A three-sodium-to-glycine stoichiometry shapes the structural relationships of ATB0,+ with GlyT2 and GlyT1 in the SLC6 family

ATB0,+ (SLC6A14) absorbs all neutral and cationic amino acids in the distal colon and lung epithelia, and is part of the amino acid transporter branch I of the SLC6 family with GlyT1 (SLC6A9) and GlyT2 (SLC6A5), two glycine-specific transporters coupled to 2:1 and 3:1 Na+:Cl−, respectively. However, ATB0,+ stoichiometry that specifies its driving force and electrogenicity remains unsettled. Using the reversal potential slope method, here we demonstrate that ATB0,+-mediated glycine transport is coupled to 3 Na+ and 1 Cl− and has a charge coupling of 2.1 e/glycine. ATB0,+ behaves as a unidirectional transporter with limited efflux and exchange capabilities. Analysis and computational modeling of the pre-steady-state charge movement reveal higher sodium affinity of the apo-ATB0,+, and a locking trap preventing Na+ loss at depolarized potentials. A 3 Na+/ 1 Cl− stoichiometry substantiates ATB0,+ concentrative-uptake and trophic role in cancers and rationalizes its structural proximity with GlyT2 despite their divergent substrate specificity.


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A thermodynamic determination of ATB 0,+ stoichiometric coefficients 156 To establish the stoichiometric coefficients of each cosubstrate, we adapted to ATB 0,+ the reversal 157 potential slope method (e.g., Appendix 1) that successfully solved the stoichiometry of EAAT3, 158 GlyT1, and GlyT2 (Zerangue and Kavanaugh, 1996; Roux and Supplisson, 2000). For this, it was 159 first necessary to alter the intracellular composition of oocytes by micoinjection and find a set of 160 intracellular concentrations able to reduce the excessive driving force of ATB 0,+ and shift its reversal 161 potential below +50 mV. 162 Initial attempts with a small nanoliter-volume injection of glycine (1 M) sufficient to evoke an 163 outward currents in GlyT1-expressing oocytes, failed to reverse ATB 0,+ nor GlyT2 Figure 4A,B. Fur-164 ther addition of NaCl in the pipette solution that strongly reduced ATB 0,+ driving-force as indicated 165 by the lower amplitude of the transport current evoked by a second glycine application, triggers 166 small outward currents at −20 mV in GlyT2-and ATB 0,+ -expressing oocytes (Figure 4A,B).  ally, we selected injection parameters that evoked robust and steady outward currents in ATB 0,+ -168 expressing oocytes ( Figure 4C). These transporter-mediated currents are isolated by subtraction 169 with MT Figure 4CD, and are bidirectional, with a stable and measurable reversal potential (Fig-170 ure 4D, blue triangles)) that is sensitive to extracellular manipulation of each cosubstrate concen-171 tration.

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In agreement, we measured a charge coupling ( T ) of 2.08 e/glycine from the slope of the linear 183 relationship between the time-integral of the transport current and 14 C glycine uptake, thus con-184 firming the tight electrogenic coupling of ATB 0,+ transport-cycle (Figure 5-Figure Supplement 1A).  nally, an apparent turnover rate ( = 18 s −1 ) was estimated from the linear relationship between max 186 and max (29.7 s −1 ), after correction for the ratio of glycine-coupled/glycine-uncoupled charges (see   Figure 3A. 190 An external gate controls the access and locking of Na + sites in the apo-ATB 0,+ 191 Evidence from the shift in Q-Vs ( Figure 3E) as well as from the abnormal rectification of the glycine-192 EC 50 at negative potentials ( Figure 2D) suggests an higher sodium affinity of the apo outward-facing 193 conformation of ATB 0,+ , and more complex allosteric interactions with Na + than for GlyT2. There-194 fore, we examined in more details the Na + -and voltage-dependence of ATB 0,+ and GlyT2 charge-195 movement.

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The Figure 6A shows a marked and unexpected reduction in the envelope of ATB 0,+ -PSSCs for a 197 tenfold reduction in sodium concentration. The upper range of the voltage steps was extended to 198 +110 mV in order to catch evidence of saturation, such as the current-crossing as the charge move-199 ment approached saturation but not the current peak-amplitude ( Figure 6A). Traces in Figure 6A 200 show that a marked current-crossing at 100 mM and 50 mM Na + that is strongly reduced at 20 mM 201 and absent at 10 mM.

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According to a minimal two-state Hill model for multiple Na + proposed for GAT1 (Mager et al. 203 (1996, 1998)), and corresponding to the scheme #1 in  210 Then we tested a sequential, linear model that preserves Hill formalism for simplicity, with a 211 single and voltage-dependent binding step for multiple Na + with a charge , but includes a gating 212 step controlling the access to or priming of Na + -sites for binding (schemes #2 and #3,  (Figure 6E), indicating that Na + binding in the 231 absence of voltage is energetically favorable for ATB 0,+ while not for GlyT2 Figure 6F. 232 Finally, we challenged the model #3 to reproduce the highly asymmetric and biphasic time 233 courses of ATB 0,+ transient current using the fitted equilibrium constants and charges of Figure 6B 234 (see material and method). Figure 6F shows simulations that effectively recapitulate the Na + dy-235 namics of ATB 0,+ PSSCs, supporting further the gating and locking mechanisms of scheme #3. As expected from their difference in driving force, the basal glycine efflux was lower for ATB 0,+ 246 and GlyT2 compared to GlyT1 (Figure 7,A,B). Application of glycine stimulated exchange of GlyT1-247 expressing oocytes as shown by the 8-fold increase in efflux rate ( Figure 7A,B), while only slightly 248 in GlyT2 and ATB 0,+ -expressing oocytes (Figure 7A,B). 249

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Here, we establish a new 3 Na + , 1 Cl − , 1 amino acid stoichiometry for ATB 0,+ that supports its unidi-251 rectional and concentrative uptake properties, and provides a rationale for its structural proximity 252 with GlyT2 in the SLC6 family.  260 We used the reversal potential slope-ratio method that is based on the zero-flux equation (Ap- an oocyte effective water-volume of 600 nL, that is sufficient to left-shift the current reversal po-270 tential to +32.8 mV or +33.1 mV for a 2 or 3 Na + -coupling, respectively, which is in agreement with 271 the average value of 32.5 mV in Figure 5D. 272 The average reversal potential slope of 28.6 mV determined for a ten-fold reduction in [Gly] is 273 close to the theoretical slope of 29.6 mV for two charges at 25°C (Equation 6), and confirms that 274 they are both energetically coupled to the transport cycle. The slope values for Na + (84.6 mV/decade) 275 and Cl − (32 mV/decade) are also closed to the theoretical values for 3 Na + /gly and 1 Cl − /gly (88.4 and 276 28.6 mV/decade, respectively), thus establishing a 3 Na + /1Cl − /1 glycine stoichiometry for ATB 0,+ .

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Does ATB 0,+ need such a large driving force ? 278 The increase in driving force provided by a third Na + substantiates the "highly-concentrative" adjec- should not be a limiting factor for ATB 0,+ scavenging function.

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Upregulation of ATB 0,+ has been detected in twelve types of solid cancers (Sikder et al., 2017, 308 2020) suggesting that its overexpression is the main mechanism to increase the uptake rate of 309 glutamine or glycine, because of the dynamic cis and trans competition when ATB 0,+ is exposed to

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Not surprisingly, the Q-Vs of ATB 0,+ and GlyT2 confirms their common ionic coupling as they run 327 parallels indicating similar charge displacement, which is almost one charge higher (+0.88 e) than 328 for GlyT1, thus suggesting a net contribution of the third Na + . Both transporters have Hill coeffi-329 cient above 2, indicating a cooperativity of the three Na + sites. However, ATB 0,+ large difference 330 in 1∕2 (+51 mV, Figure 3D) and lower microscopic Kd (23 vs. 80 mM, Figure 6B-C)) relative to GlyT2 dependency is unexpecte, it is nevertheless not unique as a similar reduction in slope and max was 336 reported for GAT3, yet uninterpreted with a kinetic model (Sacher et al., 2002). We show that addi-337 tion of a gating and locking steps with asymmetric charges solve the apparent Na + -dependency of 338 ATB 0,+ Q-V. A dynamic equilibrium between the two Na + -unbinding paths, with either its extracellu-339 lar release or locking in the transporter as the external gate close, and a small imbalance between 340 the valency of the two paths predict the reduction in max at low Na + concentrations. Conversely, 341 the locking step plays little role for GlyT2 because of its lower affinity favored gate closure of Na + -342 unbound transporters. It can be speculated that Na + -locking in ATB 0,+ , by preventing Na + release, 343 stabilize Na + -bound conformation at depolarized potentials.   Organon. We used choline + or Tris + (2-amino-2-hydroxymethyl-1,3-propanediaol) and gluconate − 420 or methyl-sulfonate − as substitutes for Na + and Cl − , respectively. (2) k is the Boltzmann constant, and T the absolute temperature.

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In the Figure 6C, sets of four Q-Vs were measured at different Na + concentrations for each
med. is estimated using the FindRoot and Nintegrate functions of Mathematica 12.

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To simulate the time course of the relaxation currents in Figure 6F, we used the NDSolve function (Mathematica 12) and numerically integrates the three differential equations that defined the dynamics of the four-state model U UBB (Figure 6-Figure Supplement 1A  M ac ac a n.

Mus m.
N a n o ra n a p .   Figure 1A shows that ATB 0,+ (blue, n = 17) sequences are more divergent during Vertebrates evolution than GlyT1 (green, n = 16) and GlyT2 (red, n = 16). The cumulative count (+1 if all sequences shared the same residue) starts at the first shared-position in the alignment (R57 for ATB 0,+ , R309 for GlyT2 and R40 for GlyT1). (C) Area-proportional Venn diagrams of the number of shared and private residues for mGlyT1, mGlyT2 and mATB 0,+ based on the sequences alignment shown in Figure 1-Figure Supplement 2). GlyT2 shares more identity with ATB 0,+ (n = 95) than with GlyT1 (n = 76) for all species examined            Figure 1A, using the human SERT structure as a template for TMs delimitation (Coleman et al., 2016). (B) Radar plot of the cumulative number of sequence identity for each glycine-transporter pairs (ATB 0,+ -GlyT2 (purple), GlyT2-GlyT1 (cyan), ATB 0,+ -GlyT1 (yelow)). Pair identity shared with PROT are excluded as indicated in the legend  recorded at the onset of voltage steps (from +50 mV and −130 mV by decrements of 10 mV) and normalized by the peak amplitude for a representative ATB 0,+ -expressing oocyte. Each trace is normalized by the absolutepeak current (the trace at V = −40 mV is not included). Middle panel: same as left in the presence of 1 mM -MT (blue traces) to block ATB 0,+ -charge movement. The gray traces represent currents recorded with the same protocol for a representative non injected oocyte. Right panel: the charge movement of ATB 0,+ (orange trace) for a single step at 50 mV after -MT subtraction. (B) Cm is given by the slope of the linear charge-voltage relationships and increases in oocytes expressing transporter membrane capacitance (Cm) of non-injected oocytes (209±3 nF, n = 33) with oocytes expressing GlyT1+ORG24598 (227±7 nF, n = 11), GlyT2 +ORG25543 (242.8±5.2 nF, n = 10), and ATB 0,+ + -MT320.6±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 ATB 0,+ (n = 50; a group of 34 ATB 0,+ -expressing oocytes was added by estimating the slope of the Q-V at hyperpolarized potentials). max for oocytes expressing ATB 0,+ and hold at V =−40 mV. An apparent turnover rate ( =18 s −1 ) is derived from the max / max slope (29.7 s −1 , R 2 =0.848, p=3.77 10 −12 ) using the equation = max max , with z =2.08 e and z =1.26 e as determined in A and Figure 3D). The shade area corresponds to the 95 % confidence interval. The model shows the three kinetic schemes tested here to fit ATB 0,+ -and GlyT2-Q-Vs as function of Na + . In glycine-free media, outward facing transporters are assumed to be either Na + -unbound (U , U) or Na + -bound (B, B ). The UB scheme (#1) corresponds to the binary Hill model proposed by Mager et al. (1996) 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 , and the equivalent charge . For simplicity, we define , 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 and 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 B state (Figure 6-Figure Supplement 4). Positive voltage steps trigger gate closure from Na + -unbound (U) and Na + -bound state (B). (B) The max and normalized Q-V equations are tabulated for each scheme, with the Δ values estimated from the fit. max for scheme #3 is conditioned by equality or inequality of the valencies of the Na + -release and Na + -locking paths