D-Serine Is a Substrate for Neutral Amino Acid Transporters ASCT1/SLC1A4 and ASCT2/SLC1A5, and Is Transported by Both Subtypes in Rat Hippocampal Astrocyte Cultures

N-methyl-D-aspartate (NMDA) receptors play critical roles in synaptic transmission and plasticity. Activation of NMDA receptors by synaptically released L-glutamate also requires occupancy of co-agonist binding sites in the tetrameric receptor by either glycine or D-serine. Although D-serine appears to be the predominant co-agonist at synaptic NMDA receptors, the transport mechanisms involved in D-serine homeostasis in brain are poorly understood. In this work we show that the SLC1 amino acid transporter family members SLC1A4 (ASCT1) and SLC1A5 (ASCT2) mediate homo- and hetero-exchange of D-serine with physiologically relevant kinetic parameters. In addition, the selectivity profile of D-serine uptake in cultured rat hippocampal astrocytes is consistent with uptake mediated by both ASCT1 and ASCT2. Together these data suggest that SLC1A4 (ASCT1) may represent an important route of Na-dependent D-serine flux in the brain that has the ability to regulate extracellular D-serine and thereby NMDA receptor activity

Values for multiple components of inhibition by L-glutamine and t-Pro of [³H]L-serine and [³H]D-serine transport into rat hippocampal astrocyte cultures.

<p>Values for multiple components of inhibition by L-glutamine and t-Pro of [³H]L-serine and [³H]D-serine transport into rat hippocampal astrocyte cultures.</p

Exchange of [³H]L-serine by rat hippocampal astrocyte cultures.

<p>After loading of cells with [³H]L-serine, amino acids were added to evoke exchange of [³H]L-serine into the supernatant as described in Materials and Methods. (A) Time course of exchange under control conditions (no added amino acid) and after addition of 1mM L-serine or 1mM t-Pro. Note the lower maximal effect of t-Pro. Data are from a single experiment that was repeated twice. (B) L-serine-evoked exchange is dependent on sodium; * p<0.01, t-test, n = 7 per group. (C) Concentration-response curves for exchange evoked by L- and D-serine, L-glutamine and t-Pro. Values are expressed as a percentage of the exchange caused by 1mM L-serine after subtraction of exchange in the absence of added amino acids. Curves were fitted using GraphPad Prism with a one-site fit. Data are from a single experiment that was repeated 3–14 times. EC₅₀ and maximal effect values are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156551#pone.0156551.t004" target="_blank">Table 4</a>. (D) Effects of L-glutamine and t-Pro and their combination on exchange. Values are expressed as a percentage of the exchange caused by 1mM L-serine after subtraction of exchange in the absence of added amino acids; * p<0.01, t-test, n = 3 per group.</p

EC₅₀ and maximum effect values for amino acid-evoked exchange of [³H]L-serine from rat hippocampal astrocyte cultures.

<p>EC₅₀ and maximum effect values for amino acid-evoked exchange of [³H]L-serine from rat hippocampal astrocyte cultures.</p

Transport of [³H]D-serine and [³H]L-serine by rat hippocampal astrocyte cultures.

<p>Transport was conducted as described in Materials and Methods. (A,B) Transport of [³H]D-serine (A) or [³H]L-serine (B) over a range of substrate concentrations in the presence and absence of sodium. Data are from a single experiment that was repeated an additional 2 times for [³H]D-serine and 7 times for [³H]L-serine. Curves were fitted using GraphPad Prism (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156551#pone.0156551.t001" target="_blank">Table 1</a> for mean K_m and V_max values). The sodium-dependent transport was calculated by subtracting transport in the presence of sodium from that in buffer where sodium was replaced by equimolar choline. (C,D) Dependence of [³H]D-serine (C) or [³H]L-serine (D) transport on extracellular sodium. Experiments were conducted in sodium-containing (Total) or choline chloride-containing (zero Na) buffer at a substrate concentration of 0.5 μM. For [³H]D-serine transport n = 3, for [³H]L-serine transport n = 12.</p