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.
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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

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Figure 8

M128R causes membrane distortion.

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M128R causes membrane distortion. (A) The amplitude of the steady-state ...
(A) The amplitude of the steady-state leak currents of M128R and P290R are larger than that of hEAAT1 and M128K. For a representative current trace, see Figure 7A. ***P < 0.001; ****P < 0.0001. One-way ANOVA tests (Brown-Forsythe) were performed F(4, 21.18) = 71.77, ****P < 0.0001. The exact numbers of cells (n) used are indicated. (B) MD simulations revealing membrane deformation when the M128R side chain faces the lipid bilayer, caused by the interactions of M128R (stick representation, gray) with lipid headgroups (an example shown in stick representation, green). Water molecules within 5 Å of the lipids are shown using a yellow surface representation. (C) M128R interaction with lipid headgroups results in the recruitment of water molecules into the lipid bilayer. The density of water averaged over last 200 ns of a representative simulation trajectory is shown in blue surface. (D) To identify the ions carrying such leak currents, the anion in the recording buffer (Cl–) was changed to a more permeant anion (NO3–), or (E) the concentration of Na+ was increased 10-fold. Reverse potentials (Erev) for hEAAT1, P290R, M128R, M128K, and uninjected cells were measured, and the changes in Erev upon alternation in anion (D) or cation (E) components of the recording buffers are presented as net shift of Erev. One-way ANOVA tests (Brown-Forsythe) were performed for Erev shifts in anion F(4, 12.25) = 48.68, *P < 0.05, ***P < 0.001; and in cation F(4, 24.51) = 35.09, ****P < 0.0001. The exact numbers of cells (n) used are indicated.