Glutamate transporter-associated anion channels adjust intracellular chloride concentrations during glial maturation

Abstract

Astrocytic volume regulation and neurotransmitter uptake are critically dependent on the intracellular anion concentration, but little is known about the mechanisms controlling internal anion homeostasis in these cells. Here we used fluorescence lifetime imaging microscopy (FLIM) with the chloride-sensitive dye MQAE to measure intracellular chloride concentrations in murine Bergmann glial cells in acute cerebellar slices. We found Bergmann glial [Cl ] to be controlled by two opposing transport processes: chloride is actively accumulated by the Na -K -2Cl cotransporter NKCC1, and chloride efflux through anion channels associated with excitatory amino acid transporters (EAATs) reduces [Cl ] to values that vary upon changes in expression levels or activity of these channels. EAATs transiently form anion-selective channels during glutamate transport, and thus represent a class of ligand-gated anion channels. Age-dependent upregulation of EAATs results in a developmental chloride switch from high internal chloride concentrations (51.6 ± 2.2 mM, mean ± 95% confidence interval) during early development to adult levels (35.3 ± 0.3 mM). Simultaneous blockade of EAAT1/GLAST and EAAT2/GLT-1 increased [Cl ] in adult glia to neonatal values. Moreover, EAAT activation by synaptic stimulations rapidly decreased [Cl ] . Other tested chloride channels or chloride transporters do not contribute to [Cl ] under our experimental conditions. Neither genetic removal of ClC-2 nor pharmacological block of K -Cl cotransporter change resting Bergmann glial [Cl ] in acute cerebellar slices. We conclude that EAAT anion channels play an important and unexpected role in adjusting glial intracellular anion concentration during maturation and in response to cerebellar activity. GLIA 2017;65:388-400.

Current carried by the Slc26 family member prestin does not flow through the transporter pathway

“…Consistent with this possibility, expression of prestin at high levels in HEK cells has a major effect on its resting potential. A similar role has been established in the leakage conductance of EAAT1/2 in glial cells in the cerebellum, that show developmental expression, reducing intracellular chloride52.…”

Glial and Neuronal Glutamate Transporters Differ in the Na+ Requirements for Activation of the Substrate-Independent Anion Conductance

“…The role of the anion channel in regulating cellular activity has been supported by EAAT5-mediated regulation of rod bipolar cells in the retina (Veruki et al, 2006). Recent studies suggest that the anion channels associated with the glial glutamate transporters, EAAT1 and EAAT2, are involved in regulation of glial intercellular chloride concentrations, potentially influencing crosstalk between glutamatergic and GABAergic neurotransmission (Untiet et al, 2017). At this point, all experiments testing the ability of EAAT-mediated anion currents to affect cellular activity have always been performed in the presence of glutamate and no data exist that support a role for the EAAT-mediated regulation of cellular activity in the absence of glutamate.…”

Stratification of astrocytes in healthy and diseased brain

“…Molecular pathways Reference Ion homeostasis K 1 buffering and homeostasis Na 1 -K 1 pump, NKA Na 1 -K 1 -Clco-transporter 1 NKCC1/SLC12A2 (operational at high K 1 loads) Inward rectifier K 1 channels K ir 4.1 Connexins Cx43, Cx30 (41,51,115,122,132,164,171,172,192,225,254) Clhomeostasis GABA A receptors Anion channels, ClC-2, Volume-regulated anion channels VRAC/SWELL1 Best1 Clchannels Na 1 -K 1 -Clco-transporter 1 NKCC1/SLC12A2 (24,65,107,173,230) H 1 homoeostasis and control of extracellular pH Na…”