A novel splice variant of the Excitatory Amino Acid Transporter 5: Cloning, immunolocalization and functional characterization of hEAAT5v in human retina

• Published in: Neurochemistry international 2016-12, Vol.101, p.76-82
• Main Authors: Lee, A., Stevens, M.G., Anderson, A.R., Kwan, A., Balcar, V.J., Pow, D.V.
• Format: Article
• Language: English
• Subjects: Alternate splicing; EAAT5; Glutamate; Photoreceptors; Retina; Transporter
• ISSN: 0197-0186; 1872-9754
• DOI: 10.1016/j.neuint.2016.10.013

Excitatory Amino Acid Transporter 5 (EAAT5) is abundantly expressed by retinal photoreceptors and bipolar cells, where it acts as a slow glutamate transporter and a glutamate-gated chloride channel. The chloride conductance is large enough for EAAT5 to serve as an “inhibitory” glutamate receptor. Our recent work in rodents has shown that EAAT5 is differentially spliced and exists in many variant forms. The chief aim of the present study was to examine whether EAAT5 is also alternately spliced in human retina and, if so, what significance this might have for retinal function in health and disease. Retinal tissues from human donor eyes were used in RT-PCR to amplify the entire coding region of EAAT5. Amplicons of differing sizes were sub-cloned and analysis of sequenced data revealed the identification of wild-type human EAAT5 (hEAAT5) and an abundant alternately spliced form, referred to as hEAAT5v, where the open reading frame is expanded by insertion of an additional exon. hEAAT5v encodes a protein of 619 amino acids and when expressed in COS7 cells, the protein functioned as a glutamate transporter. We raised antibodies that selectively recognized the hEAAT5v protein and have performed immunocytochemistry to demonstrate expression in photoreceptors in human retina. We noted that in retinas afflicted by dry aged-related macular degeneration (AMD), there was a loss of hEAAT5v from the lesioned area and from photoreceptors adjacent to the lesion. We conclude that hEAAT5v protein expression may be perturbed in peri-lesional areas of AMD-afflicted retinas that do not otherwise exhibit evidence of damage. The loss of hEAAT5v could, therefore, represent an early pathological change in the development of AMD and might be involved in its aetiology. •We have identified a novel splice variant of EAAT5 in human retina, referred to as hEAAT5v which contain an additional exon.•hEAAT5v is a functional glutamate transporter and abundantly expressed in photoreceptors.•In retinas afflicted with AMD, hEAAT5v is lost from the lesioned area and from photoreceptors adjacent to the lesion.•Loss of hEAAT5v may represent an early pathological change in the development of AMD and might be involved in its aetiology.