CLC-3 is an intracellular chloride transport protein known to reside on the endosomes and synaptic vesicles, which plays a role in cancer cell proliferation. It is a member of the CLC gee family that encodes Cl– channels and Cl–/H+ exchangers, and is predominantly expressed in membranes of the endosomal system. It may also be present in synaptic vesicles and synaptic-like microvesicles (SLMVs).
CLC-3 assists in acidification of endosomes and other compartments by shunting the electrical current of the vessicular V-type H+-ATPase. Also, CLC-3 either changes the vesicular voltage or lead to luminal chloride accumulation, as directly shown for the lysosomal 2Cl–/H+-exchanger, CLC-7.
CLC protein structure
CLC has three highly conserved Cl– binding sites, which feature a partial positive charge formed by amino acid residues located in the N-terminal portion of specific alpha-helices. In the crystal structure, Cl– could be found at three specific sites made up by these amino acids: (1) an internal site (Sint) in contact with the intracellular environment, (2) a central site (Scen) buried in the membrane bilayer, and (3) an external site (Sext) in contact with the extracellular solution.
Both Sint and Scen are occupied by Cl–, whereas Sext is occupied by the negatively charged side-chain of a conserved glutamate (E148) named Gluext. In Scen, Cl– ions are coordinated mainly by residues S107 and Y445 also called Sercen and Tyrcen, respectively. Following a mutation or protonation of E148, a Cl– occurs in Sext, which renders CLC gating proton-dependency.
Mutation of this glutamate residue abolishes voltage and Chloride-dependent gating in CLC channels and uncouples Cl–/H+ exchange, turning the proteins into passive chloride conductors.
CLC-3 plays a role in Glioblastoma multiforme
Glioblastoma multiforme, the major malignant glioma cell, expresses three members of the CLC family, CLC-2, CLC-3 and CLC-5, however, CLC-3 in particular is the a critical regulator of cell volume changes associated with cycle. There are evidences that CLC-3 is involved in neuronal excitability, proliferation and migration. It has been shown to have an important role in the invasiveness of human glioma cells as CLC-3 is abundantly expressed in the cytoplasmic membrane and in intracellular vesicles of glioma cells.
A knockdown of CLC-3 reduces resting outwardly-rectifying chloride current in glioma cells, suggesting that CLC-3 mediates resting chloride current in the plasma membrane and thus, may be involved in cell shrinkage during invasion.
Activation and inhibition of CLC-3 in glioma cells
CLC-3 activity is regulated through phosphorylation via CaMKII. Intracellular infusion of autoactivated CaMKII via patch pipette enhanced chloride currents3-fold. This regulation of CaMKII was however inhibited by autocamtide-2 related inhibitory peptide, a CaMKII-specific inhibitor.