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. 2019 Jul 23;116(32):16003–16011. doi: 10.1073/pnas.1906223116

Fig. 4.

Fig. 4.

The aphid amino acid carrier ApNEAAT1 transports the NEAAs proline, serine, alanine, glycine, and cysteine. (A) Uptake of various radiolabeled amino acids (10 µM) into ApNEAAT1-expressing and water-injected (control) oocytes measured in the absence of extracellular Na+ at pH 5.5. n = 20. ***P < 0.001; **P < 0.01; NS (not significant), P > 0.05 vs. water (2-way, unpaired t tests). (B) ApNEAAT1-specific, concentration-dependent proline uptake. Uptake into water-injected oocytes was subtracted from total uptake. Curve is fitted to Michaelis–Menten kinetics [Km = 179 ± 33 µM; Vmax = 120 ± 6 pmol.oocyte−1.(40 min)−1; r2 = 0.986]. n = 20. (C) Proline uptake in the absence (control) and presence of amino acids or analogs (all 10 mM except Tyr which is 2.5 mM). All are l-isomers unless indicated otherwise. ApNEAAT1-specific uptake is calculated by subtraction of uptake into water-injected oocytes and is expressed as percent control (that in the absence of inhibitor). Tau, taurine. n = 16–20. ***P < 0.001 vs. control; all other bars are P > 0.05 vs. control (1-way ANOVA with Sidak’s posttest). (D) Trans-stimulation of proline ([5 mM]i) efflux via ApNEAAT1 and PAT2 (rat slc36a2) by various extracellular amino acids (10 mM) was measured under Na+-free conditions at extracellular pH 5.5 (10 min). n = 4–5. ***P < 0.001; NS, P > 0.05 vs. water (2-way ANOVA with Tukey’s posttest). (E) Proline uptake in Na+-free conditions over the pH range 5.0–8.0. n = 20. The only significant difference found within each group was in ApNEAAT1-specific uptake: pH 6.5 vs. pH 5.5, P = 0.046 (2-way ANOVA with Tukey’s posttest). (F) Proline-associated inward current in PAT2-expressing but not ApNEAAT1-expressing or uninjected (control) oocytes as measured by 2-electrode voltage clamp. Oocytes were clamped at resting VM (−30 mV), superfused with Na+-free, pH 5.5 buffer and exposed to proline (0.1 to 1 mM). Mean data are shown in (SI Appendix, Table S1) and for ApNEAAT1 in the Inset. (Inset) As a direct comparison, proline uptake via ApNEAAT1 was measured under the same conditions as current measurement. ***P < 0.001.