Na+ interactions with the neutral amino acid transporter ASCT1

Abstract

The alanine, serine, cysteine transporters (ASCTs) belong to the solute carrier family 1A (SLC1A), which also includes the excitatory amino acid transporters (EAATs) and the prokaryotic aspartate transporter GltPh. Acidic amino acid transport by the EAATs is coupled to the co-transport of three Na(+) ions and one proton, and the counter-transport of one K(+) ion. In contrast, neutral amino acid exchange by the ASCTs does not require protons or the counter-transport of K(+) ions and the number of Na(+) ions required is not well established. One property common to SLC1A family members is a substrate-activated anion conductance. We have investigated the number and location of Na(+) ions required by ASCT1 by mutating residues in ASCT1 that correspond to residues in the EAATs and GltPh that are involved in Na(+) binding. Mutations to all three proposed Na(+) sites influence the binding of substrate and/or Na(+), or the rate of substrate exchange. A G422S mutation near the Na2 site reduced Na(+) affinity, without affecting the rate of exchange. D467T and D467A mutations in the Na1 site reduce Na(+) and substrate affinity and also the rate of substrate exchange. T124A and D380A mutations in the Na3 site selectively reduce the affinity for Na(+) and the rate of substrate exchange without affecting substrate affinity. In many of the mutants that reduce the rate of substrate transport the amplitudes of the substrate-activated anion conductances are not substantially affected indicating altered ion dependence for channel activation compared with substrate exchange.

Keywords: ASCT; Amino Acid Transport; Chloride Channel; EAAT; Electrophysiology; GltPh; Membrane Transport; Na+ Coupling; Site-directed Mutagenesis.

FIGURE 1.

The structure of a Glt_Ph protomer, and sequence alignments of ASCTs, EAATs, and Glt_Ph. A, Glt_Ph protomer (Protein Data Bank code 2NWX) shown in the plane of the membrane, with the trimerization domain (TM1, -2, -4, and -5) in gray and the transport domain: TM3 (light brown), TM6 (blue), TM7 (orange), TM8 (purple), HP1 (yellow), and HP2 (red). l-Aspartate is shown in stick representation, and two bound Na⁺ ions are shown as light blue spheres. Close up view of the Na1 and Na2 binding sites (B), and proposed Na3 site (C) are shown, with residues targeted for mutation in this study shown in stick representation and labeled with Glt_Ph numbering. The protein has been rotated for ease of visualization of each of the Na⁺ binding sites. Images were made using PyMol (43). D, sequence alignment of parts of TM3, HP2, TM7, and TM8 in EAAT1–3, ASCT1–2, and Glt_Ph, where conserved residues are highlighted in black, and mutated residues are highlighted by yellow boxes.

FIGURE 2.

Mutations of Asp-467 in the proposed Na1 site affect substrate binding and transport. A, current-voltage relationship elicited by 300 μm l-serine in Cl⁻ containing buffer (open squares) and NO₃⁻ containing buffer (closed squares), at pH 7.5 in wild type ASCT1. Sample currents in response to 100-ms voltage jumps from −30 to +60 mV (top panel depicts protocol) at 1 mm l-serine and 96 mm NaNO₃ are shown for ASCT1 (B) and D467T (C) (lower panels). Imemb refers to the membrane potential, and V refers to the applied voltage. Concentration-response curves are shown for l-serine (D) and Na⁺ (E) in ASCT1 (closed triangles), D467N (circles), D467S (closed squares), D467T (diamonds), and D467A (open squares) at +60 mV. l-Serine concentrations were varied in a NO₃⁻ based buffer with 96 mm NaNO₃. Na⁺ titrations were performed with 1 mm l-serine and NMDG⁺ as the substitute cation and leak currents were subtracted from baseline measurements. F, l-[³H]serine uptake into oocytes expressing wild type and mutant ASCT1 transporters. Oocytes were incubated in Cl⁻ containing buffer with 10 μm l-[³H]serine at room temperature, pH 7.5, for 10 min. Values presented are mean ± S.E., see Table 1 for n values.

FIGURE 3.

Mutation of Gly-422 in the proposed Na2 site alters Na⁺ binding. Concentration-response curves are shown for l-serine (A) and Na⁺ (B) in wild type ASCT1 (triangles) and G422S (open triangles). l-Serine concentrations were varied in a NO₃⁻ based buffer with 96 mm NaNO₃. Na⁺ titrations were performed with 1 mm l-serine and NMDG⁺ as the substitute cation. C, a sample current in response to 100 ms voltage jumps from −30 to +60 mV (top panel depicts protocol) at 1 mm l-serine and 96 mm NaNO₃ is shown for G422S (lower panel). Imemb refers to the membrane potential, and V refers to the applied voltage. D, l-[³H]serine uptake into oocytes expressing wild type and G422S mutant ASCT1 transporters. Oocytes were incubated in Cl⁻ containing buffer with 10 μm l-[3H]serine at room temperature, pH 7.5, for 10 min. Values presented are mean ± S.E., see Table 1 for n values.

FIGURE 4.

Mutations in the proposed third Na⁺ site affect Na⁺ and substrate binding, and translocation. l-Serine concentration-response curves are shown for ASCT1 (A, C, and E; closed triangles), T124A (A; open circles), T125A (A; open diamonds), D380N (C; closed circles), D380S (C; closed triangles), D380A (C; open squares), D380N/D467S (E; diamonds), and D380N/E465L (E; closed circles). l-Serine concentrations were varied in a NO₃⁻ based buffer with 96 mm NaNO₃. Na⁺ concentration-response curves are shown for ASCT1 (B, D, and F; closed squares), T124A (B; open circles), T125A (B; open diamonds), D380N (D; closed circles), D380S (D; closed triangles), D380A (D; open squares), D380N/D467S (F; diamonds), and D380N/E465L (F; closed circles). Na⁺ titrations were performed with 1 mm l-serine and NMDG⁺ as the substitute cation. G, a sample current in response to 100-ms voltage jumps from −30 to +60 mV (top panel depicts protocol) at 1 mm l-serine and 96 mm NaNO₃ is shown for D380N/D467S (lower panel). Imemb refers to the membrane potential, and V refers to the applied voltage. H, l-[³H]serine uptake into oocytes expressing wild type and mutant ASCT1 transporters. Oocytes were incubated in Cl⁻ containing buffer with 10 μm l-[³H]serine at room temperature, pH 7.5, for 10 min. Values presented are mean ± S.E., see Table 1 for n values.

FIGURE 5.

MD simulations of an ASCT1 homology model with mutations to the Na1 and Na3 sites. A, the ASCT1 model after 20 ns of simulations, showing the l-serine substrate (green sticks), as well Na⁺ (cyan sphere) bound at Na1, Na2, and Na3 sites. B, the Na1′ and Na2 sites occupied in wild type ASCT1, in the absence of a Na⁺ at Na3, after 20 ns of simulations. The Na⁺ bound at Na2 moves closer to Na1′ and are now coordinated by the Asp-380 and Asp-467 side chains. C, the new coordination of Na⁺ at Na3 in the D380A mutant transporter, in which we see the flipping of the Glu-465 side chain to coordinate a Na⁺ at Na3 in one of the chains of ASCT1. D, the new binding site of the Na⁺ bound at Na1′ in the D467S mutant transporter without a Na⁺ bound at Na3, after 20 ns of simulations. This Na⁺ is now coordinated by Ser-467 and Asp-380 side chains. E, the time evolution of the distance between the Ser-467 side chain oxygen and the Na1 ion in the D467S transporter with all ligands bound. Black represents chain A, red chain B, and green chain C. In all cases the Na⁺ bound at Na1 leaves its binding site.