Cloning and Functional Identification of a Neuronal Glutamine Transporter

Abstract

Glutamine is the preferred precursor for the neurotransmitter pool of glutamate, the major excitatory transmitter in the mammalian central nervous system. We have isolated a complementary DNA clone (designated GlnT) encoding a plasma membrane glutamine transporter from glutamatergic neurons in culture, and its properties have been examined using the T7 vaccinia system in fibroblasts. When GlnT is transfected into CV-1 cells, L-glutamine is the preferred substrate. Transport is Na ؉ -dependent and inhibited by ␣-methylaminoisobutyric acid, a specific inhibitor of neutral amino acid transport system A. Kinetic analysis of glutamine uptake by GlnT is saturable, with a Michaelis constant (K m ) of 489 ؎ 88 M at pH 7.4. Glutamine uptake mediated by GlnT is pH-sensitive with a 5-fold greater efficiency of uptake at pH 8.2 than at pH 6.6. Only the maximal velocity of transport increases without a significant change in K m . The distribution of GlnT mRNA and protein in the central nervous system is widespread and is expressed on neurons that use glutamate as their neurotransmitter. In cultured cerebellar granule cells, GlnT is expressed only on neurons and is absent from astrocytes. GlnT expression increases concomitantly with the morphologic and functional differentiation of these cells in vitro, consistent with its role of supplying glutamatergic neurons with their neurotransmitter precursor. GlnT is the first member of the system A family of neutral amino acid transporters with 11 putative membrane-spanning domains and is a potential target to modulate presynaptic glutamatergic function.

Cloning of an Amino Acid Transporter with Functional Characteristics and Tissue Expression Pattern Identical to That of System A

“…This transporter, designated ATA2, is distinct from the recently cloned brain-specific glutamine transporter GlnT (12). Because GlnT was the first cloned amino acid transporter with the ability to transport the system A-specific model substrate MeAIB, we identify GlnT as ATA1 in this paper.…”

“…The transport activity of ATA2 increases markedly when the extracellular pH is higher than 7.5 and decreases markedly when the extracellular pH is lower than 7.5. ATA1 also exhibits this characteristic (12). Based on the amino acid sequence, ATA1 and ATA2 are related to the recently cloned amino acid transport system N (SN1), a Na ϩ -and H ϩ -coupled glutamine transporter (22).…”

Glutamine Uptake by Neurons: Interaction of Protons with System A Transporters

“…We have recently found that the currents associated with SN1 are largely if not entirely uncoupled from transport (F. A. Chaudhry, M. Kavanaugh, and R. H. Edwards, unpublished observations), raising the possibility that currents associated with closely related System A transporters are also uncoupled (Reimer et al, 2000;Sugawara et al, 2000;Varoqui et al, 2000;Yao et al, 2000). To reassess the relationship of currents to transport by both SA1 and SA2, we first measured uptake of the prototypic System A substrate 3 H-MeAIB.…”

“…Together with its expression on astrocytes, these observations suggest that SN1 confers the efflux of glutamine from glia required to sustain the glutamineglutamate cycle. SN1 also shows strong sequence similarity to the proteins responsible for another classical amino acid transport system, System A (Reimer et al, 2000;Sugawara et al, 2000;Varoqui et al, 2000;Yao et al, 2000).…”

Brain edema in acute liver failure and chronic liver disease: Similarities and differences

“…Briefly, the glutamate released into the synaptic space by presynaptic neurons induces post-synaptic signaling through glutamate receptors, and is cleared by astrocytic excitatory amino acid transporters (EAAT1 and EAAT2, (Gegelashvili et al, 2007 andRothstein et al, 1994)), and then converted into glutamine through GS. The non-neuroactive glutamine is released into the extracellular space through astrocyte glutamine transporters (N and ASC-system transporter SN-1, SN-2 and ASCT2 (Chaudhry et al, 1999, Cubelos et al, 2005and Dolińska et al, 2004), and taken up by neurons (sodium-coupled amino acid transporter, SAT/ATA (Varoqui et al, 2000)), where it is converted back to glutamate via the action of PAG, in order to replenish the neurotransmitter pool. Therefore, astrocytic glutamate uptake prevents glutamate accumulation in the synaptic cleft and over-excitation of the postsynaptic neuronal receptors (Fig.…”