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David V. Pow, Aven Lee, Ashley R. Anderson, Nigel L. Barnett, Melissa G. Stevens; "big Eaat5" A Novel Glutamate Transporter In The Human Retina?. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6544.
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EAAT5 is a glutamate transporter that is expressed by bipolar neurons and photoreceptors in the retina. EAAT5 has previously been thought to exist only as one form. This study sought to determine if EAAT5 might be alternately spliced.
Human retinas (n=6) from donors aged beteween44 and 88 years of age were obtained from the Queensland eye bank. PCR was used to amplify the entire coding region of EAAT5. Bands of differing sizes were isolated on agarose gels, inserted into plasmids and transformed into E. Coli. Plasmid DNA was subsequently extracted, the inserts excised and positive clones were sequenced in both directions. Sequence data was subsequently used to facilitate production of a specific antibody against a synthetic peptide.
We demonstrate the presence of an abundant alternately spliced form of EAAT5 in the human retina, which contains an insertion between exons 7 and 8. This form represents about a third of all EAAT5 mRNA in the human retina. An antibody was raised against a unique sequence in the insert between exons 7 and 8. This antibody demonstrated that this novel form of EAAT5 (which we describe as "Big EAAT5") was abundantly expressed at the protein level as assessed by Western blotting. Analysis of retinas from donors of differing ages suggested that there was no overt change in Big EAAT5 expression relative to full length EAAT5 expression with age
Based upon key structural features we propose that this variant form is likely to be a functional glutamate transporter but may potentially exhibit distinct properties relative to the originally described form of EAAT5.
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