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

Transport by SA1 and SA2 depends on membrane potential. a, HeLa cells expressing SA2 with the vaccinia virus-T7 polymerase system (filled circles) show greater uptake of ³H-MeAIB at pH 8 than cells expressing vector alone (open circles);n = 3. b, Expression of SA2 confers saturable transport of ³H-MeAIB in the presence of either Na⁺ or Li⁺ (120 mm). The K_m for MeAIB at pH 8 in the presence of Na⁺ is 1.54 ± 0.28 mm(V_max, 4.04 ± 0.40 nmol/3 min) and 9.62 ± 1.34 mm(V_max, 3.74 ± 0.68 nmol/3 min) in the presence of Li⁺; n = 3.c, Na⁺ and to a lesser extent Li⁺ but not choline support the uptake of³H-MeAIB by SA2 (left panel).Filled bars indicate HeLa cells transfected with the rat SA2 cDNA, and open bars indicate cells transfected with the vector alone. Transport by SA2 tolerates the replacement of Cl⁻ by SCN⁻ (right panel); n = 3. d,Depolarization by 60 mm KCl (with 60 mm NaCl in the presence or absence of the K⁺ ionophore valinomycin) reduces the uptake of ³H-MeAIB by HeLa cells expressing SA2 (left panel) and SA1 (right panel); n = 3.

Currents associated with SA1 and SA2.a, Alanine (A), MeAIB (m), and glutamine (Q) (all 1 mm) produce inward currents in oocytes expressing SA2 and held at −50 mV, pH 8. Glutamine and alanine produce currents considerably larger than MeAIB. b, Oocytes expressing SA1 exhibit the largest currents in response to addition of alanine, followed by glutamine and MeAIB. c, Currents associated with SA2 are inhibited by low pH_o. d,Uninjected oocytes show no significant currents induced by these amino acids. e–g, Different amino acids (all at 1 mm) produce currents of different magnitudes in oocytes expressing SA2 (e, f) and SA1 (g) in Na⁺(e) or Li⁺ (f, g). The responses are normalized to the currents induced by alanine. The letters refer to the standard single-letter amino acid code, with m representing MeAIB,g representing GABA, c representing cystine, and t representing taurine. h,Relationship of SA2 currents induced by glutamine to membrane potential. Replacement of Na⁺ by choline dramatically reduces the size of the currents produced by 1 mm glutamine. However, the currents produced by 1 mm glutamine in Li⁺ almost equal the currents produced by 1 mm glutamine in Na⁺. i, Current–voltage relationship of oocytes expressing SA2 in NaCl and Na gluconate, with and without addition of 1 mm glutamine. Replacement of chloride by gluconate has little effect on these unsubtracted currents and in particular on their reversal potential. However, glutamine shifts the reversal potential in the positive direction, consistent with electrogenic amino acid transport.

System A Transporters SA1 and SA2 do not translocate protons. a, Charge–flux ratios were determined in oocytes expressing SA1 and SA2 held at −60, −10, and +30 mV from the total current generated over 10 min by 1 mm³H-alanine in individual oocytes and the total accumulation of radiolabeled substrate by the same individual oocytes over the same interval. Currents and ³H-alanine uptake by uninjected oocytes was subtracted. The ratios approximate 1 at all potentials in the case of SA1, consistent with the net inward movement of one charge along with each amino acid. In the case of SA2, the charge–flux ratios exceed 1; n = 3 for each condition. b, c, PS120 cells expressing SA2 (b) and SN1 (c) were loaded with the pH-sensitive dye BCECF-AM, and pH_i was measured by ratiometric imaging at 440 and 490 nm. Glutamine (Q) at 10 mm produces no change in the pH_i of cells expressing SA2 (b), whereas 1 mmdramatically increases the pH_i of cells expressing SN1 (c). Because PS120 cells expressing SN1 are much more acidic at baseline (pH_i ∼6.5) (c) than cells expressing SA2 (b), we have used 15 mm ammonium chloride to demonstrate that we can easily detect an increase in pH_i under both circumstances.d, Simultaneous measurement of currents and pH_i in oocytes expressing SA2 shows that although 1 mm glutamine induces inward currents, it does not change pH_i. Thus, transport by SA2 does not involve H⁺ translocation.

Protons compete with Na⁺ for binding to System A transporters. a, b, Increasing Na⁺ concentrations saturate the uptake of³H-MeAIB by HeLa cells expressing SA2 (a). At 4 μm MeAIB, theK_m for Na⁺ at pH_o 7 greatly exceeds the K_m at pH_o 8. Increasing MeAIB concentrations also saturate uptake by SA2 (b), with a difference inV_max but not K_mbetween pH_o 7 and 8. Representative experiments are shown, and the average of multiple experiments ± SEM is compiled in Table 2. c, d, SA1 demonstrates saturation of³H-MeAIB transport in HeLa cells by Na⁺(c) and MeAIB (d). TheK_m for Na⁺ is higher at pH_o 7 than pH_o 8, similar to SA2. However, it is more clear that pH_o has little effect on theV_max of SA1 at high Na⁺. pH_o also affects the V_max but not K_m for MeAIB (Table 2).

Ordered binding of substrates to SA1.A, The currents induced in an oocyte expressing SA1 by different concentrations of alanine in the presence of 6 mmNa⁺ (left) as a function of holding potential. The same oocyte in 96 mm Na⁺(right) exhibits no change inI_max but the K_mdrops considerably, suggesting that Na⁺ binds before amino acid. b, Currents induced in an oocyte expressing SA1 by different concentrations of Na⁺ in the presence of 0.1 mm alanine (A) (left). The same oocyte in 1 mm alanine (right) shows a much largerI_max and a differentK_m, supporting the ordered binding of first Na⁺ and then amino acid. The average of multiple experiments ± SEM is compiled in Table 3.

SA2 mediates a Na⁺ conductance in the absence of amino acid. a, In the absence of external amino acid, oocytes expressing SA2 exhibit currents that depend on Na⁺ and Li⁺. The shift in reversal potential depends on the Na⁺concentration and varies by 40 mV from 0–96 mmNa⁺ (b). c,Oocytes expressing SA1 exhibit smaller cation-dependent currents, and uninjected oocytes exhibit none (d);n = 3 for all conditions.

Expression of SA2 by central neuronal populations.a–f,In situ hybridization of coronal sections from the rat brain with ³⁵S-labeled antisense SA2 RNA (a–c) and sense RNA (d–f). All brain regions show specific hybridization signal, but labeling is particularly pronounced in the hippocampus and cerebellum.g–i, Visualized by dark-field illumination, all layers of the cortex contain cells labeled by SA2 antisense RNA (g). In the hippocampus (h), principal cells in the pyramidal and granule cell layers are strongly stained. Scattered positive cells suggestive of interneurons also occur in other layers of CA1 (i). j–m, Related H⁺-driven amino acid transporters differ in their distribution within the cerebellar cortex. j, Byin situ hybridization with specific antisense RNA, SA2 strongly labels the Purkinje cell layer (p).k, SA1 labels the granule cell layer (g). l, The closely related System N transporter SN1 shows expression by glial-like cells in the granule cell layer. m, The more distantly related vesicular GABA transporter shows expression by inhibitory neurons in the molecular layer (m) and Purkinje cells.