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).

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