Functional Properties and Cellular Distribution of the System A Glutamine Transporter SNAT1 Support Specialized Roles in Central Neurons

Abstract

Glutamine, the preferred precursor for neurotransmitter glutamate and GABA, is likely to be the principal substrate for the neuronal System A transporter SNAT1 in vivo. We explored the functional properties of SNAT1 (the product of the rat Slc38a1 gene) by measuring radiotracer uptake and currents associated with SNAT1 expression in Xenopus oocytes and determined the neuronal-phenotypic and cellular distribution of SNAT1 by confocal laser-scanning microscopy alongside other markers. We found that SNAT1 mediates transport of small, neutral, aliphatic amino acids including glutamine (K 0.5 Ϸ 0.3 mM), alanine, and the System A-specific analogue 2-(methylamino)isobutyrate. Amino acid transport is driven by the Na ؉ electrochemical gradient. The voltage-dependent binding of Na ؉ precedes that of the amino acid in a simultaneous transport mechanism. Li ؉ (but not H ؉ ) can substitute for Na ؉ but results in reduced V max . In the absence of amino acid, SNAT1 mediates Na ؉ -dependent presteady-state currents (Q max Ϸ 9 nC) and a nonsaturable cation leak with selectivity Na ؉ , Li ؉ > > H ؉ , K ؉ . Simultaneous flux and current measurements indicate coupling stoichiometry of 1 Na ؉ per 1 amino acid. SNAT1 protein was detected in somata and proximal dendrites but not nerve terminals of glutamatergic and GABAergic neurons throughout the adult CNS. We did not detect SNAT1 expression in astrocytes but detected its expression on the luminal membranes of the ependyma. The functional properties and cellular distribution of SNAT1 support a primary role for SNAT1 in glutamine transport serving the glutamate/GABA-glutamine cycle in central neurons. Localization of SNAT1 to certain dopaminergic neurons of the substantia nigra and cholinergic motoneurons suggests that SNAT1 may play additional specialized roles, providing metabolic fuel (via ␣-ketoglutarate) or precursors (cysteine, glycine) for glutathione synthesis.

Transport model of the human Na+-coupled l-ascorbic acid (vitamin C) transporter SVCT1

“…3D), indicating that the second Na ϩ must bind after the ascorbate-binding step. In contrast, considering a carrier model in dynamic equilibrium, we should expect that saturating L-ascorbic acid would always drive the transporter at its maximal rate were L-ascorbic acid bound last (11,17,19,31). Our data are not consistent with a random-binding model in which case I max for either substrate would be independent of the cosubstrate concentration.…”

“…Maximal charge (Q max ) was conserved upon progressive removal of Na ϩ , whereas the voltage midpoint (V 0.5 ) shifted to more hyperpolarized potentials. Pre-steady-state currents have been observed for several other Na ϩ -coupled (1,9,14,17,19,27) or H ϩ -coupled (8,12,16,21,22,25) solute transporters. The results of model simulation have led investigators to attribute pre-steady-state currents to two steps in the transport cycle, namely reorientation of the empty charged carrier and binding/dissociation of the driving ion partway within the plane of the membrane electric field (and in some cases also to the binding/dissociation of the ion at the internal face).…”

“…B: comparison of Q Asc (gray bar) and 22 Na ϩ accumulation (filled bar) over 5 min in oocytes expressing SVCT1 (mean Ϯ SD, n ϭ 6) superfused with 50 mM 22 Na ϩ plus 1 mM L-ascorbic acid, after subtraction of Q Asc (19.3 Ϯ 5.7 pmol) and 22 Na ϩ accumulation (157 Ϯ 294 pmol) measured in control oocytes (n ϭ 3). The Na ϩ /Q Asc ratio was 2.0 Ϯ 0. higher concentrations of the cosubstrate (11,17,19,31). In contrast, for a consecutive carrier model, the K 0.5 values are expected to rise as cosubstrate concentration is increased.…”

“…We determined the Na ϩ -L-ascorbic acid coupling stoichiometry by measuring radiotracer fluxes under voltage clamp (Ϫ50 mV), a method we have described elsewhere for other Na ϩ -coupled transporters (15,18,19,36). We used L-[1-14 C]ascorbic acid at final specific activity 0.3-3 GBq/mmol and 22 Na at a final specific activity 0.34 MBq/mg (both from Perkin-Elmer Life Sciences).…”

A Critical Role for System A Amino Acid Transport in the Regulation of Dendritic Development by Brain-derived Neurotrophic Factor (BDNF)

“…SNAT1 is found primarily in the brain, SNAT2 is expressed ubiquitously in mammalian tissues, and SNAT4 is restricted almost exclusively to the liver (8). A role has been proposed for SNAT1 in supplying glutamatergic and GABAergic neurons with glutamine, the preferred precursor for the synthesis of the neurotransmitters glutamate and GABA (9,10). However, recent data have revealed that SNAT1 is not observed in axon terminals but is localized almost exclusively to the somatodendritic compartment of glutamatergic and GABAergic neurons (11), indicating that the physiological role of SNAT1 in the brain remains controversial.…”