The many faces of the EAAT transporter family, or- the controllers of “OFF” (2024)

EAATs work as symporters by transporting one K⁺ ion out of the cell and simultaneously taking up a glutamate neurotransmitter/molecule (aspartate can also be transported) and three Na⁺ ions as well as an H⁺ into the cell in exchange (Alleva et al., 2022), see Fig. 1 (adapted from Freidman et al., 2020). This transport is dependent on an electrochemical gradient of sodium ions, and is facilitated by homo- or heterotrimers of the EAATs at the cell membrane (Kovermann et al., 2022).
EAATs are not only glutamate transporters, but also anion channels (Cl⁺) that open in response to transitions within the glutamate transport cycle (Otis & Jahr, 1998).

Figure 1: Transport of glutamate and anions by EAATs

Recently published data suggest that EAAT5, a retina specific member of the EAAT family, functions not only as a glutamate importer but also as a glutamate-gated Cl^--channel, particularly in cone photoreceptors (Lukasiewcz et al., 2021).
When glutamate is taken up into glial cells by the EAATs, it is converted to glutamine and subsequently transported back into the presynaptic neuron, converted back into glutamate, and taken up into synaptic vesicles by action of the VGLUTs. This process is named the glutamate–glutamine cycle (Andersen & Schousboe, 2023), see Figure 2.

The EAAT family consists so far of 5 members, EAAT1 to EAAT5, all with a different glutamate uptake kinetic and a different degree of chloride permeability and distribution (Todd & Harding, 2020), see Table 1.

EAAT1:
EAAT1, also referred to as GLAST-1, is expressed throughout the CNS, and is highly expressed in astrocytes and Bergmann glia in the cerebellum. In the retina, EAAT1 is expressed in Müller cells.
Robust EAAT expression, in particular EAAT1, is a widely used marker of adult neural stem cell (NSC) phenotype (Rieskamp et al., 2023).
We offer several KO-validated antibodies for the detection of EAAT1.

Figure3: Indirect immunostaining of EAAT1 in mouse cerebellum of wildtype (WT) and knockout (KO) animals with rabbit polyclonal anti-EAAT1 (cat. no. 250 113, dilution 1:5000; red).Courtesy: Yun Zhou and Niels Christian Danbolt, Dept. of Anatomy, Institute of Basic Medical Sciences, University of Oslo

Figure5: Indirect immuno-staining of EAAT2 in the neocortex of heterozygote (+/-) and knockout (-/-) mice (cat. no. 250 203, dilution 1:2000; red). Courtesy: Yun Zhou and Niels Christian Danbolt, Dept. of Anatomy, Institute of Basic Medical Sciences, University of Oslo

Figure7: Immunoblottting of EAAT3 in synaptic membrane fraction of rat brain (LP1) with rabbit polyclonal anti-EAAT3 (cat. no. 250 313).

Figure7: Immunoblottting of EAAT3 in synaptic membrane fraction of rat brain (LP1) with rabbit polyclonal anti-EAAT3 (cat. no. 250 313).

​Figure 8:Indirect immunostaining of a formaldehyde fixed mouse cerebellum section (saggital) rabbit anti-EAAT4 antibody (cat. no. 250 413, dilution 1:500, red) and guinea pig anti-Calbindin antibody (cat. no. 214 318, dilution 1:500, green). Nuclei have been visualized by DAPI staining (blue).

Figure9: Indirect immunostaining of a formaldehyde fixed mouse cerebellum section (coronal) rabbit anti-EAAT4 antibody (cat. no. 250 413, dilution 1:500, red) and guinea pig anti-Calbindin antibody (cat. no. 214 318, dilution 1:500, green). Nuclei have been visualized by DAPI staining (blue).

Figure 10: Indirect immunostaining of EAAT5 in mouse retina of wildtype (WT) and knockout (KO) animals (cat. no. 250 504, dilution 1:2000; red). The tissue was immersion fixed with 4% formaldehyde with an antigen retrieval with 1% SDS according to (Gehlen et al. 2021). Courtesy: Christoph Aretzweiler-von Schwartzenberg and Frank Müller, Institute of Biological Information Processing, Molecular and cellular physiology (IBI-1), Forschungszentrum Jülich, Germany

Figure 10: Indirect immunostaining of EAAT5 in mouse retina of wildtype (WT) and knockout (KO) animals (cat. no. 250 504, dilution 1:2000; red). The tissue was immersion fixed with 4% formaldehyde with an antigen retrieval with 1% SDS according to (Gehlen et al. 2021). Courtesy: Christoph Aretzweiler-von Schwartzenberg and Frank Müller, Institute of Biological Information Processing, Molecular and cellular physiology (IBI-1), Forschungszentrum Jülich, Germany

Figure 11: Indirect immunostaining of a formalin fixed paraffin embedded (FFPE) mouse ileum section with guinea pig anti-EAAT5 antibody (cat. no. 250 504, dilution 1:1000, DAB; brown). Nuclei have been visualized by hematoxylin staining (blue).

Figure 11: Indirect immunostaining of a formalin fixed paraffin embedded (FFPE) mouse ileum section with guinea pig anti-EAAT5 antibody (cat. no. 250 504, dilution 1:1000, DAB; brown). Nuclei have been visualized by hematoxylin staining (blue).