Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a leukodystrophy characterized by myelin vacuolization and caused by mutations in MLC1 or GLIALCAM. Patients with recessive mutations in either MLC1 or GLIALCAM show the same clinical phenotype. It has been shown that GLIALCAM is necessary for the correct targeting of MLC1 to the membrane at cell junctions, but its own localization was independent of MLC1 in vitro. However, recent studies in Mlc1(-/-) mice have shown that GlialCAM is mislocalized in glial cells. In order to investigate whether the relationship between Mlc1 and GlialCAM is species-specific, we first identified MLC-related genes in zebrafish and generated an mlc1(-/-) zebrafish. We have characterized mlc1(-/-) zebrafish both functionally and histologically and compared the phenotype with that of the Mlc1(-/-) mice. In mlc1(-/-) zebrafish, as in Mlc1(-/-) mice, Glialcam is mislocalized. Re-examination of a brain biopsy from an MLC patient indicates that GLIALCAM is also mislocalized in Bergmann glia in the cerebellum. In vitro, impaired localization of GlialCAM was observed in astrocyte cultures from Mlc1(-/-) mouse only in the presence of elevated potassium levels, which mimics neuronal activity. In summary, here we demonstrate an evolutionary conserved role for MLC1 in regulating glial surface levels of GLIALCAM, and this interrelationship explains why patients with mutations in either gene (MLC1 or GLIALCAM) share the same clinical phenotype.

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ClC-2 is a Cl(-) channel that belongs to the CLC family of chloride channel/transport proteins. ClC-2 molecular role is not clear, and Clcn2 knockout mice develop blindness, sterility, and leukodystrophy by unknown reasons. ClC-2 is associated in the brain with the adhesion molecule GlialCAM, which is defective in a type of leukodystrophy, involving ClC-2 in the homeostasis of myelin. To get more insight into the functions of ClC-2, we have identified in this work the three ClC-2 orthologs in zebrafish. clcn2a and clcn2b resulted from the teleost-specific whole genome duplication, while clcn2c arose from a gene duplication from clcn2b. The expression patterns in adult tissues and embryos of zebrafish clcn2 paralogs support their subfunctionalization after the duplications, with clcn2a being enriched in excitable tissues and clcn2c in ionocytes. All three zebrafish clc-2 proteins interact with human GLIALCAM, that is able to target them to cell junctions, as it does with mammalian ClC-2. We could detect clc-2a and clc-2b inward rectified chloride currents with different voltage-dependence and kinetics in Xenopus oocytes, while clc-2c remained inactive. Interestingly, GlialCAM proteins did not modify clc-2b inward rectification. Then, our work extends the repertoire of ClC-2 proteins and provides new tools for structure-function and physiology studies.

Key pointsr We have characterized the zebrafish clc-k and barttin proteins, demonstrating that they form a protein complex mediating chloride flux in a similar manner to their mammalian counterparts.r As in mammals, in zebrafish, clc-k and barttin are basically expressed in the kidney. Contrary to what is found in mammals, in zebrafish both proteins show an apical localization in the kidney.r We have generated the first knockout in zebrafish of a CLC protein. Lack of clc-k in zebrafish resulted in embryonic lethality, possibly caused by a reduction in total chloride content. As a consequence, there is an up-regulation of other chloride channels and other regulatory mechanisms such as renin or the uro-guanylin receptor in the kidney. r barttin is mislocalized in vivo when clc-k is not present, indicating that there is a mutual dependence of the protein expression and localization between barttin and clc-k proteins.Abstract ClC-K/barttin channels are very important for salt transport in the kidney. This function can be clearly seen since mutations in CLCNKB or BSND cause Bartter's syndrome types III and IV, respectively. Working with the freshwater teleost zebrafish, we characterized the genes homologous to the mammalian chloride channel ClC-K and its obligate subunit barttin and we obtained and studied clc-k knockout zebrafish. The zebrafish clc-k/barttin proteins are very similar to their mammalian counterparts, and both proteins are necessary to mediate chloride currents. Localization studies indicated that both proteins are exclusively expressed in the apical membranes of zebrafish kidney tubules. Knockout of clc-k resulted in embryonic lethality. These animals showed barttin mislocalization and a reduction in whole-body chloride concentration, Carla Pérez received her degree in Biotechnology from the University of Vic, Spain. She obtained her Master's Degree in Neuroscience from the University of Barcelona, Spain, and joined the Physiology and Pathology of The Functional Relationship Between Glia and Neurons Group at the same University. There, she obtained her PhD by studying the physiology of zebrafish ClC chloride channels and their associated diseases. Aida Castellanos received her degree in Biology from the University of Girona, Spain. She subsequently got her Master's Degree in Neuroscience from the University of Barcelona, Spain, and then joined the Neurophysiology Group at the same University. After getting her PhD in the field of somatosensation and pain, she currently holds a postdoctoral position at the Physiology Group of the University of Barcelona, where she studies the physiopathological relationship between glia and neurons, with a particular interest in rare diseases. C. P.-Rius and A. Castellanos contributed equally to this work.3970 C. Pérez-Rius and others J Physiol 597.15as well as up-regulation of the expression of other chloride channels and renin, and an increase in the kidney expression of the uroguanylin receptor. Our results indicate that apical kidney chloride reabsorption through c...