Disruption of an EAAT-mediated chloride channel in a Drosophila model of ataxia

Abstract

Patients with Type 6 episodic ataxia (EA6) have mutations of the excitatory amino acid transporter EAAT1 (also known as GLAST), but the underlying pathophysiological mechanism for EA6 is not known. EAAT1 is a glutamate transporter expressed by astrocytes and other glia, and it serves dual function as an anion channel. One EA6-associated mutation is a P > R substitution (EAAT1P > R) that in transfected cells has a reduced rate of glutamate transport and an abnormal anion conductance. We expressed this EAAT1P > R mutation in glial cells of Drosophila larvae and found that these larvae exhibit episodic paralysis, and their astrocytes poorly infiltrate the CNS neuropil. These defects are not seen in Eaat1-null mutants, and so they cannot be explained by loss of glutamate transport. We instead explored the role of the abnormal anion conductance of the EAAT1P > R mutation, and to do this we expressed chloride cotransporters in astrocytes. Like the EAAT1P > R mutation, the chloride-extruding K+-Cl‒ cotransporter KccB also caused astroglial malformation and paralysis, supporting the idea that the EAAT1P > R mutation causes abnormal chloride flow from CNS glia. In contrast, the Na+-K+-Cl‒ cotransporter Ncc69, which normally allows chloride into cells, rescued the effects of the EAAT1P > R mutation. Together, our results indicate that the cytopathology and episodic paralysis in our Drosophila EA6 model stem from a gain-of-function chloride channelopathy of glial cells.