Ataxia-linked SLC1A3 mutations alter EAAT1 chloride channel activity and glial regulation of CNS function
Qianyi Wu, … , Donald J. van Meyel, Renae M. Ryan
Qianyi Wu, … , Donald J. van Meyel, Renae M. Ryan
Published February 15, 2022
Citation Information: J Clin Invest. 2022;132(7):e154891. https://doi.org/10.1172/JCI154891.
View: Text | PDF
Research Article Neuroscience

Ataxia-linked SLC1A3 mutations alter EAAT1 chloride channel activity and glial regulation of CNS function

Abstract

Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system (CNS). Excitatory amino acid transporters (EAATs) regulate extracellular glutamate by transporting it into cells, mostly glia, to terminate neurotransmission and to avoid neurotoxicity. EAATs are also chloride (Cl–) channels, but the physiological role of Cl– conductance through EAATs is poorly understood. Mutations of human EAAT1 (hEAAT1) have been identified in patients with episodic ataxia type 6 (EA6). One mutation showed increased Cl– channel activity and decreased glutamate transport, but the relative contributions of each function of hEAAT1 to mechanisms underlying the pathology of EA6 remain unclear. Here we investigated the effects of 5 additional EA6-related mutations on hEAAT1 function in Xenopus laevis oocytes, and on CNS function in a Drosophila melanogaster model of locomotor behavior. Our results indicate that mutations resulting in decreased hEAAT1 Cl– channel activity but with functional glutamate transport can also contribute to the pathology of EA6, highlighting the importance of Cl– homeostasis in glial cells for proper CNS function. We also identified what we believe is a novel mechanism involving an ectopic sodium (Na+) leak conductance in glial cells. Together, these results strongly support the idea that EA6 is primarily an ion channelopathy of CNS glia.

Authors

Qianyi Wu, Azman Akhter, Shashank Pant, Eunjoo Cho, Jin Xin Zhu, Alastair Garner, Tomoko Ohyama, Emad Tajkhorshid, Donald J. van Meyel, Renae M. Ryan

×

Figure 5

Rescue assay in Drosophila demonstrates the hEAAT1 Cl– channel is essential for CNS function.

Options: View larger image (or click on image) Download as PowerPoint
Rescue assay in Drosophila demonstrates the hEAAT1 Cl– channel is essent...
(A) Representative trajectories of crawling paths of L1 larvae in 180 seconds, with velocity heatmap. Paths of 9 larvae are shown for each genotype where dEAAT1-null animals were rescued by WT hEAAT1 or mutations of hEAAT1 known to reduce Cl– channel function (S103V and K114L) or increase it (P98G and P392V). Scale bar: 5 mm. (B–D) Quantification in bar graphs of mean speed (B), total path length (C), and the beeline distance from origin (D) for larvae over 60 seconds of continuous tracking. The exact numbers of animals (n) used for panels B–D are hEAAT1 (44), S103V (51), K114L (44), P98G (58), and P392V (97). One-way ANOVA tests (Brown-Forsythe) were performed for mean speed F(4, 106.5) = 7.029, *P < 0.05, **P < 0.01; for total path length F(4, 74.75) = 26.08, ****P < 0.0001; and for beeline distance F(4, 73.08) = 27.28, ****P < 0.0001. (E) Representative images of immunohistochemistry for hEAAT1 show that S103V, K114L, P98G, and P392V mutations do not appear to affect the expression of hEAAT1 nor its distribution to astrocyte processes within CNS neuropil. Panels show a single optical confocal section within the ventral nerve cord of dEaat1-null L1 larvae with astrocyte-specific expression (with alrm-Gal4) of hEAAT1 or S103V, K114L, P98G, or P392V. At least 5 animals were dissected, immunostained, and examined for each genotype. Scale bar: 10 μm.