Cloning, transport properties, and differential localization of two splice variants of GLT‐1 in the rat CNS: Implications for CNS glutamate homeostasis

Abstract

At least two splice variants of GLT-1 are expressed by rat brain astrocytes, albeit in different membrane domains. There is at present only limited data available as to the spatial relationship of such variants relative to the location of synapses and their functional properties. We have characterized the transport properties of GLT-1v in a heterologous expression system and conclude that its transport properties are similar to those of the originally described form of GLT-1, namely GLT-1alpha. We demonstrate that GLT-1alpha is localized to glial processes, some of which are interposed between multiple synapse types, including GABAergic synapses, whereas GLT-1v is expressed by astrocytic processes, at sites not interposed between synapses. Both splice variants can be expressed by a single astrocyte, but such expression is not uniform over the surface of the astrocytes. Neither splice variant of GLT-1 is evident in brain neurons, but both are abundantly expressed in some retinal neurons. We conclude that GLT-1v may not be involved in shaping the kinetics of synaptic signaling in the brain, but may be critical in preventing spillover of glutamate between adjacent synapses, thereby regulating intersynaptic glutamatergic and GABAergic transmission. Furthermore, GLT-1v may be crucial in ensuring that low levels of glutamate are maintained at extrasynaptic locations, especially in pathological conditions such as ischemia, motor neurone disease, and epilepsy.

Expression of multiple glutamate transporter splice variants in the rodent testis

“…[21][22][23] To study the expression of GLAST1a, GLAST1b, GLT1b and GLT1c in testis, PCR primers specific for each splice variant were synthesized (Table 1).…”

“…22,24 Briefly, testes were fixed with 4% paraformaldehyde in 0.1 mol l 21 sodium phosphate buffer, then dehydrated through a graded series of water/ ethanol solutions, cleared in xylene and embedded in paraffin wax. 19 Serial sections (8 mm in thickness) were cut on a Leica rotary microtome and mounted onto silanated microscope slides.…”

Oxidative and excitotoxic insults exert differential effects on spinal motoneurons and astrocytic glutamate transporters: Implications for the role of astrogliosis in amyotrophic lateral sclerosis

“…Proteins were transferred to nitrocellulose membranes, which were blocked with 5% skim milk for 2 h and incubated overnight at 4°C with specific rabbit primary antibodies directed against either EAAT1 (1:10,000) or EAAT2 (1:50,000). The rabbit primary antibodies against EAAT1 and EAAT2 were kind gifts from Professors Niels C. Danbolt (University of Oslo, Norway; EAAT1 antibody as described earlier) and David V. Pow (The University of Newcastle, Australia; EAAT2 antibody directed against the amino acid residues 12-26 as described in Sullivan et al, 2004), respectively. Following washing, membranes were incubated with HRP-conjugated goat anti-rabbit secondary antibodies (1:1,000) for 3 h. Proteins were visualized using enhanced chemiluminescence.…”

EAAT2 regulation and splicing: relevance to psychiatric and neurological disorders

“…The sequence that makes up exon 11 in GLT1 is part of the 3 0 -UTR of GLT1b and GLT1c. 24,26 suggesting that any functional difference between the two isoforms likely involves different cellular localization patterns or participation in different signaling pathways. A third functional GLT1 variant encoding yet another novel C-terminus, referred to as GLT1c, was recently identified in the rat and human retina.…”

“…Another study demonstrated that GLT1 was expressed in glial membranes that interdigitated between synapses, whereas GLT1b was excluded from the tips of astrocytes between synapses. 26 These investigators suggested that GLT1b may be important in controlling extrasynaptic glutamate but may not regulate glutamate uptake at the synapse. A subsequent report by an independent group confirmed that GLT1 mRNA is present in astrocyte processes, whereas GLT1b mRNA is found primarily in the cell bodies.…”