A Novel System A Isoform Mediating Na+/Neutral Amino Acid Cotransport

Abstract

A cDNA clone encoding a plasma membrane alaninepreferring transporter (SAT2) has been isolated from glutamatergic neurons in culture and represents the second member of the system A family of neutral amino acid transporters. SAT2 displays a widespread distribution and is expressed in most tissues, including heart, adrenal gland, skeletal muscle, stomach, fat, brain, spinal cord, colon, and lung, with lower levels detected in spleen. No signal is detected in liver or testis. In the central nervous system, SAT2 is expressed in neurons. SAT2 is significantly up-regulated during differentiation of cerebellar granule cells and is absent from astrocytes in primary culture. The functional properties of SAT2, examined using transfected fibroblasts and in cRNA-injected voltage-clamped Xenopus oocytes, show that small aliphatic neutral amino acids are preferred substrates and that transport is voltage-and Na ؉ -dependent (1:1 stoichiometry), pH-sensitive, and inhibited by ␣-(methylamino)isobutyric acid (MeAIB), a specific inhibitor of system A. Kinetic analyses of alanine and MeAIB uptake by SAT2 are saturable, with Michaelis constants (K m ) of 200 -500 M. In addition to its ubiquitous role as a substrate for oxidative metabolism and a major vehicle of nitrogen transport, SAT2 may provide alanine to function as the amino group donor to ␣-ketoglutarate to provide an alternative source for neurotransmitter synthesis in glutamatergic neurons.

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

“…Expression of SA2 using a vaccinia virus-T7 polymerase system confers increased uptake of 3 H-MeAIB relative to untransfected cells (Fig. 2a), as previously reported for SA2 and SA1 (Reimer et al, 2000;Sugawara et al, 2000;Yao et al, 2000). MeAIB saturates transport by SA2 with a K m 1.6 Ϯ 0.33 mM, n ϭ 3 (Fig.…”

“…MeAIB saturates transport by SA2 with a K m 1.6 Ϯ 0.33 mM, n ϭ 3 (Fig. 2b), substantially higher than the 0.15-0.5 mM K m reported for SA1 (Reimer et al, 2000;Sugawara et al, 2000;Yao et al, 2000). Very similar to SA1, MeAIB flux mediated by SA2 tolerates substitution of Na ϩ by Li ϩ but not choline and replacement of chloride by thiocyanate (Fig.…”

“…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with SN1 are largely if not entirely uncoupled from transport (F. A. Chaudhry, M. Kavanaugh, and R. H. Edwards, unpublished observations), raising the possibility that currents associated with closely related System A transporters are also uncoupled (Reimer et al, 2000;Sugawara et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.…”

“…Similar to SN1, uptake by SA1 [also known as SAT2 or ATA2 Yao et al, 2000)] and SA2 [also known as GlnT or ATA1 Wang et al, 2000)] is inhibited by low pH, but unlike SN1, the System A transporters do not couple H ϩ translocation to amino acid flux. As a result, transport by SA1 and SA2 is electrogenic rather than electroneutral, and the coupled currents do not reverse at resting membrane potential.…”

“…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).…”