prieur F. Diaz J. Sokoloff P. CLIC6, a member of the intracellular chloride channel family, interacts with dopamine D(2)-like receptors.Brain Res. Mol. Brain Res. 2003; 117: 47-57Crossref PubMed Scopus (53) Google Scholar, 25Ferofontov A. Strulovich R. Marom M. Giladi M. Haitin Y. Inherent flexibility of CLIC6 revealed by crystallographic and solution studies.Sci. Rep. 2018; 8: 6882Crossref PubMed Scopus (10) Google Scholar, 26Nishizawa T. Nagao T. Iwatsubo T. Forte J.G. Urushidani T. Molecular cloning and characterization of a novel chloride intracellular channel-related protein, parchorin, expressed in water-secreting cells.J. Biol. Chem. 2000; 275: 11164-11173Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). CLIC6 is present in the conserved gene cluster ACD (AML/CLIC/DSCR1-like) in chromosome 21 and is the longest known isoform of CLIC proteins (23Friedli M. Guipponi M. Bertrand S. Bertrand D. Neerman-Arbez M. Scott H.S. et al.Identification of a novel member of the CLIC family, CLIC6, mapping to 21q22.12.Gene. 2003; 320: 31-40Crossref PubMed Scopus (30) Google Scholar, 25Ferofontov A. Strulovich R. Marom M. Giladi M. Haitin Y. Inherent flexibility of CLIC6 revealed by crystallographic and solution studies.Sci. Rep. 2018; 8: 6882Crossref PubMed Scopus (10) Google Scholar, 26Nishizawa T. Nagao T. Iwatsubo T. Forte J.G. Urushidani T. Molecular cloning and characterization of a novel chloride intracellular channel-related protein, parchorin, expressed in water-secreting cells.J. Biol. Chem. 2000; 275: 11164-11173Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Other CLIC proteins present in the ACD gene cluster are CLIC4 (chromosome 4) and CLIC5 (chromosome 6), which also indicate that these channels might have overlapping distribution and functional properties in cells. CLIC6 shares the structural homology with other CLIC proteins as well as the glutathione s-transferase superfamily (5Gururaja Rao S. Ponnalagu D. Patel N.J. Singh H. Three decades of chloride intracellular channel proteins: from organelle to organ physiology.Curr. Protoc. Pharmacol. 2018; 80: 11.21.11-11.21.17Crossref Scopus (33) Google Scholar, 27Gururaja Rao S. Ponnalagu D. Sukur S. Singh H. Sanghvi S. Mei Y. et al.Identification and characterization of a Bacterial homolog of chloride Intracellular Channel (CLIC) protein.Sci. Rep. 2017; 7: 8500Crossref PubMed Scopus (18) Google Scholar). Though the gene was discovered over 2 decades ago (23Friedli M. Guipponi M. Bertrand S. Bertrand D. Neerman-Arbez M. Scott H.S. et al.Identification of a novel member of the CLIC family, CLIC6, mapping to 21q22.12.Gene. 2003; 320: 31-40Crossref PubMed Scopus (30) Google Scholar, 24Griffon N. Jeanneteau F. Prieur F. Diaz J. Sokoloff P. CLIC6, a member of the intracellular chloride channel family, interacts with dopamine D(2)-like receptors.Brain Res. Mol. Brain Res. 2003; 117: 47-57Crossref PubMed Scopus (53) Google Scholar), evidence of the ability of CLIC6 to form an ion channel is still lacking. A genome-wide association study recently associated CLIC6 with psoriasis (28Yin X. Low H.Q. Wang L. Li Y. Ellinghaus E. Han J. et al.Genome-wide meta-analysis identifies multiple novel associations and ethnic heterogeneity of psoriasis susceptibility.Nat. Commun. 2015; 6: 6916Crossref PubMed Scopus (145) Google Scholar), lung function (29Kichaev G. Bhatia G. Loh P.R. Gazal S. Burch K. Freund M.K. et al.Leveraging polygenic functional enrichment to Improve GWAS Power.Am. J. Hum. Genet. 2019; 104: 65-75Abstract Full Text Full Text PDF PubMed Scopus (482) Google Scholar), accelerated aging associated with alcohol use (30Luo A. Jung J. Longley M. Rosoff D.B. Charlet K. Muench C. et al.Epigenetic aging is accelerated in alcohol use disorder and regulated by genetic variation in APOL2.Neuropsychopharmacology. 2020; 45: 327-336Crossref PubMed Scopus (51) Google Scholar), and opioid targets in cancer treatment (31Nishizawa D. Terui T. Ishitani K. Kasai S. Hasegawa J. Nakayama K. et al.Genome-wide association study identifies candidate loci associated with opioid analgesic requirements in the treatment of cancer Pain.Cancers (Basel). 2022; 14: 4692Crossref PubMed Scopus (3) Google Scholar). CLIC6 also known as parchorin when overexpressed in the LLC-PK1 kidney cell line potentiated the efflux of Cl− from cells. The Cl− flux was observed only when Cl− was depleted from the extracellular solution implicating CLIC6 in cellular Cl− transportation (26Nishizawa T. Nagao T. Iwatsubo T. Forte J.G. Urushidani T. Molecular cloning and characterization of a novel chloride intracellular channel-related protein, parchorin, expressed in water-secreting cells.J. Biol. Chem. 2000; 275: 11164-11173Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). In contrast, cotransfection of CLIC6 with dopamine D3-receptors in CHO cell lines fails to present any CLIC6-mediated Cl− fluxes raising concerns about the ability of CLIC6 to form an ion channel (24Griffon N. Jeanneteau F. Prieur F. Diaz J. Sokoloff P. CLIC6, a member of the intracellular chloride channel family, interacts with dopamine D(2)-like receptors.Brain Res. Mol. Brain Res. 2003; 117: 47-57Crossref PubMed Scopus (53) Google Scholar). However, in both cell systems, ectopic overexpression forced CLIC6 to localize to the plasma membrane of CHO and LLC-PK1 cells, and Cl− flux measurements were done by Cl− sensors such as MQAE and SPQ, respectively (24Griffon N. Jeanneteau F. Prieur F. Diaz J. Sokoloff P. CLIC6, a member of the intracellular chloride channel family, interacts with dopamine D(2)-like receptors.Brain Res. Mol. Brain Res. 2003; 117: 47-57Crossref PubMed Scopus (53) Google Scholar, 26Nishizawa T. Nagao T. Iwatsubo T. Forte J.G. Urushidani T. Molecular cloning and characterization of a novel chloride intracellular channel-related protein, parchorin, expressed in water-secreting cells.J. Biol. Chem. 2000; 275: 11164-11173Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). In this study, we set out to characterize CLIC6 and its ability to form ion channels in cell membranes. Since CLIC proteins are known to form ion channels on ectopic expression in cell lines (32Milton R.H. Abeti R. Averaimo S. DeBiasi S. Vitellaro L. Jiang L. et al.CLIC1 function is required for beta-amyloid-induced generation of reactive oxygen species by microglia.J. Neurosci. 2008; 28: 11488-11499Crossref PubMed Scopus (104) Google Scholar, 33Averaimo S. Abeti R. Savalli N. Brown L.J. Curmi P.M. Breit S.N. et al.Point mutations in the transmembrane region of the clic1 ion channel selectively modify its biophysical properties.PLoS one. 2013; 8e74523Crossref PubMed Scopus (20) Google Scholar) and planar bilayers (27Gururaja Rao S. Ponnalagu D. Sukur S. Singh H. Sanghvi S. Mei Y. et al.Identification and characterization of a Bacterial homolog of chloride Intracellular Channel (CLIC) protein.Sci. Rep. 2017; 7: 8500Crossref PubMed Scopus (18) Google Scholar, 34Littler D.R. Assaad N.N. Harrop S.J. Brown L.J. Pankhurst G.J. Luciani P. et al.Crystal structure of the soluble form of the redox-regulated chloride ion channel protein CLIC4.FEBS J. 2005; 272: 4996-5007Crossref PubMed Scopus (101) Google Scholar, 35Singh H. Ashley R.H. Redox regulation of CLIC1 by cysteine residues associated with the putative channel pore.Biophys. J. 2006; 90: 1628-1638Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 36Singh H. Ashley R.H. CLIC4 (p64H1) and its putative transmembrane domain form poorly selective, redox-regulated ion channels.Mol. Membr. Biol. 2007; 24: 41-52Crossref PubMed Scopus (66) Google Scholar, 37Singh H. Cousin M.A. Ashley R.H. Functional reconstitution of mammalian 'chloride intracellular channels' CLIC1, CLIC4 and CLIC5 reveals differential regulation by cytoskeletal actin.FEBS J. 2007; 274: 6306-6316Crossref PubMed Scopus (84) Google Scholar), we probed for evidence for CLIC6 to form an ion channel. On ectopic expression in HEK-293 cells, we found that CLIC6 is an anion channel that is sensitive to pH and redox regulation. Our qPCR analysis demonstrated that CLIC6 is abundant in lung and brain tissues as compared to other organs. Accordingly, we describe here the properties of CLIC6 in mouse lung epithelial (MLE) cells. Our data for the first time show that CLIC6 forms a functional ion channel that preferentially allows chloride over other anions. As an ectopic expression of CLIC6 in cell lines result in its localization to the plasma membrane (24Griffon N. Jeanneteau F. Prieur F. Diaz J. Sokoloff P. CLIC6, a member of the intracellular chloride channel family, interacts with dopamine D(2)-like receptors.Brain Res. Mol. Brain Res. 2003; 117: 47-57Crossref PubMed Scopus (53) Google Scholar, 25Ferofontov A. Strulovich R. Marom M. Giladi M. Haitin Y. Inherent flexibility of CLIC6 revealed by crystallographic and solution studies.Sci. Rep. 2018; 8: 6882Crossref PubMed Scopus (10) Google Scholar), we overexpressed CLIC6 in HEK-293 cells (Fig. 1). CLIC6 was cloned with an N terminus Flag tag, and the expression of the protein was verified in HEK-293 cells by Western blot (Fig. 1A). Transfected HEK-293 cells were also probed with anti-FLAG antibodies. As shown in Figure 1A, upon ectopic expression in HEK-293 cells, CLIC6 is expressed near the plasma membrane where it colocalizes with the wheat germ agglutinin indicating its possible presence near the plasma membrane. To measure the channel activity, we performed a whole-cell patch-clamp on HEK-293 cells transfected with CLIC6 and GFP in NMDG-Cl solutions (Fig. 1, B–I). We observed a large current at positive holding potentials (Fig. 1, C and D). CLIC6 in HEK-293 cells exhibits fast gating, which is voltage-dependent (V1/2 = 14.062 mV). The fast gate closes at negative membrane voltages and opens upon depolarization to positive voltages (Fig. 1, C and D). The reversal potential (Er) for CLIC6 was −40 mV, the same value as the holding potential used in the voltage step protocol that is consistent with the predicted reversal potential to chloride (−0.6 mV) calculated in the ion conditions described in Figure 1B. IAA-94 is a known blocker of CLIC proteins (27Gururaja Rao S. Ponnalagu D. Sukur S. Singh H. Sanghvi S. Mei Y. et al.Identification and characterization of a Bacterial homolog of chloride Intracellular Channel (CLIC) protein.Sci. Rep. 2017; 7: 8500Crossref PubMed Scopus (18) Google Scholar, 38Landry D.W. Akabas M.H. Redhead C. Edelman A. Cragoe Jr., E.J. Al-Awqati Q. Purification and reconstitution of chloride channels from kidney and trachea.Science. 1989; 244: 1469-1472Crossref PubMed Scopus (150) Google Scholar, 39Marten I. Zeilinger C. Redhead C. Landry D.W. al-Awqati Q. Hedrich R. Identification and modulation of a voltage-dependent anion channel in the plasma membrane of guard cells by high-affinity ligands.EMBO J. 1992; 11: 3569-3575Crossref PubMed Scopus (75) Google Scholar). The analysis of the instantaneous current amplitude at the different voltage steps indicated the presence of a constitutive Cl− selective current, which probably originated from CLIC6 as IAA-94 blocked these currents (Fig. 1D). Addition of 10 μM IAA-94 in bath solution led to a significant block at positive holding potentials (Fig. 1, D and F). The peak current at +100 mV was blocked by 48 ± 5% on the addition of IAA-94 (Fig. 1E). Surprisingly, we did not observe any block of CLIC6 on negative potentials. In parallel, nontransfected HEK-293 cells lacking CLIC6 were also obtained under the same recording conditions (Fig. S1). Although Cl− currents were present in nontransfected HEK-293 cells, they were not blocked by IAA-94 (Fig. S1, C and D). To determine the voltage dependence of the CLIC6 currents, the conductance (G) was plotted as a function of the voltage and fitted to the Boltzmann equation (Fig. 1F). CLIC6 shows enhanced activity on positive holding potentials as compared to the negative holding potentials as shown by G/V graph (Fig. 1F). To confirm the voltage dependency at positive membrane potentials, voltage steps ranging from −100 to +100 mV were applied followed by a voltage step to −40 mV to elicit tail currents (Figs. 1G, and S2). The tail current as a function of membrane potential was obtained by measuring the peak tail current (at 1.025 s) before and after the addition of 10 μM IAA-94. Independent of the traditional patch-clamp approach, we also probed HEK-293 cells transfected with CLIC6 by using an automated patch-clamp approach with SyncroPatch 384i (Nanion technologies (Fig. 1I). Each well of the SyncroPatch 384i has one aperture which is connected to an individual head stage of the amplifier. Hence, each well is designated as an independent experiment (n = 1). With PatchControl 384i, all the parameters including seal resistance, capacitance, and series resistance were determined from individual wells after the application of a test pulse. All parameters are monitored in real-time and can be recorded for individual experiments for each electrode. We successfully recorded currents from 13 individual HEK-293 cells transfected with CLIC6 (Fig. 1I). We obtained the current–voltage (I-V) relationship using a step protocol (Fig. 1C). The whole-cell currents presented rectification at positive holding potentials (Fig. 1J) similar to traditional whole-cell approach (Fig. 1D). Next, we tested whether single-channel currents can also be blocked by IAA-94. We recorded CLIC6 single-channel activity in cell-attached configuration (where the chloride composition was 130 mM for the pipette and 4.2 mM for the cytoplasm) at +100 mV and −100 mV for 100 s and subsequently added 10 μM IAA-94 in the bath solution. In single-channel recordings, we noticed two distinct substates of the channel in addition to a large current (Fig. 2A). The Po of CLIC6 decreased by 53 ± 4% and 51 ± 5% at +100 mV and −100 mV, respectively, on the addition of 10 μM IAA-94 (n = 4, Fig. 2B). We also observed a substate (at 50% level of the main opening, blue arrows in Fig. 2A) which was also reported for other CLIC proteins (35Singh H. Ashley R.H. Redox regulation of CLIC1 by cysteine residues associated with the putative channel pore.Biophys. J. 2006; 90: 1628-1638Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Our electrophysiology approaches conclusively demonstrate that CLIC6 can form a functional channel that can be blocked by IAA-94. CLIC proteins are known to form anion-selective channels. The selectivity of CLIC6 was measured by perfusing different anion or cation solutions in the bath. The pipette was kept at a constant 130 mM Cl− concentration with the NMDG-Cl solution (Fig. 3B). With 135 mM NMDG-Cl in the bath solution and 130 mM NMDG-Cl in the pipette, we observed large currents in HEK-293 cells transfected with CLIC6. The channel was highly active on depolarizing positive voltages (Fig. 3, C and D). On replacing NMDG-Cl with 135 mM NMDG-Br or NMDG-F in bath solution in the same HEK-293 cell transfected with CLIC6, we observed a significant decrease in whole-cell currents (Fig. 3C). The reversal potential (Er) for Cl− was −40 mV, and when Cl− was replaced with Br− and with F−, it was −60 mV (Fig. 3D). The activity of CLIC6 significantly dropped in Br− and F− solutions (Fig. 3C). In Br−, we observed a similar current kinetics at positive voltages as seen for Cl− ions albeit with a small current amplitude (Fig. 3, C and D). There was hardly any activity observed in the solution containing F− ions. Our results indicate that the selectivity of CLIC6 is Cl− >> Br− = F−. CLICs are known to form poorly selective ion channels. We also tested whether CLIC6 can allow potassium ions. The bath solution was replaced with KCl and potassium methyl sulfate (KMeSO4). Under KCl, a small current was observed in HEK-293 cells transfected with CLIC6 which was ablated in KMeSO4 (Fig. S3, A and B). Similar to NMDG-Cl, in KCl, CLIC6 was more active on positive voltages. In nontransfected HEK-293 cells, we did not observe larger currents as recorded in CLIC6-transfected cells when the bath solution was replaced with KCl or KMeSO4 (Fig. S3, C and D). These results suggest that CLIC6 is an IAA-94-sensitive Cl− selective ion channel. For CLIC proteins, an elegant model for the dual regulation of these channels by redox and pH has been proposed (35Singh H. Ashley R.H. Redox regulation of CLIC1 by cysteine residues associated with the putative channel pore.Biophys. J. 2006; 90: 1628-1638Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 40Warton K. Tonini R. Fairlie W.D. Matthews J.M. Valenzuela S.M. 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Distinct pH regulation of slow and rapid anion channels at the plasma membrane of Arabidopsis thaliana hypocotyl cells.J. Exp. Bot. 2005; 56: 1897-1903Crossref PubMed Scopus (20) Google Scholar). To analyze the impact of pH on CLIC6, we expressed CLIC6 in HEK-293 cells and carried out whole-cell patch-clamp recordings. On changing pH from 7.2 to 6.2, we observed a slight decrease but not a significant change in current density for CLIC6 (Fig. 4, C–G). In addition to 10 μM IAA-94 (Fig. 4, C and G), we observed a similar block of whole-cell currents in HEK-293 cells transfected with CLIC6 at pH 7.2 as observed in Figure 1, C and D. Surprisingly, the block was not significant at a lower pH 6.2 (Fig. 4, D and G) in HEK-293 cells transfected with CLIC6. Our experiments support a possibility (44Achilonu I. Fanucchi S. Cross M. Fernandes M. Dirr H.W. Rol
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