SNAT2 Amino Acid Transporter Is Regulated by Amino Acids of the SLC6 γ-Aminobutyric Acid Transporter Subfamily in Neocortical Neurons and May Play No Role in Delivering Glutamine for Glutamatergic Transmission

Abstract

System A transporters SNAT1 and SNAT2 mediate uptake of neutral ␣-amino acids (e.g. glutamine, alanine, and proline) and are expressed in central neurons. We tested the hypothesis that SNAT2 is required to support neurotransmitter glutamate synthesis by examining spontaneous excitatory activity after inducing or repressing SNAT2 expression for prolonged periods. We stimulated de novo synthesis of SNAT2 mRNA and increased SNAT2 mRNA stability and total SNAT2 protein and functional activity, whereas SNAT1 expression was unaffected. Increased endogenous SNAT2 expression did not affect spontaneous excitatory action-potential frequency over control. Long term glutamine exposure strongly repressed SNAT2 expression but increased excitatory actionpotential frequency. Quantal size was not altered following SNAT2 induction or repression. These results suggest that spontaneous glutamatergic transmission in pyramidal neurons does not rely on SNAT2. To our surprise, repression of SNAT2 activity was not limited to System A substrates. Taurine, ␥-aminobutyric acid, and ␤-alanine (substrates of the SLC6 ␥-aminobutyric acid transporter family) repressed SNAT2 expression more potently (10؋) than did System A substrates; however, the responses to System A substrates were more rapid. Since ATF4 (activating transcription factor 4) and CCAAT/enhancer-binding protein are known to bind to an amino acid response element within the SNAT2 promoter and mediate induction of SNAT2 in peripheral cell lines, we tested whether either factor was similarly induced by amino acid deprivation in neurons. We found that glutamine and taurine repressed the induction of both transcription factors. Our data revealed that SNAT2 expression is constitutively low in neurons under physiological conditions but potently induced, together with the taurine transporter TauT, in response to depletion of neutral amino acids.Central neurons express the sodium-coupled neutral amino acid transporters SNAT1 and SNAT2 (also known as SLC38A1, GlnT, SAT1, or ATA1 and SLC38A2, SAT2, or ATA2, respectively) (1-10), two of the three SLC38 gene family members (reviewed in Ref. 11) that collectively account for the System A amino acid transport activity classically described in most cell types (12, 13). System A catalyzes the unidirectional uptake of small aliphatic neutral amino acids, especially alanine, cysteine, glutamine, glycine, methionine, proline, and serine. Among these, glutamine is around 10-fold more abundant than any other amino acid in extraneuronal space (14 -16), which, combined with the high affinity of System A transporters for glutamine (K m ϭ 0.2-0.5 mM), makes glutamine the predominant substrate for System A in neurons of the brain. Glutamine, in addition to glucose, is a major precursor for glutamate (17), and many neuronal cell bodies in the brain express phosphate-activated glutaminase (18,19) and contain high levels of glutamate. Whereas System N transporters may serve the exodus of glutamine from the astrocyte (9,20,21), these observations present the ...

Dysregulation of Glutamine Transporter SNAT1 in Rett Syndrome Microglia: A Mechanism for Mitochondrial Dysfunction and Neurotoxicity

“…qPCR showed that the SNAT1 transcript level in Mecp2Ϫ/y microglia was 148 Ϯ 0.07% (n ϭ 5, p Ͻ 0.001) of that in Mecp2ϩ/y microglia. Western blot of primary microglia samples showed a cluster of SNAT1-immunoreactive bands centered ϳ55 kDa, consistent with published data (Varoqui et al, 2000;Grewal et al, 2009). SNAT1 protein levels were approximately three times more abundant in Mecp2Ϫ/y than in Mecp2ϩ/y microglia ( Fig.…”

“…The following primary antibodies (dilutions) were used: anti-MeCP2 (1: 1000; Millipore), anti-NDUFA7 (1:1000; Abcam), anti-mitofilin (1: 1000; Affinity Bioreagents), anti-GAPDH (1:3000; Millipore), and anti-␤-actin (1:1000; Abcam). The well characterized, highly specific polyclonal anti-SNAT1 was described previously (Varoqui et al, 2000;Grewal et al, 2009) and was used in 1:1000 dilution.…”

C/EBPβ and C/EBPδ transcription factors: Basic biology and roles in the CNS

“…These data suggest that C/EBPβ is a neuronal activity-dependent gene. As it will be discussed later, C/EBPβ is pleiotropic in neurons and the observations that neuronal C/EBPβ levels increase upon aminoacid deprivation (Grewal et al 2009) …”

“…Some examples are provided below but the list cannot be exhaustive. Various genes involved in neurotransmission are C/EBPβ targets including neurotransmitter synthesis enzymes such as choline acetyltransferase (Robert et al 2002) or histidine decarboxylase (Cortes-Canteli et al 2004), neurotransmitter precursors such as preprotachykinin-A (Kovacs et al 2006) or the aminoacid transporter SNAT2 which is used by neurons to take up Gln when in need of increased Glu synthesis (Grewal et al 2009). C/EBPβ targets also include neurotransmitter receptors such as metabotropic glutamate receptor 1 (Crepaldi et al 2007) or the pain-related purinergic receptor P2X3 (Ugarte et al 2012), ion channels like TRPV1 (Ugarte et al 2013) or proteins involved in receptor and ion channel trafficking like sorting nexin 27 (Wang et al 2013b).…”

A Conserved Na+ Binding Site of the Sodium-coupled Neutral Amino Acid Transporter 2 (SNAT2)

“…This process is essential for recycling the neurotransmitter glutamate (13). However, the exact contribution of SNAT2 to the glutamate-glutamine cycle is still controversially discussed (14).…”