Le Guellec B , Rousseau F , Bied M , Supplisson S .

	Fig. 1. (A) The phylogenetic tree of SLC6 transporter orthologs shows a split between the glycine-specific transporters: GlyT2 is more closely associated with ATB0,+ than with GlyT1 (n = 14, 16, 16, and 17 vertebrate orthologs (see species in SI Appendix, Fig. S1; mouse sequences of six GABA transporters [GABAT: SLC6A1, SLC6A6, SLC6A8, SLC6A11, SLC6A12, and SLC6A13] and the three monoamine transporters [MAT: SLC6A2, SLC6A3, and SLC6A4] were included as outgroups). (B) Higher sequences divergence in vertebrate orthologs of ATB0,+. Plot of the cumulative count (+1 if all orthologs shared the same residue), starting at the first shared arginine (RNNG). (C) Area-proportional Venn diagram distribution of shared and unique residues of mice sequences for the three glycine transporters (SI Appendix, Fig. S4). (D) Amino acid transport shows a remarkable divergence in substrate specificity between ATB0,+ and GlyT1-2. Individual bars represent the current evoked by each amino acid in oocytes expressing GlyT1 (200 µM, n = 2 to 4; Left), GlyT2 (200 µM, n = 4; Middle), or ATB0,+ (1 mM, n = 6 to 7; Right). Amino acids are identified by a single letter code, except sarcosine (Sa) and GABA (γ). Current amplitudes are normalized by the glycine response. (Insets) Application of all amino acids except glycine (A, C, S, L, F, N, K, Q, H, V, T, Y, R, M, I, W, P, E, and D at 200 µM each) do not evoke a current compared to glycine (3.8 mM) in GlyT1-expressing oocytes (2.4%, n = 6, P < 0.001) and GlyT2-expressing oocytes (2.35%, n = 10, P = 0.002). Error bars indicate SEM; paired t test: ***P < 0.001, **P < 0.01, *P < 0.05; ns, not significant.
	Fig. 2. (A) Glycine evokes large inward currents in ATB0,+ -expressing oocytes. Current–voltage relationships (I–Vs) for three glycine concentrations (20 µM [square], 200 µM [triangle], and 2 mM [circle]). (B) SimiIar I–Vs for ATB0,+, GlyT2, and GlyT1 at saturating glycine concentrations (ATB0,+[blue, 2 mM], n = 10, slope = 0.92% mV– 1, R2=1.0, P<2×10−16; GlyT2 [red, 200 µM], n = 11, slope = 1.07% mV– 1, R2=0.998, P<2×10−16; and GlyT1 [green, [Gly]=200 µM], n = 11, slope =0.87% mV– 1, R2=0.998, P=1.78×10−13 ). Glycine currents were normalized by the absolute amplitude at –40 mV, and linear regressions were performed on the –140 to –40 mV voltage range. (C) Glycine dose–response curve for ATB0,+ at VH=−40mV (blue circles, n = 32). Individual curves were fitted by the equation IGly=Imax/(1+EC50/[Gly]o) and then normalized by the maximal amplitude (Imax). Glycine EC50 of ATB0,+ (189.0 ±5.6 µM, n = 21) is higher than for GlyT1 (28 µM, green dashed line) and GlyT2 (25 µM, red dashed line) as previously determined in ref. 9. (D) Anomalous voltage dependency of glycine EC50 of ATB0,+ at hyperpolarized potentials (blue circles, n = 6). Solid and dashed lines are fits to the equation Aeza ξ V+Bezb ξ V (ξ=F/(RT)) , for ATB0,+(A =66.2, B =133.9, za =1.76, zb =–0.185). The curves for GlyT2 (dashed red line, A = 43, B =21.6, za =1.54, zb = 0) and GlyT1 (dashed green line, A =20.5, B =21.7, za =0.7, zb =0) are shown for comparison (9).
	Fig. 3. (A) Combined violin and box plots of transport current amplitudes recorded at –40 mV for different saturating glycine concentrations, i.e., 200 µM for GlyT1 (245.2 ±17.3 nA, n = 85) and GlyT2 (250.6 ±29.7 nA, n = 48) and 1 to 2 mM for ATB 0,+ (1,450.0±98.8 nA, n = 65). (B) Representative PSSCs recorded at the on and off of voltage steps in oocytes expressing GlyT1 (Left; green), GlyT2 (Middle; red), and ATB0, (Right; blue) expressing oocytes. The range of voltage steps varied from +110 mV (ATB0,+ ) or +50 mV (GlyT1 and GlyT2) to –150 mV in decrements of 10 mV. For clarity, only seven traces are shown, from +50 mV to –130 mV in decrements of 30 mV from an holding potential of –40mV. Transporter currents were isolated by subtracting traces recorded with the same voltage step protocol in the presence of specific inhibitors (10 µM ORG24598 [GlyT1], 5 µM ORG25543 [GlyT2], and 1 mM α-MT [ATB0,+]). All residual steady-state components have been subtracted for clarity, and Insets are scaled to the maximal amplitude recorded at the onset of the voltage steps. (C) The charge movement is the time integral of the relaxation currents plotted as a function of voltage (Q-V). Q-Vs for ATB0,+ (blue, n = 38), GlyT2 (red, n = 53), and GlyT1 (green, n = 14)) were fitted with a Boltzmann equation and then normalized by Qmax. Combined violin and box plot distributions of the Boltzmann equation parameters with (D) zmax, (E) V1/2, and (F) Qmax for GlyT1 (green, n = 14), GlyT2 (red, n = 53), and ATB0,+(blue, n = 38).
	Fig. 4. (A) Representative current traces in voltage-clamp oocytes expressing ATB0,+ and held at VH=−20 mV for two applications of glycine (200 µM performed before and after intracellular microinjection; arrow, 20 to 40 nL) of (Top) glycine (1 M) or (Bottom) NaCl (0.1 M) and glycine (1 M). Currents were normalized by the amplitude of the first glycine application (IGly1 ). (B) (Top) Schematic of a typical I–V protocol before and after microinjection of 16 to 20 nL of a solution containing 0.5 M NaCl and glycine. ATB0,+-mediated currents are isolated by subtraction in the presence of α-MT (500 µM). (Bottom) Normalized ATB0,+ I–Vs for two applications of glycine (200 µM) performed before (blue triangles pointing down) and after (orange triangles pointing down) microinjection of 18 nL of a solution containing glycine (0.5 M) and NaCl (0.5 M) which generates only outward currents (orange triangles pointing up). I–V subtractions are indicated in the legend. Data are mean ± SEM for six oocytes. Amplitudes were normalized by the absolute amplitude of the glycine current recorded before injection. (C-E) Shifts in reversal potentials of three I–Vs recorded in ATB0,+-expressing oocytes microinjected with 13 to 23 nL of a concentrated (0.5 M) NaCl + glycine solution for 100-fold change in (C) glycine concentration or 10-fold change in (D) Cl−and (E) Na+concentrations. Currents recorded with the same protocol in the presence of α-MT (500 µM or 1 mM at low Na+) were subtracted. (F) Semilogarithmic plot of reversal potentials for the experiments described in C–E as a function of each cosubstrate concentration ([S]o). The slopes of the reversal potential per decade concentration change were calculated for glycine (orange, 28.6 mV/decade, R2=0.873, P<2.47×10−11, n = 8), Cl−(green, 32.5 mV/decade, R2=0.754, P=2.96×10−6, n = 6), and Na+(red, 84.6 mV/decade, R2=0.983, P<2×10−16, n = 14) with the 95% confidence interval shown in shaded areas. (G) Combined violin and box plot distributions of ATB0,+ reversal potential slopes and stoichiometric coefficients for glycine, Cl−, and Na+.
	Fig. 5. (A) Representative traces of ATB0,+ -PSSCs recorded at the onset of the voltage steps (from +110 mV to –150 mV in decrements of 20 mV; VH=−40 mV) in glycine-free solution containing the indicated Na+ concentration. ATB0,+-specific PSSCs were isolated by subtraction of traces recorded in the presence of α-MT (1 mM); all residual endogenous currents have been subtracted for clarity. Normalized Q-Vs for (B) ATB0,+(n = 14, ±SEM) and (C) GlyT2 (n=7,±SEM) for the four Na+ concentrations shown in A, using the same color code. Datasets are the average of four Q-Vs normalized by the Qmax value for [Na+] =100 mM. The solid lines correspond to the fit of the four-state scheme (SI Appendix, Fig. S9A, scheme 3) depicted above, with the gating, Na+-binding, and Na+-locking steps detailed in SI Appendix, Fig. S9A. The green and blue arrows represent two exit steps from B state that are activated during depolarization, whereas the purple arrows identify steps activated by hyperpolarizing voltage steps. The dashed lines of the Q-Vs are extrapolations of the model outside the experimental voltage range. (D) The apparent charge displacement plotted as function of [Na+] is calculated according to SI Appendix, Eq. 9, for different Vmax (SI Appendix, Fit Procedures). (E) The median voltage (SI Appendix, Fit Procedure, Eq. 10) plotted as a function of [Na+] is estimated as shown in SI Appendix, Fig. S14. (F) Model prediction of ATB0,+-PSSCs for the same conditions as in A. The rate constants are described in SI Appendix, Fit Procedures. The voltage independent rate constants are k120=79s– 1, k230=88 mol– 1 s– 1, and k340=23 s– 1, with dα=0.36, dβ=0.42, and dγ=0.7. The off rate constants are calculated using the equations detailed in SI Appendix, Fig. S9B with zα, zβ, zγ, α0, γ0, and Kd and nH values shown in B.
	Fig. 6. The 3-Na+ coupling limits glycine efflux but not ATB0,+ heteroexchange. (A) Glycine efflux was measured in voltage-clamped oocytes held at –60 mV and microinjected with [14C]-glycine. The outflow of the perfusion solution was collected in the absence (basal; left traces) or presence of glycine (1 mM, glycine-stimulated exchange; right traces) for oocytes expressing GlyT1 (Top; green traces), GlyT2 (Middle; red traces), or ATB0,+ (Bottom; blue traces). Histogram bars show glycine efflux in basal condition and during glycine application measured for each transporter in this experiment. The diagrams illustrate the two components of glycine efflux in these experiments: a transporter-specific pathway that allows exchange in the presence of extracellular glycine and a nonspecific component for glycine leakage by endogeneous transporters of Xenopus oocytes. Data summarized for (B) Glycine-stimulated efflux and (C) basal efflux. ORG24598 inhibited a large fraction of the basal efflux in GlyT1-expressing oocytes, whereas ORG25543 had no significant effect on the basal glycine efflux in GlyT2-expressing oocytes. No inhibitor could be tested for ATB0,+ as its basal efflux was already low. (D–F) We used the high-affinity coagonist site of GluN1-GluN2B NMDARs as a dynamic sensor of the juxtamenbrane glycine concentration produced by transporter-mediated efflux. Indeed, glutamate (2 µM) evoked no or a small current when applied alone in basal condition with oocytes coexpressing GluN1-GluN2B subunits of NMDAR and one of the glycine transporters (dashed traces); glutamate was applied in 0.3 mM BaCl2 (i.e., Ca2+- and Mg2+-free) Ringer’s solution. Currents were normalized by the amplitude of the current evoked by 2 µM Glu + 0.3 µM glycine represented by a vertical ticked bar. However, after glycine uptake (~2 min application of glycine [1 mM] + d-APV [50 µM] in normal Ringer’s solution; SI Appendix, Fig. S16, Inset), glutamate evoked much larger NMDAR currents in GlyT1-coexpressing oocytes, and the potentiation persisted for more than an hour of continuous washing (SI Appendix, Fig. S16). The glutamate-evoked NMDAR current was blocked by NFPS (10 µM, 96.7% inhibition) (E), a fast acting and reversible GlyT1 inhibitor, and by ORG24598 (10 µM, 96.7 ±0.6% inhibition, n = 4) but with slower kinetics. In contrast, glutamate-evoked NMDAR current did not change markedly after glycine uptake in GlyT2- and ATB0,+ -coexpressing oocytes (D and F). (F) Summary of the change in GluN1/GluN2B NMDAR currents evoked by 2 µM Glu applied alone in basal condition and after glycine uptake for oocytes coexpressing GlyT1, GlyT2, and ATB0,+. Current amplitudes were normalized by the absolute amplitude of the current evoked by 2 µM Glu + 0.3 µM d-serine. (G–I) Finally, we tested whether ATB0,+ could gate NMDAR activation by heteroexchange of glycine with leucine. (G) In basal condition, the transport current evoked by leucine (200 µM, cyan trace) in ATB0,+-coexpressing oocytes had much higher amplitude than the NMDAR current evoked by glutamate (2 µM, black trace). Coapplication of leucine with glutamate shows that transporter and receptor currents are independent, with minimal potentiation of NMDARs (magenta). After glycine uptake, ATB0,+-mediated transport current evoked by leucine was reduced by 0.64-fold (cyan traces, from 1.74 to 1.15 µA) while there was little potentiation of the glutamate-evoked current when applied alone (1.66-fold increase, black traces, from 102 to 169 nA). However, coapplication of leucine and glutamate enhanced 32.8-fold the NMDAR response (magenta traces, from 174 nA to 5.74 µA), indicating glycine/leucine heteroexchange. (H) The potentiation of NMDAR current by heteroexchange leucine/glycine was blocked by αMT, an ATB0,+ inhibitor (1 mM, 92.7 ±1.4% inhibition, n = 4). (I) Summary of the change in GluN1-GluN2B NMDAR currents induced by Leu/Gly heteroexchange (IGlu+Leu–ILeu) in basal condition and after glycine uptake for oocytes coexpressing ATB0,+. Currents were normalized by the amplitude measured with 0.3 µM d-serine. Error bars indicate SEM; paired t test: ***P < 0.001, **P < 0.01, *P < 0.05.
	Fig. S1. Phylogenetic tree of the SLC6 amino acid transporters. The phylogenetic tree of Fig. 1A with the species name of each Vertebrate-ortholog for ProT (n=14), GlyT1 (n =16), GlyT2 (n = 16) and ATB0,+ (n = 17); mouse sequences of six GABA transporters (GABAT: SLC6A1,6,11,12,13), and the three monoamine transporters (MAT:SLC6A2,3,4) were included in the phylogenetic analysis as outgroups.
	Fig. S2. Sequence identities in transmembrane segments. Fraction of sequence identity in the putative transmembrane segments (TMs) of all orthologs shown in Fig. 1A. The human SERT structure was used as a template for TMs delimitation (3).
	Fig. S3. Sequence identities among vertebrates orthologs of ATB0,+, GlyT2 and GlyT1. Radar plot of the cumulative number of sequence identities for each glycine-transporter pair (ATB0,+-GlyT2 (purple), GlyT2-GlyT1 (cyan), ATB0,+-GlyT1 (yelow)). Pair identity shared with PROT are excluded as indicated in the legend.
	Fig. S4. Sequence alignments of mATB0,+, mGlyT2 and mGlyT1. The color code indicates amino acids common for the three transporters (gray, n = 220), and the identity shared between ATB0,+ and GlyT2 (purple, n = 95), ATB0,+ and GlyT1 (orange, n = 43), GlyT1 and GlyT2 (cyan, n = 76). The blue lines represent the putative transmembrane segments based on hSERT structure
	Fig. S5. ATB0,+ transport current is glycine-, Na+- and Cl−-dependent. Glycine applications (red bar: [Gly]=200 µM) does not evoked a current in Na+-free (left trace) and Cl−-free solution (right trace) with ATB0,+-expressing oocytes held at VH = −40 mV. Na+ was substituted by choline (left trace, solid bar) and Cl− was substituted by gluconate (right trace, solid bar).
	Fig. S6. Expression of ATB0,+ increases the linear capacitance of Xenopus oocytes. (A) Left panel: normalized traces of capacitive currents (linear and PSSCs) recorded at the onset of voltage steps (from +50 mV to −130 mV by decrements of 10 mV for a representative ATB0,+-expressing oocyte. Each trace is normalized by the absolute peak current (the trace at VH = −40 mV is not included). Middle panel: same as left in the presence of 1 mM α-MT (blue traces) to block ATB0,+-charge movement. The gray traces represent currents recorded with the same protocol for a representative non-injected oocyte. Right panel: the charge movement of ATB0,+ (orange trace) for a single step at 50 mV after α-MT subtraction. (B) Average Q-Vs of the linear component of capacitive currents in non-injected oocytes (gray, 209 ± 3 nF, n = 33), and oocytes expressing GlyT1 + ORG24598 (green, 227 ± 7 nF, n = 11), GlyT2 + ORG25543 (red, 242.8 ± 5.2 nF, n = 10), and ATB0,+ + α-MT (blue, 320.6 ± 11.7 nF, n = 16). (C) Combined violin and box plots of the distribution of linear membrane capacitance of non-injected oocytes (n = 33), and oocytes expressing GlyT1 (n = 11), GlyT2 (n = 10) and ATB0,+ (n = 50; a group of 34 ATB0,+-expressing oocytes was added by estimating the slope of the Q-V at hyperpolarized potentials).
	Fig. S7. ATB0,+ charge-coupling and turnover rate. (A) linear relationship between glycine uptake and the time integral of the glycine-evoked current for 30-120 s application of 14C-glycine (50 µM, each circle represents a single oocyte expressing ATB0,+). The slope (zT =2.08 e, R2=0.982, p=2.16 10−12), which is the charge coupling per glycine, is in agreement with the 3 Na+/1 Cl− stoichiometry. (B) linear relationship between Imax ([Gly] = 1 to 2 mM) and Qmax for oocytes expressing ATB0,+. An apparent turnover rate (λ = 18 s−1) is derived from the Imax/Qmax slope (29.7 s−1, R2= 0.848, p = 3.77 10−12) using the equation λ =zmax zT Imax Qmax, with zT =2.08 e and zδ=1.26 e). The shaded areas correspond to the 95 % confidence interval.
	Fig. S8. Microinjection of NaCl with glycine is required to reverse GlyT2 but not GlyT1. (A) Representative current traces recorded in voltage-clamped oocytes expressing GlyT1 (top trace, green) or GlyT2 (bottom, red), and held at VH = −20 mV for two applications of glycine (200 µM before and after intracellular microinjection (arrow: 20 to 40nl). Left panels: microinjection of glycine (1M) reverses GlyT1 as shown by a slow rise of the outward current (Iout), but not GlyT2. Right panels: microinjection of NaCl (0.1M) and glycine (1M) increases Iout in GlyT1- and reverses GlyT2. Currents are normalized by the first glycine application (IGly(1)). (B) Summary data for experiments as shown in (A) and Fig. 4A. The bar histograms show the amplitude of Iout (left) normalized by IGly(1) and the relative current evoked by the second glycine application (IGly(2)/IGly()1, right) (mean ±SEM, n are indicated in parenthesis; *** p<0.001, ** p<0.01, * p<0.05; Wilcoxon rank-sum test with Bonferroni correction for multiple comparison).
	Fig. S9. A four-state sequential model for the charge movement of ATB0,+ and GlyT2. (A) The model shows the three kinetic schemes tested here to fit ATB0,+- and GlyT2-Q-Vs as function of Na+. In glycine-free media, outward facing transporters are assumed to be either Na+-unbound (Uc , U) or Na+-bound (B, Bc). The UB scheme (#1) corresponds to the binary Hill model proposed by (4) for GAT1 and is limited to a single binding step for multiple Na+. It is defined by 3 parameters: the microscopic dissociation constant Kd, the Hill coefficient nH, and the equivalent charge zβ. For simplicity, we define βNa,V as the equilibrium constant of the binding step as function of Na+ and voltage. Schemes #2 and #3 include a gating step controlling the access of Na+ to its binding sites. The equilibrium constants are αV and γV for the gating and locking steps, respectively. In the scheme #2, gate closure occurs only in the unbound state (U) whereas it is independent of Na+-binding in scheme #3. At negative potentials all transporters are in state B (fig:S5)). Positive voltage steps trigger gate closure from Na+-unbound (U) and Na+-bound state (B). (B) The zmax and normalized Q-V equations are tabulated for each scheme, with the ∆AICc values estimated from the fit. zmax for scheme #3 conditioned by equality or inequality of the valencies of the Na+-release and Na+-locking paths
	Fig. S10. Decay time course of ATB0,+ transient currents. A Semi-log plot of the 80 to 1 % decay of PSSCs for 3 voltages steps (−120 mV, +10 mV and +90 mV) from vH (−40 mV) for the four concentrations of Na+ indicated (same traces as in Fig. 5 A). Currents were sampled at 20 kHz, and decimated by a factor 5 for clarity. B Examples of fit with a mono (red) and bi-exponential (blue) time course for currents recorded at −120 and +50 mV steps. C Amplitude-weighted time constant
	Fig. S11. The equivalent charge displacement of ATB0,+ is Na+-dependent. (A-B) Q-Vs for GlyT2 (A, n = 1) and ATB0,+ (B, n = 7) for different Na+ concentrations as indicated in the legend panel. The solid lines correspond to the individual fits using a Boltzmann equation, normalized by Qmax for Na+ = 100 mM . (C-D) The solid lines represent plots of the first derivative of the Boltzmann fits shown in A-B for GlyT2 (C) and ATB0,+ (D). The dashed lines are the first derivative of the Hill equation (scheme #1 in S9) with the parameters nH = 1.9, zmax = 0.55 and Kd = 67.4 mM for ATB0,+ and nH = 2.04, zmax =1.13 and Kd = 290 mM for GlyT2
	Fig. S12. Global fits of Q-V(Na) with scheme 1 and 2. (A-D) Global fits of the Q-Vs of ATB0,+ (top) and GlyT2 (bottom) as a function of the Na+ concentration for the kinetic schemes 1 (A), and 2 (B), as described in S9.
	Fig. S13. Distribution of the four states of scheme 3 at different Na+ concentrations. The distribution of the four-state (Uc, U, B, Bc ) of ATB0,+ (left panels) and GlyT2 (right panels) plotted as a function of voltage for the Na+ concentration indicated on the left, using the set of parameters in Fig. 5B-C. The closed circles represent the fraction of the B state at VH= −40 mV.
	Fig. S14. Predicted Qmax convergence at low Na+ concentration at positive voltage. Prediction from scheme 3 shows a reduction of the apparent zmax, and therefore Qmax at low Na+ concentration (Fig. 5D) . Here, we show the extrapolation of ATB0,+- and GlyT2-Q-V(s) calculated for a large, not experimentally accessible, voltage range (−0.4 to 1 V), using the same set of parameters depicted in Figs. 6B-C. As expected for a sequential model, the equilibrium equation of scheme 3 predicts that all four Q-Vs converge to 1 at extreme positive potentials.
	Fig. S15. Na+-dependency of the median voltage. The Q-Vs of ATB0,+ (top) and GlyT2 (bottom) are plotted for four Na+ concentrations as indicated, using the fit parameters of scheme 3. The median voltage (Vmed. ) for equal charge distribution is estimated by numerical integration as described in methods and indicated on the voltage axis of each plot. The darker and lighter shaded areas represent the negative and positive integrals, respectively
	Fig. S16. After glycine uptake, GlyT1-mediated efflux becomes an efficient and prolonged source of coagonist for NMDA receptor activation by glutamate alone. Application of glutamate (2µM) evoked small amplitude NMDAR current in basal conditions (trace a) but becames much larger after (traces b-e) glycine uptake (inset on top) for an oocyte coexpressing GluN1/GluN2B subunits of NMDAR and GlyT1. The potentiation of NMDAR activation by GlyT1-mediated glycine efflux persisted for more than an hour after uptake, indicating small rate of glycine efflux. The holding potential was changed as indicated in the legend, and the glycine accumulation produced by 2 min uptake was estimated from the time-integral of the transport current divided by the Faraday’s constant. Assuming an oocyte water volume of 700 nl, this uptake would increase the intracellular glycine concentration by 1.2 mM.
	Fig. S17. A minimal model for ATB0,+ transport cycle predicts the anomalous voltage-dependence of glycine EC50. A we used a simple four-state model to fit a set of 5 glycine I-Vs obtained with the same ATB0,+-expressing oocyte (1.8 × 1011 transporters) before and after microinjection of 23 nl of NaCl+glycine (0.5M). We used a water volume of 700 nl to estimate the intracellular concentrations produced by microinjection, with initial values in the upper range because of the accumulation already produced by the large glycine current (3.1µA at −40 mV, corresponding to a glycine flux of 15.4pmol s−1). The table of concentrations includes the theoretival reversal potential of ATB0,+. The purpose of this model was to examine the voltage dependence of the EC50, assuming that the rate controlling glycine binding (k1f,k3b) and unbinding (k1b,k3f) are voltage independent. Adding an intrinsic valence of the transporter (zc) that does not contribute to the net charge carried during a cycle was necessary to reduce and balance the valence of the translocation steps for the fully loaded and empty carrier. The limiting rate constant at low and high glycine concentrations are indicated with blue (k1f) and red (k4f) arrows, respectively. B-C glycine I-Vs before (B) and after (C) intracellular microinjection. ATB0,+ steadystate currents were isolated by subtraction using αMT (1 mM) as described in Fig 4.B. The solid lines correspond to the fit using the concentrations for each cosubstrates in the table in A. The rate constants are given in A. DThe EC50 estimated from the model using the rate constants in A shows ATB0,+ characteristic and unusual voltage-dependence at negative voltages

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