1.A.12 The Intracellular Chloride Channel (CLIC) Family

Proteins of the CLIC family are voltage-sensitive chloride channels found in intracellular membranes but not the plasma membranes of animal cells. They are found in human nuclear membranes, and the bovine protein targets to the microsomes, but not the plasma membrane, when expressed in Xenopus laevis oocytes. These proteins are thought to function in the regulation of the membrane potential and in transepithelial ion absorption and secretion in the kidney (Singh, 2010). They possess one or two putative transmembrane α-helical spanners (TMSs). The bovine p64 protein is 437 amino acyl residues in length and has the two putative TMSs at positions 223-239 and 367-385. The N- and C-termini are cytoplasmic, and the large central luminal loop may be glycosylated. The human nuclear protein (CLIC1 or NCC27) is much smaller (241 residues) and has only one putative TMS at position 30-36. It is homologous to the second half of p64. CLIC1 is functional as a homooligomer (probably a homotetramer) and can be expressed in bacteria. It exists in both soluble and membrane-associated forms, and the soluble form inserts into lipid bilayers in a spontaneous, pH-dependent, two state process to form active Cl- channels (Warton et al., 2002). A crystal structure for the soluble monomeric form of CLIC1 is available at 1.4 Å resolution (Harrop et al., 2001). It is structurally similar to members of the glutathione-S-transferase superfamily. It has a redox site like that of glutaredoxin. Homologues include proteins of unknown function from C. elegans and D. melanogaster, as well as several glutathione-dependent dihydroascorbate reductases from plants. Bacterial homologues are also found (i.e., the stringent starvation protein A of Neisseria meningitidis (pirB81024)).

CLIC5 was the first mitochondrial chloride channel to be identified in the inner mitochondrial membrane, while CLIC4 is located predominantly in the outer mitochondrial membrane. Gururaja Rao et al. 2020 discussed the intracellular chloride channels, their roles in pathologies, such as cardiovascular, cancer, and neurodegenerative diseases, and developments concerning their usage as theraputic targets in humans. CLICs are upregulated or downregulated in certain types of cancer, and in certain cases, during cell cycle progression, the localization and functions of the cytosolic form of the transmembrane CLIC proteins are also altered (Wang et al. 2021).

There are six human paralogues within the CLIC family, all of which encode putative chloride (anion selective) channels. CLIC1, 4, 5A and 5 have been reconstituted in artificial liposomes (Berryman et al., 2004; Singh et al., 2007). Cl- flux is sensitive to the chloride channel blocker, IAA-94. It associates with the cytoskeleton including a protein, ezrin, in apical microvilli, but may normally be in an intracellular membrane compartment (Berryman et al., 2004). Many CLICs are strongly associated with cytoskeletal proteins. CLIC1, CLIC4 and CLIC5, in planar lipid bilayers, form multiconductance channels that are almost equally permeable to Na(+), K(+) and Cl(-), suggesting that the 'CLIC' nomenclature may need to be revised. CLIC1 and CLIC5, but not CLIC4, were strongly and reversibly inhibited (or inactivated) by 'cytosolic' F-actin in the absence of any other protein. This inhibitory effect could be reversed by using cytochalasin to disrupt the F-actin. They regulate chloride ion concentration, stabilization of cell membrane potential, trans-epithelial transport, cell volume and apoptotic processes in response to cellular stress.

The crystal structures of human CLIC1 and CLIC2 have been determined. CLIC1 is a disulfide bonded dimer, but CLIC2 is a monomer with an intramolecular disulfide bond, irrespective of redox conditions (Cromer et al., 2007). CLIC2 forms pH-dependent chloride channels in vitro with higher channel activity at low pH values. The channels are subject to redox regulation. In both crystal forms, an extended loop region from the C-terminal domain, called the foot loop, inserting itself into an interdomain crevice of a neighboring molecule, is observed. The equivalent region in the structurally related glutathione transferase superfamily corresponds to the active site. This so-called foot-in-mouth interaction suggests that CLIC2 might recognize other proteins such as the ryanodine receptor through a similar interaction (Cromer et al., 2007).

The genome of Arabidopsis thaliana contains unusual members of the glutathione S-transferase (GST) superfamily with a cysteine in place of a serine at the active site. Four of these genes (at-dhar 1-4) have appreciable homology to intracellular Cl- channels (CLICs) from vertebrates and invertebrates (Elter et al., 2007). Transient expression of AtDHAR1 as the wild-type protein or as a chimera with GFP in mammalian HEK293 or Chinese hamster ovary cells generated a distinct inward rectifying conductance with characteristic biphasic kinetics but no apparent ion selectivity. Analysis of the subcellular localization of AtDHRA1::GFP showed that the bulk of the protein was located in the cytoplasm as soluble protein; however, an appreciable fraction of it could also be found in association with the non-soluble microsomal fraction. Thus, plant members of the GST superfamily are similar to those from animals and have multiple functions. The increase of ion conductance by AtDHAR1 is better explained by a CLIC-like channel activity than by a modification of endogenous channel proteins (Elter et al., 2007).

Ponsioen et al., (2009) have reported that cytosolic CLIC4 undergoes rapid but transient translocation to discrete domains at the plasma membrane upon stimulation of G13-coupled, RhoA-activating receptors, such as those for lysophosphatidic acid, thrombin and sphingosine-1-phosphate. CLIC4 recruitment is strictly dependent on Galpha13-mediated RhoA activation and F-actin integrity, but not on Rho-kinase activity; it is constitutively induced upon enforced RhoA-GTP accumulation. Membrane-targeted CLIC4 does not appear to enter the plasma membrane or modulate transmembrane chloride currents. Mutational analysis reveals that CLIC4 translocation depends on at least six conserved residues, including reactive Cys35, whose equivalents are critical for the enzymatic function of GSTs. Ponsioen et al. (2009) suggest that CLIC4 is regulated by RhoA to be targeted to the plasma membrane, where it may function not as an inducible chloride channel but rather by displaying Cys-dependent transferase activity toward a yet unknown substrate. Membrane interaction promotes transmembrane extension and oligomerization of CLIC1 (Goodchild et al., 2011).

Chloride intracellular channel protein 1 (CLIC1) exists as a soluble monomer or in an integral membrane form. The TMS implicated in membrane penetration and pore formation, comprises helix α1 and strand β2 of the N-domain of soluble CLIC1. Peter et al. (2013) reported the secondary, tertiary and quaternary structural changes of the CLIC1 TMS as it partitions between an aqueous and membrane-mimicking environment. A synthetic 30-mer peptide comprising this TMS was examined in sodium dodecyl sulfate (SDS) micelles, and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes. In the membrane, the peptide assumes a helical structure, acquisition of this secondary structure is concentration-dependent, suggesting an oligomerization event. Stable dimeric and trimeric species were identified using SDS-polyacrylamide gel electrophoresis (Peter et al. 2013).

The Chloride Intracellular Ion Channel (CLIC) family consists of six conserved proteins in humans. Members exist as both monomeric soluble proteins and integral membrane proteins where they function as chloride-selective ion channels. Structural studies showed that in the soluble form, CLIC proteins adopt a glutathione S-transferase (GST) fold with an active site exhibiting a conserved glutaredoxin monothiol motif, similar to the omega class GSTs. Al Khamici et al. 2015 demonstrated that CLIC proteins have glutaredoxin-like glutathione-dependent oxidoreductase enzymatic activity. CLICs 1, 2 and 4 demonstrate typical glutaredoxin-like activity using 2-hydroxyethyl disulfide as a substrate. Mutagenesis experiments identify cysteine 24 as the catalytic cysteine residue in CLIC1, consistent with its structure. CLIC1 reduces sodium selenite and dehydroascorbate in a glutathione-dependent manner. The drugs IAA-94 and A9C specifically block CLIC channel activity and inhibit CLIC1 oxidoreductase activity. This activity may regulate CLIC ion channel function (Al Khamici et al. 2015).Several human CLIC homologues may contribute to diseases including cancer (Leanza et al. 2013). The cellular redox environment and pH are facilitators of CLIC1 insertion into membranes, but spontaneous membrane insertion of CLIC1 is regulated by membrane cholesterol.  A GXXXG motif in CLIC1 likely serves as the cholesterol-binding domain that facilitates the protein's membrane interaction and insertion (Hossain et al. 2019).

The generalized transport reaction believed to be catalyzed by proteins of the CLIC family is:

Cl- (cytoplasm) → Cl- (intraorganellar space).



and Singh H. (2010). Two decades with dimorphic Chloride Intracellular Channels (CLICs). FEBS Lett. 584(10):2112-21.

Al Khamici, H., L.J. Brown, K.R. Hossain, A.L. Hudson, A.A. Sinclair-Burton, J.P. Ng, E.L. Daniel, J.E. Hare, B.A. Cornell, P.M. Curmi, M.W. Davey, and S.M. Valenzuela. (2015). Members of the chloride intracellular ion channel protein family demonstrate glutaredoxin-like enzymatic activity. PLoS One 10: e115699.

Averaimo, S., R. Abeti, N. Savalli, L.J. Brown, P.M. Curmi, S.N. Breit, and M. Mazzanti. (2013). Point mutations in the transmembrane region of the clic1 ion channel selectively modify its biophysical properties. PLoS One 8: e74523.

Berryman, M., J. Bruno, J. Price, and J.C. Edwards. (2004). CLIC-5A functions as a chloride channel in vitro and associates with the cortical actin cytoskeleton in vitro and in vivo. J. Biol. Chem. 279: 34794-34801.

Carlini, V., I. Verduci, F. Cianci, G. Cannavale, C. Fenoglio, D. Galimberti, and M. Mazzanti. (2020). CLIC1 Protein Accumulates in Circulating Monocyte Membrane during Neurodegeneration. Int J Mol Sci 21:.

Cianci, F. and I. Verduci. (2021). Transmembrane Chloride Intracellular Channel 1 (tmCLIC1) as a Potential Biomarker for Personalized Medicine. J Pers Med 11:.

Cromer, B.A., M.A. Gorman, G. Hansen, J.J. Adams, M. Coggan, D.R. Littler, L.J. Brown, M. Mazzanti, S.N. Breit, P.M. Curmi, A.F. Dulhunty, P.G. Board, and M.W. Parker MW. (2007). Structure of the Janus protein human CLIC2. J. Mol. Biol. 374: 719-731.

Duncan, R.R., P.K. Westwood, A. Boyd, and R.H. Ashley. (1997). Rat brain p64H1, expression of a new member of the p64 chloride channel protein family in endoplasmic reticulum. J. Biol. Chem. 272: 23880-23886.

Edwards, J.C., C. Cohen, W. Xu, and P.H. Schlesinger. (2006). c-Src control of chloride channel support for osteoclast HCl transport and bone resorption. J. Biol. Chem. 281: 28011-28022.

Elter, A., A. Hartel, C. Sieben, B. Hertel, E. Fischer-Schliebs, U. Lüttge, A. Moroni, and G. Thiel. (2007). A plant homolog of animal chloride intracellular channels (CLICs) generates an ion conductance in heterologous systems. J. Biol. Chem. 282: 8786-8792.

Goodchild, S.C., C.N. Angstmann, S.N. Breit, P.M. Curmi, and L.J. Brown. (2011). Transmembrane Extension and Oligomerization of the CLIC1 Chloride Intracellular Channel Protein upon Membrane Interaction. Biochemistry 50: 10887-10897.

Gururaja Rao, S., N.J. Patel, and H. Singh. (2020). Intracellular Chloride Channels: Novel Biomarkers in Diseases. Front Physiol 11: 96.

Halpin, S.F. (2004). Brain imaging using multislice CT: a personal perspective. Br J Radiol 77SpecNo1: S20-26.

Hansen, A.M., Y. Qiu, N. Yeh, F.R. Blattner, T. Durfee, and D.J. Jin. (2005). SspA is required for acid resistance in stationary phase by downregulation of H-NS in Escherichia coli. Mol. Microbiol. 56: 719-734.

Harrop, S.J., M.Z. DeMaere, W.D. Fairlie, T. Reztsova, S.M. Valenzuela, M. Mazzanti, R. Tonini, M.R. Qiu, L. Jankova, K. Warton, A.R. Bauskin, W.M. Wu, S. Pankhurst, T.J. Campbell, S.N. Breit, and P.M. Curmi. (2001). Crystal structure of a soluble form of the intracellular chloride ion channel CLIC1 (NCC27) at 1.4-Å resolution. J. Biol. Chem. 276: 44993-5000.

Hossain, K.R., D.R. Turkewitz, S.A. Holt, L. Herson, L.J. Brown, B.A. Cornell, P.M.G. Curmi, and S.M. Valenzuela. (2019). A conserved GXXXG motif in the transmembrane domain of CLIC proteins is essential for their cholesterol-dependant membrane interaction. Biochim. Biophys. Acta. Gen Subj 1863: 1243-1253. [Epub: Ahead of Print]

Hu, H., R.J. Howard, U. Bastolla, E. Lindahl, and M. Delarue. (2020). Structural basis for allosteric transitions of a multidomain pentameric ligand-gated ion channel. Proc. Natl. Acad. Sci. USA 117: 13437-13446.

Kleba, B., T.R. Clark, E.I. Lutter, D.W. Ellison, and T. Hackstadt. (2010). Disruption of the Rickettsia rickettsii Sca2 autotransporter inhibits actin-based motility. Infect. Immun. 78: 2240-2247.

Landry, D, S. Sullivan, M. Nicolaides, C. Redhead, A. Edelman, M. Field, Q. al-Awqati, and J. Edwards. (1993). Molecular cloning and characterization of p64, a chloride channel protein from kidney microsomes. J. Biol. Chem. 268: 14948-14955.

Leanza, L., L. Biasutto, A. Managò, E. Gulbins, M. Zoratti, and I. Szabò. (2013). Intracellular ion channels and cancer. Front Physiol 4: 227.

Meng, X., G. Wang, C. Viero, Q. Wang, W. Mi, X.D. Su, T. Wagenknecht, A.J. Williams, Z. Liu, and C.C. Yin. (2009). CLIC2-RyR1 interaction and structural characterization by cryo-electron microscopy. J. Mol. Biol. 387: 320-334.

Murthi P., Stevenson JL., Money TT., Borg AJ., Brennecke SP. and Gude NM. (2012). Placental CLIC3 is increased in fetal growth restriction and pre-eclampsia affected human pregnancies. Placenta. 33(9):741-4.

Nishizawa, T., T. Nagao, T. Iwatsubo, J.G. Forte, and T. Urushidani. (2000). Molecular cloning and characterization of a novel chloride intracellular channel-related protein, parchorin, expressed in water-secreting cells. J. Biol. Chem. 275: 11164-11173.

Peretti M., Angelini M., Savalli N., Florio T., Yuspa SH. and Mazzanti M. (2015). Chloride channels in cancer: Focus on chloride intracellular channel 1 and 4 (CLIC1 AND CLIC4) proteins in tumor development and as novel therapeutic targets. Biochim Biophys Acta. 1848(10 Pt B):2523-31.

Peter B., Polyansky AA., Fanucchi S. and Dirr HW. (2014). A Lys-Trp cation-pi interaction mediates the dimerization and function of the chloride intracellular channel protein 1 transmembrane domain. Biochemistry. 53(1):57-67.

Peter, B., N.C. Ngubane, S. Fanucchi, and H.W. Dirr. (2013). Membrane mimetics induce helix formation and oligomerization of the chloride intracellular channel protein 1 transmembrane domain. Biochemistry 52: 2739-2749.

Peter, B., S. Fanucchi, and H.W. Dirr. (2014). A conserved cationic motif enhances membrane binding and insertion of the chloride intracellular channel protein 1 transmembrane domain. Eur Biophys. J. 43: 405-414.

Ponsioen B., van Zeijl L., Langeslag M., Berryman M., Littler D., Jalink K. and Moolenaar WH. (2009). Spatiotemporal regulation of chloride intracellular channel protein CLIC4 by RhoA. Mol Biol Cell. 20(22):4664-72.

Singh, H. and R.H. Ashley. CLIC4 (p64H1) and its putative transmembrane domain form poorly selective, redox-regulated ion channels. Mol. Membr. Biol. 24: 41-52.

Tulk, B.M., P.H. Schlesinger, S.A. Kapadia, and J.C. Edwards. (2000). CLIC-1 functions as a chloride channel when expressed and purified from bacteria. J. Biol. Chem. 275: 26986-26993.

Valenzuela, S., D.K. Martin, S.B. Por, J.M. Robbins, K. Warton, M.R. Bootcov, P.R. Schofield, T.J. Campbell, and S.N. Breit. (1997). Molecular cloning and expression of a chloride ion channel of cell nuclei. J. Biol. Chem. 272: 12575-12582.

Wang, H., J. An, S. He, C. Liao, J. Wang, and B. Tuo. (2021). Chloride intracellular channels as novel biomarkers for digestive system tumors (Review). Mol Med Rep 24:.

Warton, K., R. Tonini, W.D. Fairlie, J.M. Matthews, S.M. Valenzuela, M.R. Qiu, W.M. Wu, S. Pankhurst, A.R. Bauskin, S.J. Harrop, T.J. Campbell, P.M.G. Curmi, S.N. Breit, and M. Mazzanti. (2002). Recombinant CLIC1 (NCC27) assembles in lipid bilayers via a pH-dependent two-state process to form chloride ion channels with identical characteristics to those observed in Chinese hamster ovary cells expressing CLIC1. J. Biol. Chem. 277: 26003-26011.

Wu, X., R. Altman, M.A. Eiteman, and E. Altman. (2014). Adaptation of Escherichia coli to elevated sodium concentrations increases cation tolerance and enables greater lactic acid production. Appl. Environ. Microbiol. 80: 2880-2888.


TC#NameOrganismal TypeExample

Organellar chloride (anion selective) channel, p64 (outwardly rectifying) (437 aas and 1 TMS). CLIC5 was the first mitochondrial chloride channel to be identified in the inner mitochondrial membrane, while CLIC4 is located predominantly in the outer mitochondrial membrane. Gururaja Rao et al. 2020 discussed the intracellular chloride channels, their roles in pathologies, such as cardiovascular, cancer, and neurodegenerative diseases, and developments concerning their usage as theraputic targets in humans.


CLIC5 or p64 of Bos taurus


Nuclear chloride channel-27, NCC27 or CLIC1 (Br- > Cl- > I-) (241 aas).  CLIC1 has two charged residues, K37 and R29, in its single TMS which are important for the biophysical properties of the channel (Averaimo et al. 2013).  A putative Lys37-Trp35 cation-pi interaction stabilizes the active dimeric form of the CLIC1 TMS in membranes (Peter et al. 2013).  This channel may play a role in cancer (Peretti et al. 2014).  A positively charged motif at the C-terminus of the single TMS enhances membrane partitioning and insertion via electrostatic contacts.  It also functions as an electrostatic plug to anchor the TMS in membranes and is involved in orientating the TMS with respect to the cis and trans faces of the membrane (Peter et al. 2014).  The CLIC1 protein accumulates in the circulating monocyte membrane during neurodegeneration (Carlini et al. 2020). The involvement of CLIC1 protein functions in physiological and in pathological conditions has been reviewed (Cianci and Verduci 2021).


CLIC1 or NCC27 of Homo sapiens

1.A.12.1.3Organellar chloride channel, CLIC-5A (251 aas; 2 TMSs; one of six homologous human genes) (95% identical to 1.A.12.1.1 but lacks the N-terminal 185 residues.) It associates with the cortical actin cytoskeleton (Berryman et al., 2004).


CLIC-5A of Homo sapiens (Q53G01)

1.A.12.1.4Organellar chloride channel CLIC-6 (704 aas) [The C-terminal half (residues 400-704) resembles a CLIC channel; the N-terminal half (residues 104-356) resembles a repeated C-terminal region of the bovine Na+/Ca2+,K+ exchanger (TC #2.A.19.4.1) as well as several other bacterial and eukaryotic proteins]. AnimalsCLIC-6 of Homo sapiens (Q96NY7)

The Janus protein, CLIC2. The 3-D structure of its water soluble form has been determined at 1.8 Å resolution (Cromer et al., 2007). CLIC2 interacts with the skeletal ryanodine receptor (RyR1) and modulates its channel activity (Meng et al., 2009).


CLIC2 of Homo sapiens (O15247)


Chloride intracellular channel protein 4, CLIC4.  Regulates the histamine H3 receptor (Maeda et al., 2008)) Discriminates poorly between anions and cations (Singh and Ashley, 2007). 76% identical to CLIC5; it may play a role in cancer (Peretti et al. 2014). CLIC5 was the first mitochondrial chloride channel to be identified in the inner mitochondrial membrane, while CLIC4 is located predominantly in the outer mitochondrial membrane. Gururaja Rao et al. 2020 discussed the intracellular chloride channels, their roles in pathologies, such as cardiovascular, cancer, and neurodegenerative diseases, and developments concerning their usage as theraputic targets in humans.


CLIC4 of Homo sapiens (Q9Y696)


Intracellular Cl- channel-3 (CLIC3). The 3-d structure is known (3FY7). This protein is associated with pregnancy disorders (Murthi et al., 2012). 


CLIC3 of Homo sapiens (O95833)


TC#NameOrganismal TypeExample

TC#NameOrganismal TypeExample
1.A.12.2.1The plant Cl- intracellular channel protein DHAR1 (glutathione dehydrogenase/dehydroascorbate reductase) (Elter et al., 2007)PlantsDHAR1 of Arabidopsis thaliana (NP_173387)

Putative Glutathione S-transferase.  Pore formation has not been demonstrated in prokaryotes.


Probable glutathione S-transferase of Leptospira interrogans


TC#NameOrganismal TypeExample

The bacterial CLIC homologue, stringent starvation protein A, SspA (212 aas; 0 TMSs) [N-terminal Trx domain; C-terminal glutathione S-transferase (GST) domain].  May be involved in acid (Hansen et al. 2005) and sodium ion tolerance (Wu et al. 2014).


Stringent starvation protein A of E. coli (P0ACA3)


Glutathione S-transferase, YfcF of 214 aas.  Pore formation has not been demostrated.


YfcF of E. coli