Chloride Intracellular Channel (CLIC) proteins are ubiquitous in multicellular
organisms and often coexist in soluble and integral membrane forms. They can
autoinsert into membranes to form molecular components of intracellular ion
channels. Soluble CLICs are structurally similar to Q-type glutathione-S-transferases
(GSTs), but the structure of the membrane proteins remains elusive.
In this study, soluble, recombinant, human CLIC1, rat brain CLIC4 (p64Hl) and
human CL1C5A, a splice variant of p64 (CLIC5B), were expressed as cleavable Histagged proteins and incorporated into voltage-clamped planar lipid bilayers. They
inserted spontaneously with or without an intact His-tag to form redox-regulated ion
channels. All showed well-defined sublevels with similar reversal potentials and
ionic selectivities. Redox titration in vitro using a glutathione coupled buffer system
led to a sequential reduction in conductance as oxidation progressed on the
extracellular or "luminal" side. These changes were fully reversible, suggesting that
intracellular redox potential may play a role in modulating CLIC channel activity.
The channels were sensitive to the cysteine-reactive compounds NEM and DTNB.
Furthermore, CLIC1 and CLIC4 activity was inhibited by their respective affinitypurified antibodies.
The putative pore forming region was identified based on previous work and CLIC
channels appear to contain at least 4 subunits each with a single transmembrane
domain (TMD). In this simple model, the luminal side of each subunit contains a
single cysteine residue located just before the putative pore entrance. Consistent with
these ideas, truncated proteins comprising the first 58 residues of CLIC1, or the first
61 residues of CLIC4 (sufficient in each case to contain the putative TMD),
autoinserted into bilayers to form redox-sensitive ion channels and could be blocked
by cysteine reactive compounds applied from the luminal side. The truncated
proteins showed reduced conductances, and were non-selective between anions and
CLIC5A was originally identified in an ezrin-containing cytoskeletal complex. The
hypothesis that actin regulates the channel activity of CLICs was tested. Actin
polymerisation on the cytosolic side (but not the luminal side) led to near-complete
channel closure, restricting openings to minor substate levels of CLIC1 and CLIC5A
(there was no effect on CTIC4). Addition of actin-destabilising agents such as
latrunculin B and cytochalasin B inhibited or reversed the effect. CLIC1 also
colocalised with F-actin in both HEK-293 cells and neuroblastoma cells (N2a).
These studies indicated that CLIC5A and CL1C1 channel function may be regulated
by the cortical actin cytoskeleton, providing a new mechanism to regulate localised
ion flux in cells. Unusually, the interaction may be direct, without any intermediate
or adapter protein.
CLICs also interact with dynamin I and cytoskeletal proteins in vitro. Additional in
vitro studies were carried to map the CLIC binding site to the proline-rich domain
(PRD) of dynamin I. This interaction also occurs in vivo, since dynamin 1 and CLICs
form a complex in nerve terminals as previously shown by co-immunoprecipitation.
In agreement, immunofluorescence studies in rat cerebellar granule cells showed
significant co-localization between dynamin I and CLICs. The CLIC proteins
showed distinct sub cellular localizations. CLIC1 and CLIC4 are also localised in
nerve terminals as shown by co-localization with the synaptic vesicle (SV) marker
synaptophysin. The localisation of CLICs in nerve terminals and their interaction
with dynamin suggests a possible role in SV recycling.
In summary, CLICs can form poorly-selective, oligomeric ion channels modulated
by luminal GSH-dependent transthiolation and in some cases by actin polymerisation
from the cytosolic side. The transmembrane domain can autoinsert, but is not enough
to form channels identical to wild-type channels. These preliminary structurefunction studies have provided an overview of the molecular mechanism of
membrane CLICs. Discovery of regulatory mechanisms for CLICs may shed more
light on their functional roles in cells during processes such as the cell cycle and