Structural determinants of interaction, trafficking and function in the ClC-2/MLC1 subunit GlialCAM involved in leukodystrophy.

KEY POINTS: The extracellular domain of GlialCAM is necessary for its targeting to cell junctions, as well as for interactions with itself and MLC1 and ClC-2. The C-terminus of GlialCAM is not necessary for interaction but is required for targeting to cell junctions. The first three residues of the transmembrane segment of GlialCAM are required for GlialCAM-mediated ClC-2 activation. ABSTRACT: Mutations in the genes encoding the astrocytic protein MLC1, the cell adhesion molecule GlialCAM or the Cl(-) channel ClC-2 underlie human leukodystrophies. GlialCAM binds to itself, to MLC1 and to ClC-2, and directs these proteins to cell-cell contacts. In addition, GlialCAM dramatically activates ClC-2 mediated currents. In the present study, we used mutagenesis studies combined with functional and biochemical analyses to determine which parts of GlialCAM are required to perform these cellular functions. We found that the extracellular domain of GlialCAM is necessary for cell junction targeting and for mediating interactions with itself or with MLC1 and ClC-2. The C-terminus is also necessary for proper targeting to cell-cell junctions but is not required for the biochemical interaction. Finally, we identified the first three amino acids of the transmembrane segment of GlialCAM as being essential for the activation of ClC-2 currents but not for targeting or biochemical interaction. Our results provide new mechanistic insights concerning the regulation of the cell biology and function of MLC1 and ClC-2 by GlialCAM.