Ion channels are expressed on the plasma membrane of all cells and have also been found on the membranes of intracellular organelles. They subserve essential functions in all cells and evidence of their importance includes their high degree of conservation across species and the rapidly growing list of genetic diseases associated with ion channel defects. Ion channels are also important therapeutically and have been utilised extensively and effectively as drug targets, by the pharmaceutical industry. Early evidence suggests that CLIC1, the subject of this grant, may be a suitable therapeutic target for inflammatory disease therapy and to our knowledge is a subject of interest of both Pfizer and Aventis, who have contacted us about this protein.

CLIC1 (formerly NCC27) is a 241 amino acid ion channel protein first cloned by us because of its increased expression with macrophage (MAC) activation. We have subsequently published extensively on this protein, which was the first identified human member of a growing family of intracellular organellar ion channels, whose biological function is still incompletely understood.

a. Biological significance of CLIC1 and the CLIC family. Whilst there is still little know about the biology of CLIC proteins, evolution has provided us with an irrefutable argument as to their importance: remarkably preservation across evolution of this family. The whole CLIC family is highly conserved across a very wide range of species e.g. only three conservative substitutions between human and murine CLIC1 proteins. Clearly defined relatives can be detected in the genomes of all vertebrates sequenced so far, the chordate, Ciona intestinalis, and the invertebrates Anopheles and Drosophila and C. elegans. Even Ciona CLIC and human CLICs are about 50% identical. CLIC1 itself is located within the major histocompatibility complex (MHC), one of the most conserved and important regions in the genome. It is in close proximity to other stress response proteins including TNF-a, HSP70 proteins and proteins involved in Ag presentation.

b. Other members of the human CLIC Family. The six member mammalian family of CLIC proteins share high similarity (about 60-75%) in the “CLIC domain” but vary in their cellular and sub-cellular distribution. CLICs are associated with various intracellular membranes including the plasma and nuclear membranes and localises to granular cytoplasmic organelles, the ER membrane, large dense-core vesicles, mitochondria, TGN vesicles and secretory vesicles. CLIC4 (mtCLIC) is partly localised to mitochondria, helps maintain mitochondrial membrane potential and participates in the apoptotic response to p53.

We have published detailed electrophysiological characterisation of CLIC1. Using a combination of whole cell, single channel and macropatch recording, we have shown that CLIC1 is a Cl- dependent Cl- channel and conducts both inward and outward current, with a reversal potential around 0 mV and conductances of about 8.2 pS for inward and 15.8 pS for outward current. Despite CLIC1’s unusual characteristics, (small and with only a single putative transmembrane region), we and others have published data that definitively identify it as an ion channel. By selectively tagging either the N- or C-terminus of CLIC1, and varying the side of the membrane from which we record channel activity, we have shown that CLIC1 is a transmembrane protein that forms an integral part of the ion channel. Further, these studies indicate that the N-terminus projects outwardly and the C-terminus inwardly. Additionally, we and others have demonstrated that soluble recombinant E.coli-expressed CLIC1 protein forms ion channels in artificial lipid bilayers. Whilst bilayer studies of solubilised membranes may be prone to artifact, the situation with CLIC1 is more clear-cut. Most ion channels must be incorporated into liposomes, which then fuse with the lipid bilayer. However, with CLIC1 (and CLIC4), soluble aqueous recombinant protein enters the bilayer directly. CLIC1 has similar electrophysiological characteristics, whether transfected into CHO cells or in artificial lipid bilayers, indicating it can function as an ion channel without ancillary proteins.

An understanding of structure/function is an essential element in understanding molecule function and our group has started to unravel this in the CLIC family. Our collaborators (Prof Paul Curmi’s group) have solved the high-resolution crystallographic structure of the soluble form of CLIC1, the first high-resolution study of a member of this family and more recently the structure of CLIC4, drosophila CLIC and EXC-4. CLIC1 is a structural homolog of the glutathione-S-transferases (GST) and contains a glutathione (GSH) binding site motif but does not bind reduced GSH. CLIC1 must undergo significant conformational change from its globular, soluble structure in order to form an ion channel. Its GST-like structure provides a strong argument for the role of oxidation/reduction/GSH in the function of CLIC1 and pursuing this line of reasoning, we have shown that on oxidation, CLIC1 undergoes a reversible transition from a monomeric to a non-covalent dimeric state, due to the formation of an intra-molecular disulphide bond (Cys24-Cys59). We have determined the crystal structure of this oxidised state, and show that a major structural transition has occurred, exposing a large hydrophobic surface, which forms the dimer interface. The oxidised CLIC1 forms Cl- channels in artificial bilayers and vesicles, while a reducing environment prevents the formation of ion channels. Initial mutational studies suggested that both Cys24 and Cys59 are required for channel activity, but later, as yet unpublished studies indicate that only Cys24 is essential, whilst Cys59 acts by increasing sensitivity to the oxidative modification needed for lipid bilayer binding.

There is limited data on the biological function of vertebrate CLICs in general and CLIC1 specifically:

i) A mutation, generating a stop codon in exon five of CLIC5, is the cause of a spontaneous mutation (jitterbug) in mice, causing impaired hearing and vestibular dysfunction associated with dysmorphic stereocilia, progressive hair cell degeneration. CLIC5 is normally localised to stereocilia of cochlear and vestibular hair cells.

ii) CLIC4 is up-regulated by TNF-a, stress and p53 and has been strongly linked to in vitro control of apoptosis.

iii) CLIC1 is up-regulated by activation in MACs and we have shown that PMA and amyloid peptide induce CLIC1 on the plasma membrane of both murine microglial cells and the microglial cell line BV2. In co-cultures with neuronal cells, these activated microglia cause neuronal cell apoptosis due to release of reactive nitrogen species and TNF-a.

Structure/Function Studies

An understanding of structure/function is an essential element in understanding molecule function and our group has started to unravel this in the CLIC family. Our collaborators (Prof Paul Curmi’s group) have solved the high-resolution crystallographic structure of the soluble form of CLIC1, the first high-resolution study of a member of this family and more recently the structure of CLIC4, drosophila CLIC and EXC-4. CLIC1 is a structural homolog of the glutathione-S-transferases (GST) and contains a glutathione (GSH) binding site motif but does not bind reduced GSH. CLIC1 must undergo significant conformational change from its globular, soluble structure in order to form an ion channel. Its GST-like structure provides a strong argument for the role of oxidation/reduction/GSH in the function of CLIC1 and pursuing this line of reasoning, we have shown that on oxidation, CLIC1 undergoes a reversible transition from a monomeric to a non-covalent dimeric state, due to the formation of an intra-molecular disulphide bond (Cys24-Cys59). We have determined the crystal structure of this oxidised state, and show that a major structural transition has occurred, exposing a large hydrophobic surface, which forms the dimer interface. The oxidised CLIC1 forms Cl^- channels in artificial bilayers and vesicles, while a reducing environment prevents the formation of ion channels. Initial mutational studies suggested that