CLIC1-membrane interactions and insertion mechanism

soluble and membrane-inserted forms. The exact nature of the membrane-bound form and its significance within the wider biological function of CLIC1 are matters still receiving much attention. The structure of the CLIC1 channel is unknown, but is generally proposed to be tetrameric (Warton et al., 2002), or even a multimer of up to 16 subunits (Singh and Ashley, 2006). This is because of the

rather varying single-channel conductances noted by different research groups, which have been suggested to represent different substates of the channel (Singh and Ashley, 2006). It was also suggested that the appearance of different substates may be related to the type of lipids used in the membrane system (Singh and Ashley, 2006), or to the chloride concentrations used (Tonini et al., 2000).

The conserved putative transmembrane region (Cys24 – Val46) of CLIC1 is significantly hydrophobic, and like all of the human CLICs, but not necessarily the invertebrate CLICs, has an amphipathic character which is ideal for a membrane-traversing ion channel helix (Littler et al., 2007). The proven membrane-insertion ability and functionality of the invertebrate CLICs would tend to suggest that this amphipathic character is not an absolute requirement for channel formation and function (Littler et al., 2007). Cys24 is located at the N- cap position of helix α1, and would be present just on the outside of the membrane in the membrane-inserted conformation. Its location there was proposed, and has been shown, to regulate channel activity via redox control, with Pro25 possibly acting as a hinge region for opening and closing the channel (Harrop et al., 2001; Singh and Ashley, 2006). Channel activity of CLIC1 reconstituted in bilayers was lowered in the presence of 5 mM trans GSH or 1 mM dithiothreitol (DTT). The channels also remained open or closed longer. However, trans oxidation with oxidised glutathione (GSSG) greatly reduced channel activity, and cis GSSG had no effect on function. The thiol-reactive reagent N-ethylmaleimide (NEM) blocked channel activity from the trans side, but had no effect from the cis side, and a Cys24 mutant was insensitive to the effects of NEM. These results are somewhat in conflict with those of Littler et al. (2004), who found that oxidation was essential for dimer formation and channel activity. In their experiments, oxidation greatly increased channel activity, and this activity of oxidised CLIC1 was abolished in the presence of 5 mM DTT. Thus, the exact role of oxidation in the function of CLIC1 is still being studied.

While studies of the truncated N-domain of CLIC1 have yet to appear, work on the truncated N-domains of CLIC4 and the nematode CLIC homologue EXC-4

have shown that this portion of the protein is all that is required for function (Berry and Hobert, 2006; Singh and Ashley, 2007). Truncated CLIC4 formed channels less readily and with reduced conductance and selectivity, but nevertheless functioned reproducibly in every experiment. They demonstrated the same redox sensitivity as the full-length protein. This would indicate that the extramembranous C-domain region serves only to orient the protein and act as a concentrating vestibule for ions travelling through the channel (Singh and Ashley, 2007). A vestibule-like function would greatly aid the efficient functioning of the channel, given that a negative surface potential depletes the surface concentration of anions, as discussed in Section 1.1.1.2. It is likely that the conserved KRR motif acts as a “plug” to anchor the protein in the membrane surface and possibly to direct ions toward the channel opening.

A study on EXC-4, a Caenorhabditis elegans excretory canal cell CLIC protein showed that deletion of strand β2 in the PTM region resulted in decreased membrane localisation, and that mutation of a highly conserved Leu46 in helix α1 (also in the PTM region) to a helix-breaking Proline disrupted membrane localisation, indicating that these secondary structural elements are important for membrane insertion of CLIC proteins (Berry et al., 2003). The crystal structures of CLIC2 and trimeric CLIC4 show helix α2 to be disordered, and slight differences exist in this helix in the different crystal forms of CLIC1, suggesting a possible susceptibility to conformational change in this region leading to membrane docking (Li et al., 2006; Cromer et al., 2007). Structural rearrangement of the N-terminal domain would require the β1α1β2 supersecondary motif to detach from the rest of the protein, extend and refold into a helical membrane-traversing structure (Fanucchi et al., 2008). Inherent plasticity in the domain interface and the proven ability of the N-domain to form an all α-helical conformation show that this may certainly be possible (Harrop et

al., 2001; Littler et al., 2004). The protein may oligomerise either prior to or post-

Further to studies that showed that CLIC1 activity occurred more readily and with greater conductance at low pH (Warton et al., 2002; Tulk et al., 2002), a recent study from our laboratory investigated the structural properties and conformational stability of CLIC1 as a function of pH in the absence of membranes (Fanucchi et al., 2008). A highly populated intermediate species with a solvent-exposed hydrophobic surface was detected at acidic pH (≤ pH 5.5) under mildly denaturing conditions. An intermediate with the same properties was detected at pH 7.0 and 37 °C. Thus it was proposed that the negative potential and acidic environment encountered at the surface of the membrane could alter local and long range electrostatic interactions within the protein structure, priming it for membrane insertion via a lowering of the energy barrier for its conversion from a soluble to a membrane-bound conformation.