A three-sodium-to-glycine1
stoichiometry shapes the structural2
relationships of ATB
0,+
with GlyT23
and GlyT1 in the SLC6 family4
Bastien Le Guellec
1†
, France Rousseau
1†
, Marion Bied
1
, Stéphane Supplisson
1*
5
*For correspondence:
stephane.supplisson@bio.ens.psl.eu
(SS)
†
These authors contributed
equally to this work
1
Institut de Biologie de l’ENS (IBENS), Inserm, CNRS, École normale supérieure, PSL6
Research University, Paris, France7
8
Abstract ATB
0,+
(SLC6A14) absorbs all neutral and cationic amino acids in the distal colon and9
lung epithelia, and is part of the amino acid transporter branch I of the SLC6 family with GlyT110
(SLC6A9) and GlyT2 (SLC6A5), two glycine-specific transporters coupled to 2:1 and 3:1 Na
+
:Cl
−
,11
respectively. However, ATB
0,+
stoichiometry that specifies its driving force and electrogenicity12
remains unsettled. Using the reversal potential slope method, here we demonstrate that13
ATB
0,+
-mediated glycine transport is coupled to 3 Na
+
and 1 Cl
−
and has a charge coupling of 2.114
e/glycine. ATB
0,+
behaves as a unidirectional transporter with limited efflux and exchange15
capabilities. Analysis and computational modeling of the pre-steady-state charge movement16
reveal higher sodium affinity of the apo-ATB
0,+
, and a locking trap preventing Na
+
loss at17
depolarized potentials. A 3 Na
+
/ 1 Cl
−
stoichiometry substantiates ATB
0,+
concentrative-uptake18
and trophic role in cancers and rationalizes its structural proximity with GlyT2 despite their19
divergent substrate specificity.20
21
Introduction22
Several Na
+
-coupled amino acid transporters mediate the active uptake of glycine, which, despite23
its chemical simplicity - a nonessential amino-acid, without a side-chain or stereoisomer, and a24
membrane-impermeable zwitterion at physiological pH (Chakrabarti, 1994) - is nevertheless (1)25
a complex signaling molecule in the CNS as a fast inhibitory neurotransmitter and an agonist of26
the NR1 and NR3 subunits of the NMDA receptors (Legendre, 2001; Zeilhofer et al., 2012; Johnson27
and Ascher, 1987; Chatterton et al., 2002; Grand et al., 2018), (2) a key organic osmolyte for the28
regulation of cell volume in early embryos (Dawson et al., 1998), and (3) an essential metabolite for29
cell growth (Wang et al., 2013). Profiling studies have identified an increase in glycine one-carbon30
metabolism as an early warning signal of the rapid cell proliferation in cancers (Jain et al., 2012;31
Locasale, 2013; Amelio et al., 2014).32
Three of these glycine transporters (GlyT1, GlyT2, and ATB
0,+
) and the proline transporter (PROT)33
are clustered in the amino-acid transporters branch (I) of the Solute Carrier 6 (SLC6) family, also34
named Neurotransmitter:Sodium Symporters (NSS) (Bröer and Gether, 2012 ). GlyT1 and GlyT2 cor-35
respond to the classic, high-affinity and glycine-specific "system Gly", while ATB
0,+
lacks substrate36
specificity (Christensen, 1990) and transports a broad variety of 𝛼 and 𝛽 amino acids, D- and L-37
amino acids, amino acid derivatives, and pro-drugs (Sloan and Mager, 1999; Winkle et al., 1985;38
Hatanaka et al., 2001; Nakanishi et al., 2001; Hatanaka et al., 2002, 2004; Anderson et al., 2008).39
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This broad specificity suggests that ATB
0,+
has a distinctive substrate site from the one of GlyT140
and GlyT2, but nevertheless, we refer to ATB
0,+
as a glycine transporter for simplicity.41
GlyT1 and GlyT2 recapture and recycle glycine released at fast inhibitory synapses in the spinal42
cord, brainstem and cerebellum (Legendre, 2001; Eulenburg et al., 2005; Zeilhofer et al., 2012;43
Ankri et al., 2015). GlyT2 is a specific marker of glycinergic neurons and operates like a unidi-44
rectional glycine pump as its 3 Na
+
/1 Cl
−
stoichiometry provides an excessive driving force for45
influx (Roux and Supplisson, 2000; Supplisson and Roux, 2002). GlyT2 inactivation disrupts the cy-46
tosolic accumulation and vesicular release of glycine (Gomeza et al., 2003; Rousseau et al., 2008;47
Apostolides and Trussell, 2013), and GlyT2
−∕−
knockout mice develop a severe hypoglycinergic syn-48
drome (Gomeza et al., 2003) while mutations of human SLC6A9 cause Hyperekplexia (OMIM:#614618),49
a rare neurological disorder with exaggerated startle reflexes (Rees et al., 2006; Carta et al., 2012).50
In contrast, GlyT1 is as a bidirectional, 2 Na
+
/1 Cl
−
-coupled transporter that is expressed primar-51
ily on astrocytes and behaves as a buffer, sink, or source of extracellular glycine (Zafra et al.,52
1995; Supplisson and Bergman, 1997; Huang and Bordey, 2004; Aubrey et al., 2005, 2007; Sipilä53
et al., 2014; Shibasaki et al., 2017). However, GlyT1 concentrative uptake is sufficient to specify54
the glycinergic phenotype of retinal amacrine cell (Eulenburg et al., 2018), and critical for the cell55
volume regulation of early mouse embryos (Steeves et al., 2003; Steeves and Baltz, 2005). On56
the extracellular side, GlyT1 tunes the basal concentration and spillover of glycine (Sipilä et al.,57
2014; Ahmadi et al., 2003) that gate NMDARs activation depending on their subunits composition,58
synaptic location, brain structure, and developmental stage (Supplisson and Bergman, 1997; Sup-59
plisson and Roux, 2002; Tsai et al., 2004; Martina et al., 2005; Papouin et al., 2012; Bail et al.,60
2015; Ferreira et al., 2017; Otsu et al., 2019). GlyT1-deficit leads to an hyperglycinergic phenotype61
with hypotonia and motor disorders in GlyT1
−∕−
-knockout mice and Shocked Zebrasfish-mutant62
(Gomeza et al., 2003; Tsai et al., 2004; Cui et al., 2005; Mongeon et al., 2008; Hirata et al., 2010;63
Eulenburg et al., 2010). Mutations of human SLC6A5 caused glycine encephalopathy with normal64
serum glycine (OMIM:#617301), a severe metabolic disease that produces hypotonia and respira-65
tory failures in neonatal (Kurolap et al., 2016; Hauf et al., 2020).66
In comparison, the energetic and biophysical properties of ATB
0,+
remain undercharacterized.67
ATB
0,+
mediates electrogenic and concentrative uptakes of all neutral and cationic amino acids68
with Hill coefficients suggesting a 2 Na
+
and 1 Cl
−
stoichiometry (Sloan and Mager, 1999; Hatanaka69
et al., 2001; Nakanishi et al., 2001; Karunakaran et al., 2008). ATB
0,+
is expressed in the lung and70
distal colon epithelia, and in the mammary and pituitary glands (Villalobos et al., 1997; Sloan and71
Mager, 1999; Ugawa et al., 2001; Sloan et al., 2003; Chen et al., 2020). Mice lacking ATB
0,+
are viable72
and with normal phenotype (Babu et al., 2015; Ahmadi et al., 2018), but genome-wide association73
studies have identified SNPs in non coding regions of SLC6A14 that are linked to obesity (Suviolahti74
et al., 2003; Corpeleijn et al., 2010; Sivaprakasam et al., 2021), male infertility (Noveski et al., 2014),75
and the phenotypic variation and severity of cystic fibrosis (Sun et al., 2012; Corvol et al., 2015). In76
particular, the lack of ATB
0,+
impairs intestinal fluid secretion in the context of cystic fibrosis and is77
responsible of Meconium Ileus at birth (Ahmadi et al., 2018; Ruffin et al., 2020). Ganapthy’s group78
has demonstrated the implication of ATB
0,+
in cancers, as one of the four amino-acid transporters79
with SLC1A5, SLC7A5, SLC7A11 that are up-regulated in tumors for matching the increased demand80
in amino acids for cell growth, and more particularly for glutamine and glycine (Bhutia et al., 2014;81
Coothankandaswamy et al., 2016; Sikder et al., 2017; Sniegowski et al., 2021).82
Here, we investigated the stoichiometry and exchange mode of ATB
0,+
in order to better evalu-83
ate its driving force and transport properties under physiological and pathological conditions. An84
additional motive was to understand the raison d’être of the surprising structural hierarchy be-85
tween these three glycine transporters of the SLC6 family. We used the reversal potential of ATB
0,+
86
in order to establish its stoichiometric coefficients by a thermodynamic method. Our results revise87
the apparent consensus for a 2 Na
+
stoichiometry, and the charge/glycine and charge movement88
in glycine-free media confirmed the higher Na
+
coupling. Finally, we show that 3 Na
+
-coupling re-89
duces both the efflux and exchange of glycine in ATB
0,+
and GlyT2-expressing oocytes, a property90
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that is not shared by other 3 Na
+
-coupled transporters like the glutamate transporter EAAT2 who91
operates by a distinct elevator mechanism (Kavanaugh et al., 1997; Drew and Boudker, 2015).92
Results93
Odd phylogenetic and functional relationships of ATB
0,+
with GlyT1 and GlyT294
The position of ATB
0,+
in the phylogenetic tree of SLC6 transporters (Figure 1A), closer to GlyT295
than this one is of GlyT1, seems at odds with their marked functional divergence in terms of speci-96
ficity and affinity for glycine, as detailed below and in the next paragraph. Although the length of97
the common branch is short (0.14 for ATB
0,+
/GlyT2 vs. 0.07 for GlyT1/PROT, Figure 1A), the same98
split is nonetheless observed for all Vertebrates orthologs despite a greater divergence in ATB
0,+
99
sequences (Figure 1A-B). Indeed, the percentage of identities (ID) between orthologs is lower for100
ATB
0,+
(39 % ID) than for GlyT2 (69.7 % IDs) and GlyT1 (60.3 % ID) as shown inFigure 1B. A higher di-101
vergence is also detected in the more conserved transmembrane segments with 46.5 % vs. 77.3 %,102
and 70 % IDs, respectively Figure 1–Figure Supplement 1A. Venn diagrams of private and shared103
residues confirm that mGlyT2 shares more IDs with mATB
0,+
(n = 95) than with mGlyT1 (n = 76, Fig-104
ure 1C, Figure 1–Figure Supplement 2) and the GlyT2/ATB
0,+
pair shows a similar excess for each105
ortholog (+15.5±1.1 ID, n = 8, Figure 1–Figure Supplement 1B). PROT was included in the analysis106
to count only for IDs specific of a glycine-transporter pair (GlyT2/ATB
0,+
: 55.4±1.1 ID, GlyT2/GlyT1:107
39.9±1.5 ID, ATB
0,+
/GlyT1: 25.5±1.5 ID, n = 8, Figure 1–Figure Supplement 1B).108
The substrate site of ATB
0,+
is expected to differ substantially from the one of GlyT1 and GlyT2109
as it can accommodate a large variety of amino acids size and side chains, with aliphatic, cationic,110
hydrophobic, or hydrophilic properties. Indeed, all the natural amino acids at the exception of111
glutamate and aspartate evoke an inward current in ATB
0,+
-expressing oocytes, in contrast to the112
absolute glycine specificity of GlyT1 and GlyT2 (Figure 1D). Therefore, other factors than a common113
substrate site for glycine must explain the structural proximity of ATB
0,+
and GlyT2. Nevertheless, it114
is worth noticing that both transporters share the same intolerance for N-methyl-derivative while115
GlyT1 transports sarcosine (Figure 1D).116
ATB
0,+
glycine-EC
50
shows an anomalous voltage-dependency117
Glycine evokes inward currents in ATB
0,+
-expressing oocytes that do not reverse up to +50 mV (Fig-118
ure 2A), and are strictly dependent on Na
+
and Cl
−
(Figure 2–Figure Supplement 1). The current-119
voltage relationship is quasi-linear at saturating glycine concentration, indicating a weak voltage-120
dependency of its transport cycle similar to GlyT1 and GlyT2 (Figure 2B), with efold of 296, 313, and121
254 mV, respectively.122
In contrast, ATB
0,+
dose-response-curve for glycine shows a distinct and lower apparent affin-123
ity, with a more complex voltage-dependency than for GlyT1 and GlyT2 (Figure 2C-D) as previously124
reported in Roux and Supplisson (2000). In particular, ATB
0,+
-EC
50
shows a minimum at −20 mV125
(171±4 µM, n = 6), and then increases at both, depolarized and hyperpolarized potentials, whereas126
GlyT2- and GlyT1-EC
50
are voltage-independent in the negative range (Figure 2D, Roux and Supplis-127
son (2000)).128
On average, a saturating glycine concentration evokes ∼ 6-fold larger currents in ATB
0,+
-expressing129
oocytes than for GlyT2 and GlyT1 (1450.0±98.8 pA (n = 65) vs. 250.0±29.7 pA (n = 48) , and 245.2±17.3 pA130
(n = 85) at 𝑉
𝐻
=−40 mV for 1–2 mM (ATB
0,+
) and 200 µM (GlyT2, GlyT1), respectively, Figure 3A). There-131
fore, we examined whether the membrane-expression, charge per glycine, and turnover-rate can132
account for ATB
0,+
larger electrogenicity.133
ATB
0,+
and GlyT2 share similar charge movements134
We used the charge-movement of ATB
0,+
as a proxy of their cell-surface expression in Xenopus135
oocytes. The Figure 3B shows representative traces of the presteady-state currents (PSSCs) of136
GlyT1, GlyT2 and ATB
0,+
rthat are ecorded in glycine-free media and isolated using their specific137
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inhibitors (ORG24598, ORG25543 and 𝛼-Methyl-D,L-tryptophan (𝛼MT), respectively) to subtract the138
oocyte endogenous-currents. It could be noticed that ATB
0,+
and GlyT2 share asymmetric PSSCs,139
with large outward-currents and biphasic time-courses, whereas GlyT1-PSCCs are weakly voltage-140
dependent (Figure 3B ).141
The charge movement plotted as function of voltage (Q-V) confirms the likeness of ATB
0,+
and142
GlyT2, with the same slope and clear evidence of saturation (Figure 3C). Fits of Q-Vs with a Boltz-143
mann equation (2) show similar apparent charge (𝑧
max
) for ATB
0,+
(1.26±0.02 e, n = 38) and GlyT2144
(1.25±0.01 e, n = 53) that are almost one charge higher than for GlyT1 (0.37±0.02 e, n = 14, Figure 3D).145
The potential of half distribution (𝑉
1∕2
) is positive for ATB
0,+
(+16.2±0.9 mV, n = 38) and right-shifted146
relative to GlyT2 and GlyT1 (−35.0±0.8 mV (n = 5) and −17.3±3.3 mV (n = 14), respectively, Figure 3E).147
Finally, the average ATB
0,+
-𝑄
max
is 2.4-fold larger than for GlyT2 (44.0±2.8 nC (n = 38) vs. 18.6±1.3 nC148
(n = 53), respectively Figure 3F), thus confirming ATB
0,+
higher cell-surface expression.149
Overexpression of membrane transporters can expand the cell-surface of injected oocytes150
(Hirsch et al., 1996), we compared the linear membrane capacitance (Cm) of non-injected oocytes151
(207±3 nF, n = 33) with oocytes expressing GlyT1 (227±7 nF, n = 11), GlyT2 (243.0±5.2 nF, n = 10),152
and ATB
0,+
(301.0±6.3 nF, n = 50, p<0.001) Figure 3–Figure Supplement 1C. As the membrane ca-153
pacitance is known to be proportional to the surface area (Cm = 1 µF/cm
2
), the ∼45 % increase in154
Cm suggests a similar expansion of surface area in oocytes expressing ATB
0,+
.155
A thermodynamic determination of ATB
0,+
stoichiometric coefficients156
To establish the stoichiometric coefficients of each cosubstrate, we adapted to ATB
0,+
the reversal157
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 was159
first necessary to alter the intracellular composition of oocytes by micoinjection and find a set of160
intracellular concentrations able to reduce the excessive driving force of ATB
0,+
and shift its reversal161
potential below +50 mV.162
Initial attempts with a small nanoliter-volume injection of glycine (1 M) sufficient to evoke an163
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 indicated165
by the lower amplitude of the transport current evoked by a second glycine application, triggers166
small outward currents at −20 mV in GlyT2- and ATB
0,+
-expressing oocytes (Figure 4A,B). Eventu-167
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 subtraction169
with 𝛼MT 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.172
Using this experimental paradigm, we impaled two electrode voltage-clamp oocytes express-173
ing ATB
0,+
with a third micropipette and injected 13–23 nL of solution containing 0.5 M NaCl and174
glycine. As a steady outward current develops following injection, we constructed three I-Vs for175
each oocyte with either a change in glycine (2, 0.2, and 0.02 mM, Figure 5A), Na
+
(100, 30, and176
10 mM, Figure 5B), or Cl
−
(100, 30, and 10 mM, Figure 5C) and the current reversal potentials were177
plotted in linear-log plots for each cosubstrate (Figure 5D). Regression analysis show similar slopes178
for glycine (28.6±1.0 mV/decade, n = 8) and Cl
−
(31.5±2.7 mV/decade, n = 6), but much steeper for179
Na
+
(84.7±4.5 mV/decade, n = 11 Figure 5D). The average stoichiometric coefficients determined180
by the slope ratios (𝑛
Gly
= 1.0±0.1 , 𝑛
Cl
= 1.1±0.1 , and 𝑛
Na
= 2.9±0.2 , Figure 5E) support a 3 Na
+
, 1 Cl
−
,181
1 glycine stoichiometry for ATB
0,+
.182
In agreement, we measured a charge coupling (𝑧
T
) of 2.08 e/glycine from the slope of the linear183
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). Fi-185
nally, an apparent turnover rate (𝜆 = 18 s
−1
) was estimated from the linear relationship between 𝐼
max
186
and 𝑄
max
(29.7 s
−1
), after correction for the ratio of glycine-coupled/glycine-uncoupled charges (see187
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Figure 5–Figure Supplement 1B). Together, the charge coupling and turnover rate of ATB
0,+
well pre-188
dict the current amplitude for an average expression in oocyte (𝑄
max
= 44 nC) as 𝐼
max
= 𝑄
max
𝑧
T
𝑧
max
𝜆 =189
1307 nA), within the range reported in 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-facing193
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.196
The Figure 6A shows a marked and unexpected reduction in the envelope of ATB
0,+
-PSSCs for a197
tenfold reduction in sodium concentration. The upper range of the voltage steps was extended to198
+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 6A200
show that a marked current-crossing at 100 mM and 50 mM Na
+
that is strongly reduced at 20 mM201
and absent at 10 mM.202
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 Figure 6–Figure Supplement 1A), a ten-fold204
reduction in [Na
+
] is predicted to left-shift the Q-V without altering its slope nor 𝑄
max
(Mager et al.,205
1996). Although individual fits with a Boltzmann equation support this prediction for GlyT2 (Fig-206
ure 6–Figure Supplement 2A-B), this is not the case for ATB
0,+
as shown by the decreasing peaks of207
the Q-V first derivative (Figure 6–Figure Supplement 2C-D). As expected, the scheme #1 generates208
a poor global fit of ATB
0,+
Q-Vs as function of Na
+
, while being acceptable for GlyT2 Figure 6–Figure209
Supplement 3A).210
Then we tested a sequential, linear model that preserves Hill formalism for simplicity, with a211
single and voltage-dependent binding step for multiple Na
+
with a charge 𝑧𝛽, but includes a gating212
step controlling the access to or priming of Na
+
-sites for binding (schemes #2 and #3, Figure 6–213
Figure Supplement 1A). Gate opening and closure are voltage-dependent with 𝑧𝛼 the charge of the214
gating step (U⟷ U
𝑐
) of Na
+
unbound transporters and 𝑧𝛾 the charge displacement that lock Na
+
215
(B⟷B
𝑐
) (Figure 6–Figure Supplement 1A) .216
Because these two-path exits from the Na
+
-bound state (B) at positive potentials could carries217
asymmetric charges (𝑧𝛾>𝑧𝛼+𝑧𝛽), scheme #3 was able to solve the apparent Na
+
-dependency of 𝑧
max
218
(Figure 6D) as indicated by the overwhelming difference in Akaike criterion information (ΔAICc) tab-219
ulated in Figure 6–Figure Supplement 1B. Furthermore, the scheme #3 describes also the constant220
𝑧
max
of GlyT2 Q-V (Figure 6D), as 𝑧𝛾 ≤ 𝑧𝛼 + 𝑧𝛽. Na
+
locking in the apo outward facing conformation221
of ATB
0,+
is further facilitated by an higher affinity for Na
+
, with a 3.5 fold difference in Kd (23 and222
80 mM, for ATB
0,+
and GlyT2, respectively), whereas conversely, GlyT2 lower affinity facilitates gate223
closure primarily from the Na
+
-unbound state U (Figure 6–Figure Supplement 1C,Figure 6–Figure224
Supplement 4). As expected for a linear model, extrapolations at extreme voltage (+2 V) confirm a225
convergence to the same 𝑄
max
value Figure 6–Figure Supplement 5.226
Globally, the fit parameters for ATB
0,+
and GlyT2 in the scheme #3 show a remarkable coherency227
(Figure 6B,C), with little difference in the Hill coefficients (2.3 and 2.4, for ATB
0,+
and GlyT2 respec-228
tively). Because it is a four-state model, the median voltage (V
𝑚𝑒𝑑.
) was estimated as function of229
[Na
+
] (Figure 6D) as shown in Figure 6–Figure Supplement 6) that is positive for [Na
+
]> 40 mM for230
ATB
0,+
but always negative for GlyT2, up to 200 mM (Figure 6E), indicating that Na
+
binding in the231
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 time233
courses of ATB
0,+
transient current using the fitted equilibrium constants and charges of Figure 6B234
(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 #3.236
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![Figure 2. Weak and anomalous voltage-dependency of ATB0,+-mediated currents (A) Glycine evokes large inward currents in ATB0,+-expressing oocytes. Current-voltage (I-V)relationships of the glycine-evoked current (20µM (square), 200µM (triangle) and 2mM (circle)). (B) Linear I-Vs recorded at saturating glycine concentrations for ATB0,+ (blue, [Gly]=2mM, n = 10,slope = 0.92% mV−1, R2 = 1.000, p<2 1016), GlyT2 (red, [Gly]=200µM, n = 11, slope = 1.07% mV−1,R2 = 0.998, p<2 10−16), and GlyT1 (green, [Gly]=200µM, n = 11, slope = 0.87% mV−1, R2 = 0.998, p =1.78 1013). Currents are normalized by the absolute amplitude at −40mV and linear regressionswere performed on the −140–−40mV voltage range. (C) Glycine dose-response curve for ATB0,+(blue circles, VH = −40mV, n = 32). Individual curves were fitted by the equation: IGly = Imax1+ EC50[Gly]o ,and then normalized by the maximal amplitude (Imax). The solid line represents the fit of thenormalized equation. The glycine EC50 of ATB0,+ (ECATB0,+50 =189 ±5.6µM, n=21) is higher thanfor GlyT1 (ECGlyT150 = 28µM, green dashed line) and GlyT2 ( ECGlyT250 = 25µM, red dashed line) aspreviously determined in Roux and Supplisson (2000). (D) Anomalous voltage-dependency of ECATB0,+50 at hyperpolarized potentials (blue circles, n = 6). The solid line is the fit to the equa-tion: ECATB0,+50 = 66.2 exp (1.76 V ) + 133.9 exp (−0.185 V ). The dashed lines are fits for ECGlyT150 (20.5 exp (0.7 V ) + 21.7) and ECGlyT250 (43.0 exp (1.54 V ) + 21.6, = FRT ) previously determined in Roux and Supplisson (2000).](/figures/figure-2-weak-and-anomalous-voltage-dependency-of-atb0-376npoxy.webp)
![Figure 6. The charge movement shows evidence for an external gate controlling Na+ access and locking inapo-ATB0,+ (A) Representative traces of ATB0,+-PSSCs recorded at the onset of voltage steps (from +110mV to −150mV by decrements of20mV; VH = −40mV) in glycine-free solution containing the indicated Na+ concentration. ATB0,+-specific PSSCs are isolated bysubtraction of traces recorded in the presence of -MT (1mM ); any residual currents have been subtracted for clarity. (B-C)Normalyzed Q-Vs for ATB0,+ (B, n = 14, ±SEM) and GlyT2 (C, n = 7, ±SEM) for the four Na+ concentrations shown in A, with thesame color code. The data are average of sets of four Q-Vs normalized by the Qmax value for [Na+]=100mM. The solid linescorrespond to the fit of the four-state scheme (#3) depicted above, with a gating, Na+-binding and Na+-locking steps that aredetailed in Figure 6–Figure Supplement 1. The green and blue arrows in the model represent the two steps activated duringdepolarization while the purple arrows identifies steps activated by hyperpolarizing voltage steps. The dashed lines of the Q-Vsare extrapolations of the model outside the experimental voltage range. (D-E) The (D) median voltage (D) and apparent chargedisplacement (E) plotted as function of [Na+]. The median voltage is estimated as shown in (Figure 6–Figure Supplement 6). (F)model prediction of ATB0,+-PSSCs for the same conditions as in A. The rate constants are described in the Materials and Methodssection. The voltage independent rate constants are k120=79 s−1, k230=88mol−1 s−1 and k340=23 s−1, with d = 0.36, d = 0.42and d = 0.7. The off rate constants are back calculated using the equations, equilibrium constants and valencies detailed in Figure 6–Figure Supplement 1B. Figure 6–Figure supplement 1. A four-state sequential model for the charge movement of ATB0,+ and GlyT2 Figure 6–Figure supplement 2. The equivalent charge displacement of ATB0,+ is Na+-dependent](/figures/figure-6-the-charge-movement-shows-evidence-for-an-external-2631lhpb.webp)
