GLAST/EAAT1‐induced Glutamine release via SNAT3 in Bergmann glial cells: evidence of a functional and physical coupling

Abstract

Glutamate, the major excitatory transmitter in the vertebrate brain, is removed from the synaptic cleft by a family of sodiumdependent glutamate transporters profusely expressed in glial cells. Once internalized, it is metabolized by glutamine synthetase to glutamine and released to the synaptic space through sodium-dependent neutral amino acid carriers of the N System (SNAT3/slc38a3/SN1, SNAT5/slc38a5/SN2). Glutamine is then taken up by neurons completing the so-called glutamate/glutamine shuttle. Despite of the fact that this coupling was described decades ago, it is only recently that the biochemical framework of this shuttle has begun to be elucidated. Using the established model of cultured cerebellar Bergmann glia cells, we sought to characterize the functional and physical coupling of glutamate uptake and glutamine release. A time-dependent Na + -dependent glutamate/aspartate transporter/EAAT1-induced System N-mediated glutamine release could be demonstrated. Furthermore, D-aspartate, a specific glutamate transporter ligand, was capable of enhancing the co-immunoprecipitation of Na + -dependent glutamate/aspartate transporter and Na + -dependent neutral amino acid transporter 3, whereas glutamine tended to reduce this association. Our results suggest that glial cells surrounding glutamatergic synapses may act as sensors of neuron-derived glutamate through their contribution to the neurotransmitter turnover. Keywords: Bergmann glia, glutamate transporters, glutamate/ glutamine shuttle, glutamine transporters. EAAT 1-5, excitatory amino acid transporters; ECL, enhanced chemiluminescence reagent; EDTA, ethylenediaminetetraacetic acid; GLAST, Na + -dependent glutamate/aspartate transporter; Gln, glutamine; Glt-1, glutamate transporter-1; GS, glutamine synthetase; KA, kainate; KBP, kainate binding protein; L-AP4, L-(+)-2-amino-4-phosphonobutyric acid; L-Glu, L-glutamate; NMDA, N-methyl-D-aspartate; NP-40, Nonidet P-40; PBS, phosphate-buffer saline; PBT, phosphate-buffer saline containing 0.3% Triton; SDS-PAGE, sodium dodedecyl sulfate polyacrylamide gel electrophoresis; SNAT2-3-5, Na + -dependent neutral amino acid transporter 2-3-5; TBS, Tris-buffered saline; THA, threo-b-hydroxyaspartate; trans-ACPD, (+/-)-1-aminocyclopentane-trans-1,3-dicarboxilic acid.

Protein Kinase C Phosphorylates the System N Glutamine Transporter SN1 (Slc38a3) and Regulates Its Membrane Trafficking and Degradation

“…Subsequently, such glutamine release is sensed by MNTB principal neurons which express system A transporters consistent with an intact glutamate/GABA-glutamine cycle (63, 64). Similarly, studies on cultured Bergmann glia cells show that activation of glutamate transporters by d -aspartate results in release of glutamine (65). Altogether, these data strongly suggest functional coupling between the glutamate and glutamine transporters and existence of a glutamate/GABA-glutamine cycle.…”

Activation of sodium‐dependent glutamate transporters regulates the morphological aspects of oligodendrocyte maturation via signaling through calcium/calmodulin‐dependent kinase IIβ's actin‐binding/‐stabilizing domain

“…This observation led to the idea that glutamate homeostasis may be tightly regulated at the zones where axons and oligodendrocyte processes meet and in a fashion that may be analogous to what has been described for the tripartite synapse (Arranz et al 2008). At the tripartite synapse, a so called “glutamate/glutamine shuttle” has been described (Martinez-Lozada et al 2013; Rodriguez and Ortega 2012; Uwechue et al 2012) in which, upon its uptake, glutamate is converted to glutamine by the enzymatic activity of glutamine synthetase. Glutamine is then released via sodium-dependent neutral amino acid transporters to be re-taken up by neurons and to serve as a precursor for glutamate synthesis.…”

Astroglial glutamate transporters coordinate excitatory signaling and brain energetics

“…A variety of hypothesis-driven or yeast two-hybrid approaches were used to identify proteins that co-immunoprecipitate with one or more of the Na + -dependent glutamate transporters, including Ajuba (Marie et al, 2002), glutamate transporter associated proteins (GTRAPs) (Jackson et al, 2001; Lin et al, 2001), protein kinase Cα (González et al, 2003; González et al, 2005), a septin GTPase (Sept 2) (Kinoshita et al, 2004), syntaxin 1A (Yu et al, 2006), PSD-95 (Gonzalez-Gonzalez et al, 2008; Gonzalez-Gonzalez et al, 2009), the actin binding protein α-adducin (Bianchi et al, 2010), Na + /H + exchanger regulatory proteins (Lee et al, 2007; Sato et al, 2013), protein interacting with C kinase (PICK1) (Bassan et al, 2008), membrane-associated guanylate kinase with inverted orientation protein-1 (MAGI-1) (Zou et al, 2011), a glutamine transporter (SNAT3) (Martinez-Lozada et al, 2013), and the sodium/calcium exchanger (NCX1) (Magi et al, 2012; Magi et al, 2013). Many of these proteins directly or indirectly regulate various aspects of transporter function, including maturation, targeting, trafficking, or functional coupling of these transporters.…”