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. 2013 Mar 12;57(1):e11.
doi: 10.4081/ejh.2013.e11.

Excitatory amino acid transporter 5 is widely expressed in peripheral tissues

A Lee  1 , A R Anderson, M Stevens, S Beasley, N L Barnett, D V Pow
Affiliations

Excitatory amino acid transporter 5 is widely expressed in peripheral tissues

A Lee et al. Eur J Histochem. .

Abstract

It is routinely stated in the literature that Excitatory Amino Acid Transporter 5 (EAAT5) is a retina-specific glutamate transporter. EAAT5 is expressed by retinal photoreceptors and bipolar cells, where it serves as a slow transporter and as an inhibitory glutamate receptor, the latter role is due to the gating of a large chloride conductance. The dogma of an exclusively retinal distribution has arisen because Northern blot analyses have previously shown only modest hybridisation in non-retinal tissues. Others have re-interpreted this as indicating that EAAT5 was only present in retinal tissues. However, this view appears to be erroneous; recent evidence demonstrating abundant expression of EAAT5 in rat testis prompted us to re-examine this dogma. A new antibody was developed to an intracellular loop region of rat EAAT5. This new tool, in concert with RT-PCR and sequencing, demonstrated that EAAT5 is widely distributed at the mRNA and protein levels in many non-nervous tissues including liver, kidney, intestine, heart, lung, and skeletal muscle. We conclude that EAAT5 is a widely distributed protein. Whether it functions in all locations as a glutamate transporter, or mainly as a glutamate-gated chloride conductance, remains to be determined.

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Conflict of interest statement

Conflict of interests: the authors declare no conflict of interests.

Figures

Figure 1
Figure 1
Tissue distribution of EAAT5 mRNA. A) RNA from various rat tissues (as indicated) were reverse transcribed and used in PCR with EAAT5 (lower bands) and β-actin (upper bands) primers. An aliquot of each PCR was electrophoresed on a 2% agarose gel and visualised with ethidium bromide staining. A water control (blank) is shown. B) Densitometric analysis of EAAT5 amplification products from three separate experiments; data are shown as mean ± SD.
Figure 2
Figure 2
The EAAT5 loop epitope. A) Schematic representation of wild-type EAAT5 secondary structure with exon boundaries overlayed onto the predicted topology; an antibody was generated against an intracellular epitope of EAAT5 (as indicated) corresponding to a region encoded by exon 6 of the EAAT5 gene. B) Alignment of the EAAT5 epitope sequence against that of other EAAT members (GLAST, GLT-1a, EAAC1 and EAAT4); identical residues are highlighted in grey.
Figure 3
Figure 3
Characterization of EAAT5 loop antibody. (A) Dot blot showing the antibody against an epitope encoded by exon 6 of EAAT5 selectively detected the immunising peptide (1) but not other irrelevant peptides (2 and 3). (B) HEK293 cells were transfected with plasmids encoding GLAST, GLT-1a or EAAT5. Cell extracts were resolved by SDS-PAGE and immunoblotted with the EAAT5 loop antibody, an antibody directed the Cterminal portion of EAAT5 (C'-EAAT5), or antibodies directed against GLAST or GLT- 1a. Both the EAAT5 loop antibody and C'-EAAT5 antibody detected a band of ∼62 kDa in EAAT5 transfected cells but not in untransfected cells (UT) nor in cells transfected with GLAST or GLT-1a.
Figure 4
Figure 4
Western blot demonstrating expression of EAAT5 in various rat tissues (as indicated). A ∼62 kDa band (corresponding to wild-type EAAT5) is labelled in retina (control) and all other tissues examined. Additional smaller bands (presumed to represent tissue-specific alternate splicing of EAAT5) are also evident in retina, liver, kidney and heart. All lanes were loaded with ∼20 µg of total lysate with exception of kidney (∼60 µg input).
Figure 5
Figure 5
Immunolabelling for EAAT5 in retina (A and B), kidney (C), small intestine (D), lung (E), skeletal muscle (F) and heart (G). In retina (A) labelling is associated with somata of photoreceptors (p) as well as their outer segments (os) and inner segments (is). Strong labelling is associated with the synaptic terminals of the photoreceptors in the outer plexiform layer (OPL). Labelling is similarly evident in somata of bipolar cells (b) and processes in the inner plexiform layer (IPL). Ganglion cell somata in the ganglion cell layer (gcl) are also labelled. (B) arrow indicates labelling of photoreceptor synaptic terminals. In kidney (C), heaviest labelling is in the outer stripe (os) of the cortex, with lesser labelling in the inner stripe (is); labelling was absent from the medulla (m). In the intestine (D), labelling was associated with goblet cells (g) whilst in lung (E), labelling was associated with epithelia of the alveoli. In skeletal muscle (F) and heart (G), labelling was around the periphery of muscle fibres. Scale bars: A, C, F, G, 25 µm; B, 500 µm; D, 50 µm; E, 10 µm.
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