Figure 7(A) All four SLC6A20 protein variants (human SLC6A20 v1 and v2; mouse SLC6A20A and SLC6A20B; shown in schematic diagrams) mediate similar levels of glycine and proline transports. HEK293T cells expressing SLC6A20 protein variants were used to measure ensemble currents induced by increasing concentrations of glycine/proline in auto-patch experiments. Sample images indicate HEK293T cells expressing human SLC6A20 v1; myristoylated GFP expression, used only for this imaging but not for transport measurements, show detectable surface expression of expressed SLC6A20 proteins. Scale bar, 10 μm. Increasing concentrations of glycine/proline were sequentially applied to HEK293T cells, as shown in Appendix Fig. S7A. The indicated currents are average values from an ensemble of multiple (~20) cells in a single well. (glycine, n = 34 cells for untransfected/blank HEK293T cells, 36 for human SLC6A20-V1, 38 for human SLC6A20-V2, 48 for mouse SLC6A20A, 51 for mouse SLC6A20B, *P < 0.05, **P < 0.01, ***P < 0.001 (relative to untransfected), two-way ANOVA with Tukey's test; proline, n = 46 cells for non-transfected, 43 for hSLC6A20-V1, 31 for hSLC6A20-V2, 54 for mSLC6A20A, 54 for mSLC6A20B, *P < 0.05, **P < 0.01, ***P < 0.001 relative to untransfected, two-way ANOVA with Tukey's test). The error bars represent SEM. (B) Glycine/proline-induced currents for mouse SLC6A20A are dependent on sodium chloride, as shown by the suppression of the currents when sodium chloride is replaced with choline chloride or sodium gluconate. Each experimental condition was sequentially applied to HEK293T cells, as shown in Appendix Fig. S7B. The indicated currents are average values from an ensemble of multiple (~20) cells in a single well. (glycine, n = 21 cells for untransfected/blank, 17 for human SLC6A20-V1, 25 for human SLC6A20-V2, 33 for mSLC6A20A, 28 for mSLC6A20B ***P < 0.001 (relative to buffer not containing glycine), two-way ANOVA with Bonferroni's test; proline, n = 17 cells for untransfected, 23 for human SLC6A20-V1, 47 for SLC6A20-V2, 31 for mSLC6A20A, and 31 for mSLC6A20B, ***P < 0.001, two-way ANOVA with Bonferroni's test). The error bars represent SEM. (C) Glycine/proline-induced currents for mouse SLC6A20A do not show reversal potentials, as shown by the lack of reverse currents induced by increasing holding potentials in HEK293T cells. These experiments were performed using single HEK293T cells in a conventional patch-clamp (not auto-patch) setup. (glycine, n = 3, 7, 3, 3, 3, 3 cells for untransfected HEK293T cells, 10, 11, 5, 6, 6, 5 cells for mouse SLC6A20A, at -90, -60, -30, 0, 30, 60 mV of holding potentials, respectively; proline, n = 4, 7, 4, 5, 3, 4 cells for untransfected HEK293T cells, 3, 6, 3, 3, 3, 3 cells for mouse SLC6A20A, at -90, -60, -30, 0, 30, 60 mV of holding potentials, respectively). The error bars represent SEM. (D) Human neural progenitor cells (NPCs) and neurons differentiated from NPCs for 1-6 weeks express SLC6A20 proteins, as revealed by immunoblot analysis using the SLC6A20 antibody directed against a region of mouse SLC6A20A (aa 301-369; KATFNYENCLKKVSLLLTNTFDLEDGFLTASNLEQVKGYLASAYPSKYSEMFPQIKNCSLESELDTAVQ; Sigma) that is 83% identical to the human SLC6A20 sequence. Two different splice variants of human SLC6A20 (v1 and v2) expressed in HEK293T cells were used as positive controls. PSD-95, an abundant excitatory postsynaptic scaffold protein, was used as a maker for neuronal maturation, and β-actin was used as a loading control. Note that the band intensity of 'unboiled' SLC6A20 at 2-week time point is stronger relative to the boiled SLC6A20 band at 2 weeks, in line with that SLC6A20 is a transmembrane protein with twelve transmembrane domains that easily forms larger protein aggregates upon boiling and get stuck at the start location of western gels, decreasing the protein band intensity of SLC6A20. We did not try the unboiling of other samples at different time points because one sample was enough for proof of concept. U, untransfected HEK293T cells; V1/V2, HEK293T cells expressing human SLC6A20-v1/v2.