Alternate splicing and expression of the glutamate transporter EAAT5 in the rat retina.

Excitatory amino acid transporter 5 (EAAT5) is an unusual glutamate transporter that is expressed in the retina, where it is localised to two populations of glutamatergic neurons, namely the bipolar neurons and photoreceptors. EAAT5 exhibits two distinct properties, acting both as a slow glutamate transporter and as a glutamate-gated inhibitory receptor. The latter property is attributable to a co-associated chloride conductance. EAAT5 has previously been thought to exist only as a full-length form. We now demonstrate by PCR cloning and sequencing, the presence of five novel splice variant forms of EAAT5 which skip either partial or complete exons in the rat retina. Furthermore, we demonstrate that each of these variants is expressed at the protein level as assessed by Western blotting using splice-specific antibodies that we have generated. We conclude that EAAT5 exists in multiple spliced forms, and propose, based upon retention or absence of key structural features, that these variant forms may potentially exhibit distinct properties relative to the originally described form of EAAT5.

Localisation of novel forms of glutamate transporters and the cystine-glutamate antiporter in the choroid plexus: Implications for CSF glutamate homeostasis.

The choroid plexus is a structure within each ventricle of the brain that is composed of fenestrated vessels surrounded by secretory epithelial cells. The epithelial cells are linked by tight junctions to create a permeability barrier. The epithelial cells are derived from neuroectoderm, and are thus defined by some authors as a subtype of macroglia. Glutamate is a tightly regulated substance in the CSF, as it is in the rest of the brain. In the brain macroglia express multiple sodium dependent and independent glutamate transporters and are the main regulators of extracellular glutamate. However, the identities of the transporters in the choroid plexus and their localisations have remained poorly defined. In this study we examined the expression and distribution of multiple splice variants of classical sodium-dependent glutamate transporters, as well as the cystine-glutamate antiporter, and the PDZ protein NHERF1, (which acts as a molecular anchor for proteins such as the glutamate transporter GLAST). We identified three forms of sodium-dependent transporters (GLAST1a, GLAST1c and GLT1b) that are expressed at the apical surface of the epithelial cells, a location that matches the distribution of NHERF1 and the cystine-glutamate antiporter. We propose that this coincident localisation of GLAST1a/GLAST1c/GLT1b and the cystine-glutamate antiporter would permit the cyclical trafficking of glutamate and thus optimise the accumulation of cystine for the formation of glutathione in the choroid plexus.

Exon 4-skipping GLT-1: a new form of an abundantly expressed glutamate transporter.

GLT-1 (EAAT2) is an abundant glial glutamate transporter in the mammalian brain. It plays important roles, especially in the termination of neurotransmitter signals at excitatory synapses in grey matter. In normal brain, alternative splicing of GLT-1 has been described, where exons in the GLT-1 gene are skipped or intronic sequences spliced in to generate new sequences. This study describes the isolation of a cDNA clone encoding a new splice variant of GLT-1 where exon 4 is skipped. This novel variant was isolated by RT-PCR cloning from adult rat brain and encodes a protein of 500 amino acids (MW ~54.5 kDa). RT-PCR analysis showed that mRNA was readily detectable in various brain regions of rat, primary astrocyte cultures and in tissues such as testis, but little mRNA was detectable in retina and liver. An antibody that selectively recognizes exon-4 skipping GLT-1 revealed strong signals in Western blots and labelled grey matter astrocytes. We conclude that exon-4 skipping GLT 1 is abundantly expressed in the brain and may represent either a functional glutamate transporter or a modulator of glutamate transporter function.