Establishing Drosophila as a research model for the study of Voltage Gated Chloride Channelopathies
Creators: name: Judd-Mole, Sebastian
Publisher: name: Monash University
Year: 2017
Subjects: Uncategorized

description: Proteins of the Voltage Gated Chloride Channel (CLC) family perform a variety of roles, including the regulation of membrane excitability, transepithelial transport, cell volume regulation and the acidification of intracellular organelles. The family can be divided into two mechanistically distinct subclasses, plasma membrane Cl- channels and vesicular Cl-/H+ exchangers. The importance of these proteins to human health is demonstrated by the spectrum of diseases that arise from disruption of CLC coding genes. These include, myotonia, leukodystrophy, Bartter’s syndrome, Dent’s disease, osteopetrosis and lysosomal storage disease. Despite extensive research on the mammalian CLC family using rodent models and human cell lines, many details on the cellular roles of CLC proteins and how they link to these disorders remain unclear. It has also become apparent that some CLC proteins have multiple roles between cell types that we are yet to fully understand. This study addresses these issues by establishing Drosophila melanogaster as a dynamic research model for the CLC family. While there are nine human CLCs that segregate into three evolutionary clades, in Drosophila there are only three, CLC-a, CLC-b and CLC-c, each of which is homologous to an entire mammalian CLC branch. This makes for a powerful system whereby manipulation of one Drosophila CLC coding gene can reveal the fundamental cellular roles performed across the respective mammalian clade. In this project, CLC-c was shown to localise primarily to the apical membrane as well as to an intracellular vesicle distinct from early endosomes. CLC-c knockdown in the dorsal midline caused adult thorax defects that could be partially rescued by expression hCLC-5, indicating some functional overlap. The generation of a CLC-c null mutant revealed that CLC-c-/- flies die early in development, a response far more severe than that seen upon disruption of any one of the three rodent CLC-c homologues. Furthermore, CLC-c-/- cells generated in a heterozygous background showed an increase in acidic vesicles prior to undergoing apoptosis. CLC-b was found to have a ubiquitous expression domain and localise to late endosomes and lysosomes, as seen for its two mouse homologues CLC-6 and CLC-7. Generation of a CLC-b null mutant revealed that like its mammalian homologues, CLC-b disruption causes pigmentation defects and age related degeneration akin to the symptoms of neuronal ceroid lipofuscinosis seen in Clcn7-/- mice. Finally, a fluorescent CLC-a fusion protein localised exclusively to the basolateral membrane in most cell types, similar to its closest mammalian homologue CLC-2, and targeted modulation of CLC-a expression caused neuronal defects. However, unlike CLCN2-/- mice and humans, which show mild signs of neurodegeneration, flies homozygous for a large deletion in the CLC-a gene died soon after embryonic development, suggesting a broader role for CLC-a beyond that of hCLC-2. Collectively, the findings and tools generated within this study provide a dynamic research platform for understanding the functional roles of the CLC family members using the Drosophila model.