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D. V. Pow, A. Lee; Cloning and Identification of Multiple New Variant Forms of the Predominant Photoreceptor Glutamate Transporter EAAT5. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4138.
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© ARVO (1962-2015); The Authors (2016-present)
EAAT5 is the predominant glutamate transporter used by photoreceptors in the retina to recover glutamate released by their synaptic terminals, the associated chloride conductance potentially having a regulatory effect on glutamate release due to its effect on membrane polarisation. The study investigated whether multiple forms of EAAT5 existed.
Western blotting was performed using an antibody against the amino terminal of EAAT5. PCR analysis was performed using primers flanking the coding region of EAAT5 Multiple bands were identified. Bands were excised, inserted into plasmids, expanded in E.Coli and 50 clones sequenced. Deduced peptide sequences were subsequently used to make antibodies against newly identified variants of EAAT5.
Western blotting revealed bands corresponding to wild type EAAT5 and 5 smaller bands, suggestive of splice variants. PCR identified wild type EAAT5 and 5 splice variant forms which skipped, either completely or partially various exons, including exon 3, exon 7 exon 8, exon 9 and exon 10. The exon 8- and exon 10-skipping forms generated frame shifts that should lead to truncated proteins. Specific antibodies have been raised against exon 3 and exon 7 skipping forms and others are being characterised.
Exon-skipping forms of EAAT5 exist in the retina. Exon skipping forms of other EAATs have been implicated in the pathogenesis of some CNS diseases. Accordingly we are currently investigating the properties of these new variant forms of EAAT5 to determine if they might influence glutamate transport properties or exhibit disparities in their chloride conductances, which might influence retinal function in healthy and diseased retina
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