A new splice variant of the glutamate–aspartate transporter: Cloning and immunolocalization of GLAST1c in rat, pig and human brains

Abstract

GLAST (EAAT1) is an abundant glial glutamate transporter in the mammalian brain. It plays important roles in terminating excitatory transmission in grey matter, as well as pathophysiological roles, including protecting white matter from excitotoxic injury. In normal brain, alternative splicing of GLAST has been described: GLAST1a and GLAST1b arise from the splicing out of exons 3 and 9, respectively. This study describes the isolation of a novel cDNA clone from neonatal hypoxic pig brain, referred to as GLAST1c, where exons 5 and 6 are skipped. GLAST1c encodes a protein of 430 amino acids. RT-PCR analysis showed that GLAST1c mRNA was readily detectable in control and hypoxic pig cortex, as well as in various brain regions of rat (cortex, mid, hind and cerebellum), and human cortex, retina and optic nerve. We have raised antibodies that selectively recognize GLAST1c and demonstrate expression of this novel splice variant in astrocytes and oligodendrocytes in rat brain, pig brain and human brain, including grey and white matter. Similarly expression of GLAST1c was observed in primary astrocyte cultures and in cultured oligodendrocytes. In unstimulated astrocytes GLAST1c exhibited an intracellular peri-nuclear distribution similar to that observed when GFP-tagged GLAST1c was transfected into COS 7 cells. In astrocytes this protein rapidly redistributed to the surface upon stimulation of protein kinase with phorbol esters. We conclude that GLAST1c may represent an astrocyte and oligodendrocyte glutamate transporter, though this could not be formally validated by d-aspartate uptake studies, due to the low transfection efficiency of constructs into COS 7 cells.