Mammalian members of the Voltage Gated Chloride Channel family of Cl-/H+ exchanger proteins (CLC-3 to 7) are thought to play a range of cellular roles in transepithelial transport, endocytosis, cell volume regulation and the acidification of intracellular organelles. Mutations of these proteins in mice or humans have been shown to cause disease symptoms such as neuronal ceroid lipofuscinosis (CLC-6 and 7), osteopetrosis (CLC-7), Dents disease (CLC-5) and lysosomal storage disorders (CLC-3).
Drosophila has fewer CLC proteins, with CLC-c homologous to mammalian CLC-3, 4 and 5 and CLC-b homologous to CLC-6 and 7. As such it represents an excellent system in which to study the precise cellular role of each class of chloride channel. We have characterised the function of CLC-b and CLC-c.
A CLC-b:GFP fusion protein was found to localize to lysosomes. ClC-b null homozygotes are viable but exhibit a dramatic reduction in both lifespan and locomoter activity, plus a mild pigmentation defect. These phenotypes are reminsicent of the symptoms of neuronal ceroid lipofuscinoses caused by disruption of CLC-6 or 7 and may represent a defect in lysosomal acidification leading to lysosomal storage disorder-like pathologies. The ClC-b mutant flies are therefore a promising model for studying the molecular mechanism underlying such disorders.
A CLC-c:GFP fusion protein was shown to localize both to the apical plasma membrane and to endolysosomal vesicles. ClC-c null mutant homozygotes die in early larval development indicating that, unlike any of its three mammalian homologues, ClC-c is essential for viability. Mosaic analysis demonstrated that ClC-c homozygous mutant cells have a survival defect and are out-competed by surrounding wild type cells in the larval wing imaginal disc. Prior to undergoing apoptosis, these ClC-c mutant cells exhibit an increase in LysoTracker staining that is independent of autophagy. Therefore unlike proposed roles for its mammalian homologues in lysosomal acidification, CLC-c is more likely to be required either in the recycling of endosomes back to the plasma membrane, or the transition of late endosomes to lysosomes. A block in either function may result in an increase in acidified late endosomes, leading to the elevated LysosTracker signal observed.
Our initial charaterization of CLC-b and CLC-c function paves the way to an in-depth investigation of the molecular pathology underlying disorders such as Dent's disease and neuronal ceroid lipofucinoses, which could then be used as a platform for the identification of drugs able to restore function to defective chloride channel proteins.