Proceedings of The Physiological Society

University College London (2011) Proc Physiol Soc 24, C16 and PC16

Oral Communications

New insight into CLC-2 chloride channel function: GlialCAM, a protein defective in a leukodystrophy is a channel subunit in glial cells

E. Jeworutzki1, T. López-Hernández3, X. Gasull2, R. Estévez3,4, M. Pusch1

1. Istituto di Biofisica, National Research Council, Genova, Italy. 2. Lab. Neurophysiology, Department Physiological Sciences I, School of Medicine, University of Barcelona-IDIBAPS, Barcelona, Spain. 3. Physiology Setion, Department Physiological Sciences II, School of Medicine, University of Barcelona, Barcelona, Spain. 4. U-750, Centro de Investigaci

CLC-2 is an ubiquitously expressed inwardly rectifying chloride channel with many candidate functions. Brains of Clcn2 -/ - mice show vacuole formation in myelin sheets1. Similar vacuolar structures were found in patients which carried mutations in MLC1, a transmembrane protein of unknown function, leading to a rare leukodystrophy called MLC (megaencephalic encephalopathy with subcortical cysts) 2. Since MLC1 does not interact with CLC-2 and no mutations in the CLCN2 gene were found in MLC patients 3, a role of CLC-2 in MLC seemed to be excluded. Recently a second disease gene, GlialCAM, causing MLC was found 4. GlialCAM is a cell adhesion molecule mainly expressed in glial cells 5 and interacts directly with MLC1 4. Our biochemical approach to identify interacting partners for GlialCAM uncovered CLC-2 as a major binding partner besides MLC1 in brain. CLC-2 and GlialCAM co-localise in Bergmann glia, myelin and at astrocyte-astrocyte contacts at the end-feet. In HEK cells, we observe a shift of CLC-2 being uniformly distributed over the plasma membrane to areas of cell contacts when co-expressed with GlialCAM. Since GlialCAM alone is localised in cell contacts as well, it is likely that it acts as a carrier molecule for CLC-2. Functionally, co-expression of CLC-2 with GlialCAM resulted in a large increase of CLC-2 mediated currents in Xenopus oocytes and HEK cells. The activation of the channel becomes almost instantaneous and currents lose rectification. CLC-2/GlialCAM currents resemble those of an N-terminal deletion mutant of CLC-2 (ΔN). However, in contrast to ΔN the CLC-2/GlialCAM complex stays osmo-sensitive and reduces pH blockage at acidic conditions. Anion selectivity and Cd2+ block remain unchanged in the complex. We studied four disease causing GlialCAM mutations in the IgV domain. All of them disrupted the clustering of CLC-2/GlialCAM at cell contacts, but did not alter the ability to increase and modify CLC-2 mediated currents. In conclusion, we propose that CLC-2 is localised in cell contacts in glial cells and modulated through an interaction with the common gate by the cell adhesion molecule GlialCAM. This interaction may be important in the pathology of MLC disease and opens new insights into the role of CLC-2 in glial cells.

Where applicable, experiments conform with Society ethical requirements