Functional and Kinetic Comparison of Alanine Cysteine Serine Transporters ASCT1 and ASCT2

Neutral amino acid transporters ASCT1 and ASCT2 are two SLC1 (solute carrier 1) family subtypes, which are specific for neutral amino acids. The other members of the SLC1 family are acidic amino acid transporters (EAATs 1–5). While the functional similarities and differences between the EAATs have been well studied, less is known about how the subtypes ASCT1 and 2 differ in kinetics and function. Here, by performing comprehensive electrophysiological analysis, we identified similarities and differences between these subtypes, as well as novel functional properties, such as apparent substrate affinities of the inward-facing conformation (in the range of 70 μM for L-serine as the substrate). Key findings were: ASCT1 has a higher apparent affinity for Na+, as well as a larger [Na+] dependence of substrate affinity compared to ASCT2. However, the general sequential Na+/substrate binding mechanism with at least one Na+ binding first, followed by amino acid substrate, followed by at least one more Na+ ion, appears to be conserved between the two subtypes. In addition, the first Na+ binding step, presumably to the Na3 site, occurs with high apparent affinity (<1 mM) in both transporters. In addition, ASCT1 and 2 show different substrate selectivities, where ASCT1 does not respond to extracellular glutamine. Finally, in both transporters, we measured rapid, capacitive charge movements upon application and removal of amino acid, due to rearrangement of the translocation equilibrium. This charge movement decays rapidly, with a time constant of 4–5 ms and recovers with a time constant in the 15 ms range after substrate removal. This places a lower limit on the turnover rate of amino acid exchange by these two transporters of 60–80 s−1.


Introduction
ASCTs (alanine serine cysteine transporters) belong to the SLC1 family of membrane transport proteins. Two subtypes of this family, ASCT1 and ASCT2 (SLC1A4 and SLC1A5) are neutral amino acid transporters, in contrast to the remaining five members of the family (excitatory amino acid transporters, EAATs), which transport acidic amino acids [1][2][3][4][5][6][7]. ASCT2 was previously shown to function as a Na + -dependent neutral amino acid exchanger, which requires the presence of Na + , but does not use the Na + gradient as a driving force for amino acid uptake [2][3][4][5]8]. In this mechanism, extracellular amino acid is exchanged with intracellular amino acid, without the ability of the empty transporter to complete the transport cycle.
In previous studies, a high expression level of ASCT2 was observed in cancer cells, suggesting an association with cancer metabolism. It was suggested to directly or indirectly provide energy and nitrogen resources to rapidly growing cells, by taking up glutamine [9][10][11][12]. In addition, ASCT1 and 2 may be involved in neuronal disorders. For example, SLC1A4 is the pathogenic gene of a neurologic disorder manifesting in intellectual disability, postnatal microcephaly, spasticity and thin corpus callosum [13].
Within the SLC1 family, the various subtypes share a high sequence similarity and identity [14]. ASCT1 and 2 share 57% sequence identity and ASCTs share up to 29% sequence identity with EAATs [12,14]. The substrate binding site, however, shows distinct

Cell Culture and Transfection
HEK293 cells (American Type Culture Collection No. CRL 1573) were cultured as described previously. Cell cultures were transiently transfected with wild-type or mutant hASCT1, hASCT2 cDNAs, inserted into a modified pBK-CMV-expression plasmid. Transfection were performed according Jetprime transfection reagent and protocol (Polyplus). The cells were transfected after 24-36 h then used for electrophysiological analysis.

Voltage-Jump Experiments
Voltage jumps (−100 to +60 mV) were applied to perturb the translocation equilibrium and to determine the voltage dependence of the anion conductance. To determine ASCT-specific currents, external solution contained 140 mM NaMes in the presence of varying concentrations of amino acid substrate. The internal solution contained 130 mM NaSCN in the presence of 10 mM amino acid substrate. Competitive blocker (R)-gamma-(4biphenylmethyl)-L-proline [33] was used in control voltage jump experiments, yielding the unspecific current component, which was subtracted from the total current. Capacitive transient compensation and series resistance compensation of up to 80% was employed using the EPC-7 amplifier. Non-specific transient currents were subtracted in Clampfit software (Molecular Devices).

Rapid Solution Exchange
Fast solution exchanges were performed using the SF-77B (Warner Instruments, LLC, Holliston, MA, USA) piezo-based solution exchanger, allowing a time resolution in the 10-20 ms range. Amino acid substrate was applied through a theta capillary glass tubing (TG200-4, OD = 2.00 mm, ID = 1.40 mm. Warner Instruments, LLC, MA, USA), with the tip of the theta tubing pulled to a diameter of 350 µm and positioned at 0.5 mm to the cell [34]. For paired-pulse experiments, currents were recorded with varying interval time after removal of amino acid, starting at 10 ms.

Data Analysis
The data analysis was performed in Microsoft Excel and Microcal Origin software. Error bars are shown as mean ± stand deviation, collected from recordings of 6 to 10 cells, for statistical analysis. To determine substrate and Na + apparent K m values, non-linear curve fitting was used with a Michaelis-Menten-like equation, I = I max × [substrate]/(K m + [substrate]), where I max is the current at saturating substrate concentration.
Transient signals of piezo-based solution-exchange results were analyzed in Clampfit software (Axon Instruments) by fitting with a sum of two exponential components. I = I 1 ·exp(−t/τ rise ) + I 2 ·exp(−t/τ decay ). Here, I is the current amplitude, τ the time constant and t the time.

ASCT1 Binds Amino Acid Substrate with Higher Apparent Affinity than ASCT2 in the Outward-Facing State
To test the function of ASCTs, including apparent substrate affinities, we selected the HEK293 cell expression system for transfection with ASCT cDNAs, together with electrophysiological analysis in the whole cell recording configuration. In this system, alanine did not elicit currents in non-transfected cells. Typical currents recorded from human ASCT2 transfected cells in response to extracellular solution exchange to various concentrations of serine are shown in Figure 1A. As expected, application of serine activated the ASCT2 anion conductance in the presence of the intracellular anion, SCN − , indicating the population of the substrate-induced anion-conducting state. No steady-state currents were observed in the absence of permeating anions and at zero-trans conditions for substrate and sodium, as discussed later (Section 3.5). Inward currents were concentration dependent and were analyzed with a Michaelis-Menten-like equation, to yield the apparent K m for the substrate, serine ( Figure 1B) as 350 ± 60 µM. K m values for other amino acid substrates in comparison with ASCT1 are summarized in Figure 1C, indicating that substrate apparent affinities are 2-5 times higher in ASCT1 than in ASCT2. Consistent with previous results, L-glutamine did not generate currents in ASCT1-expressing cells. These data confirm previous results that neutral amino acids serine and alanine are major substrates for ASCTs but glutamine only binds to ASCT2. currents were observed in the absence of permeating anions and at zero-trans conditions for substrate and sodium, as discussed later (Section 3.5). Inward currents were concentration dependent and were analyzed with a Michaelis-Menten-like equation, to yield the apparent Km for the substrate, serine ( Figure 1B) as 350 ± 60 μM. Km values for other amino acid substrates in comparison with ASCT1 are summarized in Figure 1C, indicating that substrate apparent affinities are 2-5 times higher in ASCT1 than in ASCT2. Consistent with previous results, L-glutamine did not generate currents in ASCT1-expressing cells. These data confirm previous results that neutral amino acids serine and alanine are major substrates for ASCTs but glutamine only binds to ASCT2.

Leak Anion Conductance Is Less Pronounced in ASCT1 than in ASCT2
ASCT anion conductance was tested using SCNas a highly permeable anion at the intracellular side of the membrane. As expected, application of amino acid substrate at a concentration of 500 μM generated inward current, which increased with negative membrane potential (Figure 2A, voltage jump protocol illustrated in the top panel). This inward current is caused by SCNleaving the cell. Steady-state current was then plotted as a function of the membrane voltage, as shown in the I-V curves in Figure 2B,C. Serine induced the largest anion current in both transporters, while glutamine was ineffective in generating anion current in ASCT1 at any voltage tested. Interestingly, ASCT1 showed a much smaller leak anion current, relative to the substrate-induced current, than ASCT2. This current was observed in the absence of amino acid substrate, but in the presence of Na + . It is specific for ASCTs, as background was subtracted using a competitive inhibitor, which blocks the leak anion conductance.

Leak Anion Conductance Is Less Pronounced in ASCT1 than in ASCT2
ASCT anion conductance was tested using SCNas a highly permeable anion at the intracellular side of the membrane. As expected, application of amino acid substrate at a concentration of 500 µM generated inward current, which increased with negative membrane potential (Figure 2A, voltage jump protocol illustrated in the top panel). This inward current is caused by SCNleaving the cell. Steady-state current was then plotted as a function of the membrane voltage, as shown in the I-V curves in Figure 2B,C. Serine induced the largest anion current in both transporters, while glutamine was ineffective in generating anion current in ASCT1 at any voltage tested. Interestingly, ASCT1 showed a much smaller leak anion current, relative to the substrate-induced current, than ASCT2. This current was observed in the absence of amino acid substrate, but in the presence of Na + . It is specific for ASCTs, as background was subtracted using a competitive inhibitor, which blocks the leak anion conductance. currents were observed in the absence of permeating anions and at zero-trans condi for substrate and sodium, as discussed later (Section 3.5). Inward currents were co tration dependent and were analyzed with a Michaelis-Menten-like equation, to the apparent Km for the substrate, serine ( Figure 1B) as 350 ± 60 μM. Km values for amino acid substrates in comparison with ASCT1 are summarized in Figure 1C, ind ing that substrate apparent affinities are 2-5 times higher in ASCT1 than in ASCT2. sistent with previous results, L-glutamine did not generate currents in ASCT1-expre cells. These data confirm previous results that neutral amino acids serine and alanin major substrates for ASCTs but glutamine only binds to ASCT2.

Leak Anion Conductance Is Less Pronounced in ASCT1 than in ASCT2
ASCT anion conductance was tested using SCNas a highly permeable anion a intracellular side of the membrane. As expected, application of amino acid substrat concentration of 500 μM generated inward current, which increased with neg membrane potential ( Figure 2A, voltage jump protocol illustrated in the top panel). inward current is caused by SCNleaving the cell. Steady-state current was then pl as a function of the membrane voltage, as shown in the I-V curves in Figure 2B,C. S induced the largest anion current in both transporters, while glutamine was ineffecti generating anion current in ASCT1 at any voltage tested. Interestingly, ASCT1 show much smaller leak anion current, relative to the substrate-induced current, than AS This current was observed in the absence of amino acid substrate, but in the presen Na + . It is specific for ASCTs, as background was subtracted using a competitive inhi which blocks the leak anion conductance.

Intracellular Substrate Inhibits the Leak Anion Conductance in ASCT1
Next, we tested the substrate binding properties of the inward-facing state by applying amino acid from the intracellular side of the membrane. This was done using the inside-out patch configuration, allowing the application of intracellular substrate through solution exchange, as previously described for other transporters [35]. From earlier studies with the EAAT SLC1 family members, it is known that intracellular substrate application activates the anion conductance, but with a 20 times lower glutamate apparent affinity (intracellular glutamate apparent affinity is about 100 µM compared with 5 µM in the outward-facing conformation) [35]. As ionic conditions, we used SCNin the inside-out recording pipet, together with 140 mM NaMes and 10 mM serine. Thus, the amino acid binding site should be inward-facing before serine was applied to the inside-out patch ( Figure 3). As shown in Figure 3C, serine activated the anion conductance in ASCT2, resulting in negative current due to SCNoutflow from the patch pipet [3,[36][37][38]. When increasing the concentration of serine, anion currents increased in a concentration-dependent manner, saturating at high concentrations with an apparent K m of 70 ± 18 µM ( Figure 3D). In contrast, serine application to ASCT1-containing inside-out patches resulted in different behavior ( Figure 3A), with substrate blocking the anion conductance, rather than activating it. Similar behavior of blocking the leak anion current was found for intracellular application of a competitive inhibitor (data not shown). From the serine concentration dependence, an apparent K m value of 77 ± 34 µM was determined Figure 3B, when fit at low substrate concentrations. At concentrations exceeding 500 µM, a slight decrease in current was observed. The reason for this behavior is not known, although one possibility would be that ASCT1 has an allosteric binding site for the substrate. For ASCT2, these results indicate that substrate binds with slightly higher apparent affinity to the inward-facing conformation, whereas in ASCT1, apparent substrate affinities of the inward and outward-facing states are similar.
Biomolecules 2022, 12, x FOR PEER REVIEW 5 of 13 ration of 200 ms, voltage protocol shown at the top) from an ASCT1-transfected cell in the presence of extracellular NaMes and intracellular NaSCN with 10 mM serine. 400 μM (R)-gamma-(4-Biphenylmethyl)-L-proline was used to eliminate the unspecific background currents by subtraction. The anion current-voltage relationship at steady-state is plotted in (B) for ASCT1 and (C) for ASCT2.

Intracellular Substrate Inhibits the Leak Anion Conductance in ASCT1
Next, we tested the substrate binding properties of the inward-facing state by applying amino acid from the intracellular side of the membrane. This was done using the inside-out patch configuration, allowing the application of intracellular substrate through solution exchange, as previously described for other transporters [35]. From earlier studies with the EAAT SLC1 family members, it is known that intracellular substrate application activates the anion conductance, but with a 20 times lower glutamate apparent affinity (intracellular glutamate apparent affinity is about 100 μM compared with 5 μM in the outward-facing conformation) [35]. As ionic conditions, we used SCNin the inside-out recording pipet, together with 140 mM NaMes and 10 mM serine. Thus, the amino acid binding site should be inward-facing before serine was applied to the inside-out patch ( Figure 3). As shown in Figure 3C, serine activated the anion conductance in ASCT2, resulting in negative current due to SCNoutflow from the patch pipet [3,[36][37][38]. When increasing the concentration of serine, anion currents increased in a concentration-dependent manner, saturating at high concentrations with an apparent Km of 70 ± 18 μM ( Figure 3D). In contrast, serine application to ASCT1-containing inside-out patches resulted in different behavior ( Figure 3A), with substrate blocking the anion conductance, rather than activating it. Similar behavior of blocking the leak anion current was found for intracellular application of a competitive inhibitor (data not shown). From the serine concentration dependence, an apparent Km value of 77 ± 34 μM was determined Figure 3B, when fit at low substrate concentrations. At concentrations exceeding 500 μM, a slight decrease in current was observed. The reason for this behavior is not known, although one possibility would be that ASCT1 has an allosteric binding site for the substrate. For ASCT2, these results indicate that substrate binds with slightly higher apparent affinity to the inward-facing conformation, whereas in ASCT1, apparent substrate affinities of the inward and outward-facing states are similar.

ASCT Activity Shows Biphasic Na + Concentration Dependence
Evidence from molecular dynamics simulation and electrophysiology studies [1,2,5,8,[39][40][41], points to the existence of three sodium coordination sites in ASCT1 and 2. By analyzing Na + dependence of amino acid-induced anion currents, we determined the Na + apparent affinity for both subtypes over a large [Na + ] range, as shown in Figure 4. Total cation concentration was maintained at 140 mM by balancing with NMG + . As observed previously for ASCT2, apparent outward-directed currents were generated by applying amino acid substrate at very low Na + concentration, ranging from 0.1 to 1 mM for both ASCT1 and 2 ( Figure 4A,D), although the magnitude of the outward current was much smaller in ASCT1, presumably due to its lower leak anion conductance (see above). This apparent outward current is caused by a block of the tonic leak anion current (which is inward directed) by the amino acid substrate at low [Na + ]. At higher Na + concentrations, the current direction reversed to generate inward current (substrate-induced anion conductance ( Figure 4B,E)). When plotted against the Na + concentration ( Figure 4C,F), a bi-phasic current-[Na + ] relationship was observed, indicating that more than one Na + binding step is required [5,8,21]. With fitting to a two-binding site equation, as shown by the red curves in Figure 4C,F, the following K m values for Na + binding were observed: hASCT2, K Na1 = 0.3 ± 0.1 mM, K Na2 = 27 ± 8 mM; hASCT1, K Na1 = 0.3 ± 0.5 mM, K Na2 = 14 ± 3 mM, indicating slightly weaker Na + apparent affinity for the second cation binding steps in ASCT2 compared to ASCT1.

ASCT Activity Shows Biphasic Na + Concentration Dependence
Evidence from molecular dynamics simulation and electrophysiology stud [1,2,5,8,[39][40][41], points to the existence of three sodium coordination sites in ASCT1 an By analyzing Na + dependence of amino acid-induced anion currents, we determined Na + apparent affinity for both subtypes over a large [Na + ] range, as shown in Figur Total cation concentration was maintained at 140 mM by balancing with NMG + . As served previously for ASCT2, apparent outward-directed currents were generated applying amino acid substrate at very low Na + concentration, ranging from 0.1 to 1 m for both ASCT1 and 2 ( Figure 4A,D), although the magnitude of the outward current much smaller in ASCT1, presumably due to its lower leak anion conductance (see abo This apparent outward current is caused by a block of the tonic leak anion current (wh is inward directed) by the amino acid substrate at low [Na + ]. At higher Na + concen tions, the current direction reversed to generate inward current (substrate-induced an conductance ( Figure 4B,E)). When plotted against the Na + concentration ( Figure 4C,F bi-phasic current-[Na + ] relationship was observed, indicating that more than one binding step is required [5,8,21]. With fitting to a two-binding site equation, as shown the red curves in Figure 4C,F, the following Km values for Na + binding were observ hASCT2, KNa1 = 0.3 ± 0.1 mM, KNa2 = 27 ± 8 mM; hASCT1, KNa1 = 0.3 ± 0.5 mM, KNa2 = 14 mM, indicating slightly weaker Na + apparent affinity for the second cation binding st in ASCT2 compared to ASCT1.  The K m for Na + was determined from fitting dose-response curves of a two Na + binding site function. I = I max1 * [Na + ]/(K Na1 + [Na + ]) + I max2 * [Na + ]/(K Na2 + [Na + ]). For ASCT2, K Na1 0.3 ± 0.1 mM, K Na2 is 27.9 ± 8.8 mM. For ASCT1, K Na1 is 0.3 ± 0.1 mM, K Na2 is 14.0 ± 3.4 mM. In all experiments, the transmembrane potential was 0 mV.
To further test the Na + dependence of ASCT activity, we measured the substrate apparent affinities as a function of the Na + concentration. As shown in Supplementary Materials Figure S1, apparent substrate K m values decrease with increasing extracellular Na + concentration, as expected for cooperative binding of amino acid substrate and Na + and as supported by the fits to a sequential Na + -substrate-Na + binding model (solid lines in Supplementary Materials Figure S1).

ASCT1 and 2 Have Comparable Kinetics of Substrate Translocation
The kinetics of reaction steps associated with ASCT2 substrate translocation were previously studied using rapid alanine application with laser photolysis of caged-alanine [5,8,34,42]. In this previous study, transient currents induced by alanine application decayed with a time constant of 3 ms, providing information about the electrogenic steps in the translocation equilibrium [20][21][22][43][44][45]. Here, we extended these experiments using a piezo-based rapid solution exchange device, allowing the rapid application, as well as removal of amino acid. These experiments were performed in the absence of permeating anions, to isolate electrogenic steps of the process. The intracellular solution contained Na + and amino acid and, initially, the extracellular solution contained only Na + , conditions under which the amino acid binding site should be facing to the extracellular side of the membrane. Subsequently, serine was applied to the extracellular side of the membrane and later removed. As expected for electroneutral amino acid exchange, we did not observe any steady-state current. However, substantial transient inward current associated with cation binding and/or movement of the charged transport domain was caused by serine application [8,22] in both ASCT1 and 2, as shown in Figure 5A-D. The time constant for current decay was 4-5 ms after serine application. However, the kinetics of this decay could be partially limited by the rate of solution exchange, in particular for removal of the substrate. As expected, the transient currents were absent in the absence of extracellular Na + , suggesting that Na + is a requirement for the electrogenic rearrangement of the ASCT binding sites within the membrane. Upon the removal of serine, transient current was observed in the opposite, outward direction, as expected for the re-equilibration of the translocation equilibrium to the outward-facing conformation, resulting in capacitive-like charge movement (i.e., the charge that moved inward during amino acid application has to move outward upon amino acid removal). Consistently, the outward charge movement upon substrate removal was of the same magnitude as the inward charge movement upon substrate application. To further test the Na + dependence of ASCT activity, we measured the substrate apparent affinities as a function of the Na + concentration. As shown in Supplementary Materials Figure S1, apparent substrate Km values decrease with increasing extracellular Na + concentration, as expected for cooperative binding of amino acid substrate and Na + and as supported by the fits to a sequential Na + -substrate-Na + binding model (solid lines in Supplementary Materials Figure S1).

ASCT1 and 2 Have Comparable Kinetics of Substrate Translocation
The kinetics of reaction steps associated with ASCT2 substrate translocation were previously studied using rapid alanine application with laser photolysis of caged-alanine [5,8,34,42]. In this previous study, transient currents induced by alanine application decayed with a time constant of 3 ms, providing information about the electrogenic steps in the translocation equilibrium [20][21][22][43][44][45]. Here, we extended these experiments using a piezo-based rapid solution exchange device, allowing the rapid application, as well as removal of amino acid. These experiments were performed in the absence of permeating anions, to isolate electrogenic steps of the process. The intracellular solution contained Na + and amino acid and, initially, the extracellular solution contained only Na + , conditions under which the amino acid binding site should be facing to the extracellular side of the membrane. Subsequently, serine was applied to the extracellular side of the membrane and later removed. As expected for electroneutral amino acid exchange, we did not observe any steady-state current. However, substantial transient inward current associated with cation binding and/or movement of the charged transport domain was caused by serine application [8,22] in both ASCT1 and 2, as shown in Figure 5A-D. The time constant for current decay was 4-5 ms after serine application. However, the kinetics of this decay could be partially limited by the rate of solution exchange, in particular for removal of the substrate. As expected, the transient currents were absent in the absence of extracellular Na + , suggesting that Na + is a requirement for the electrogenic rearrangement of the ASCT binding sites within the membrane. Upon the removal of serine, transient current was observed in the opposite, outward direction, as expected for the re-equilibration of the translocation equilibrium to the outward-facing conformation, resulting in capacitive-like charge movement (i.e., the charge that moved inward during amino acid application has to move outward upon amino acid removal). Consistently, the outward charge movement upon substrate removal was of the same magnitude as the inward charge movement upon substrate application. Next, we performed a paired-pulse experiment, where the time interval between removal of serine to a second application of serine was varied. This experiment can test how quickly the outward-facing conformation is restored after serine removal, providing information about the amino acid exchange turnover rate of ASCTs. As shown in Figure 6A,D, the peak serine-induced inward current recovered relatively rapidly after serine removal, with complete recovery after 50-100 ms for both transporters. We next performed a fit of the recovery amplitude with an exponential function. The rate constant for recovery was 60 ± 4 s −1 for ASCT1 ( Figure 6B). and 75 ± 3 s −1 for ASCT2 ( Figure 6E), suggesting that the recovery time constant is similar for both ASCT subtypes. Furthermore, we tested time constants at varying substrate concentrations, from 200 µM to 1000 µM, showing that the recovery rate remained unchanged ( Figure 6C,F). This is expected because the re-equilibration of the exchange equilibrium should only depend on intracellular substrate concentration after removal of extracellular amino acid. using a θ tube. Experiments were performed with 140 mM Na + together with 500 μM serine extracellular side, the intracellular solution contained 130 mM NaMes and 10 mM serine constants for the decay of the transient current were τ1 = 2.3 ± 0.1 ms for serine application a 7.8 ± 0.2 ms for serine removal (ASCT1) and the charge movements Q1 = 290 fC and Q2 = 3 respectively. For ASCT2 the time constants were τ1 = 3.1 ± 0.1 ms and τ2 = 11.4 ± 0.3 ms. (B,D) S experiments but in the absence of external Na + .
Next, we performed a paired-pulse experiment, where the time interval bet removal of serine to a second application of serine was varied. This experiment ca how quickly the outward-facing conformation is restored after serine removal, prov information about the amino acid exchange turnover rate of ASCTs. As shown in F 6A,D, the peak serine-induced inward current recovered relatively rapidly after s removal, with complete recovery after 50-100 ms for both transporters. We nex formed a fit of the recovery amplitude with an exponential function. The rate consta recovery was 60 ± 4 s −1 for ASCT1 ( Figure 6B). and 75 ± 3 s −1 for ASCT2 ( Figure 6E) gesting that the recovery time constant is similar for both ASCT subtypes. Further we tested time constants at varying substrate concentrations, from 200 μM to 1000 showing that the recovery rate remained unchanged ( Figure 6C,F). This is expecte cause the re-equilibration of the exchange equilibrium should only depend on int lular substrate concentration after removal of extracellular amino acid. In addition, we tested kinetics associated with the substrate translocation step different transported amino acids. As shown in Supplementary Materials Figure S2 constants associated with recovery from serine and alanine application are in the order of magnitude, but vary slightly for amino acid and subtype, with rate constan ASCT2 being larger. Recovery rates after glutamine application are slower and no was observed for ASCT1, for which glutamine does not function as a transported strate (Supplementary Figure S2). In addition, we tested kinetics associated with the substrate translocation step using different transported amino acids. As shown in Supplementary Materials Figure S2, rate constants associated with recovery from serine and alanine application are in the same order of magnitude, but vary slightly for amino acid and subtype, with rate constants for ASCT2 being larger. Recovery rates after glutamine application are slower and no signal was observed for ASCT1, for which glutamine does not function as a transported substrate (Supplementary Figure S2).
Finally, we determined the voltage dependence of substrate-induced transient currents, at membrane potentials varying from −100 mV to +60 mV (Supplementary Materials Figure S3). Peak amplitudes of the transient current signal were plotted as a function of the membrane potential in Supplementary Materials Figure S3A,D. While voltage dependence of the peak current is weak for ASCT2, a stronger voltage dependence was observed for ASCT1, with current increasing about 1.07 fold for every −20 mV change in membrane potential (Supplementary Materials Figure S3B,E). As expected, the current increased at more negative potential, due to positive charge of the cotransported Na + and/or loaded transport domain moving inward within the membrane. Overall, these results are consistent with a previous report [8], which did not find a strong voltage-dependent behavior of amino acid induced transient ASCT2 current.

Discussion
In this report, we compare the function of the neutral amino acid transporter members of the SLC1 family, ASCT1 and 2, using detailed electrophysiological and kinetic analysis. Despite minor differences in amino acid substrate specificity, with ASCT1 unable to recognize glutamine, the general functional properties are highly conserved between the two transporters. ASCT1 and 2 both catalyze a substrate-induced anion conductance, which is uncoupled with substrate transport. While both transporters also exhibit a leak anion conductance, in the absence of amino acid substrate, this leak conductance is much less pronounced in ASCT1 compared to ASCT2. Second, both transporter subtypes exhibit a biphasic response to Na + , suggesting that the sequential Na + -amino acid-Na + binding mechanism is conserved. However, the apparent affinity of the Na + binding step following substrate binding is somewhat lower in ASCT2 compared to ASCT1. Third, apparent affinities for the amino acid substrate serine binding to the inward-facing binding site are in the sub-100 µM range. However, serine is an inhibitor of the leak anion conductance in ASCT1 when applied to the intracellular side of the membrane, in contrast to ASCT2, where it is an activator. Finally, electrogenic transport steps in the translocation reaction are conserved in ASCT1 and 2 and occur with similar rates, suggesting that the turnover rates of the two transporter subtypes for amino acid exchange are >65 s −1 .
Amino acid and Na + binding were proposed to occur in a sequential fashion, similar to the EAAT members of the family, with at least one Na + binding to the apo-form of the transporter and another Na + binding to the amino acid-bound form. This sequential binding sequence appears to be conserved in both ASCT subtypes. In addition, both transporters show the biphasic Na + dose response relationship, indicating that the Na + binding step to the empty transporter is of high affinity in both ASCT1 and 2, with an apparent K m value < 1 mM. This suggests that this cation binding step is always saturated under physiological conditions, unless [Na + ] would reach exceedingly low values. In contrast to ASCT2, the block of leak anion current by substrate at low sodium concentrations is not very pronounced, in agreement with the idea that the leak anion conductance is small in ASCT1. If there is a physiological significance for this difference in function is unknown. The [Na + ] binding step following substrate binding is of lower affinity, in the 20 mM range, although this step is also close to being saturated at physiological [Na + ]. This finding is in agreement with the hypothesis that Na + plays a modulatory role in ASCTs, as it is not providing a driving force for amino acid exchange. Overall, these data are in excellent agreement with a recent report on ASCT2 showing a Na + /glutamine stoichiometry of 2:1 and, for the first time, directly demonstrating sodium flux across the membrane during amino acid transport [46].
The binding properties for amino acid from the intracellular side (inward-facing conformation) had not been previously characterized for ASCTs using electrophysiological analysis. However, using radiolabeled substrates uptake assay with sided proteoliposomes, Indiveri and colleagues were able to determine both the internal and external K m for ASCT2 for several amino acids [32,47,48]. Interestingly, in their assay, serine had a 100-fold higher K m in the inside-out proteoliposome configuration. In contrast, our data suggest that the internal and external K m values are in the same 50-100 µM range. The reason for this discrepancy is not known. One possibility would be that Indiveri and colleagues reconstituted purified human ASCT2 in proteoliposomes, whereas our experiments were performed in HEK293 cells, which may contain regulatory factors that are absent in the purified transporter. Second, it is possible that the experimental K m values differ for activation of the anion conductance and for actual substrate transport. In any case, the potential symmetry/asymmetry of amino acid affinities of the exchanger binding sites has strong implications on our understanding of ASCT function in the physiological environment and needs to be investigated more thoroughly. Another interesting result from the inside-out patch experiments is that serine inhibits the leak anion conductance, rather than activating an anion conductance, as is the case in ASCT2.
In a previous report, it was shown that rapid amino acid application to ASCT2 resulted in inwardly-directed transient currents, most likely caused by the movement of charged Na + into the transport domain, or by structural changes of the charged transport domain due to amino acid translocation, or both [5]. While rate constants for amino acid translocation were determined in this previous work, the turnover rate was only estimated. Here, we extended this previous analysis by using a paired-pulse amino acid application protocol, allowing the determination of the recovery rate of the current upon amino acid removal. This recovery rate is a better indicator of turnover rate than the rate constant of transient current after amino acid application, since the latter only informs on electrogenic partial reactions. The recovery rate was independent of extracellular serine concentration, as expected, but changed when the nature of the amino acid substrate was varied. For serine, a recovery rate constant of 75 s −1 was obtained for ASCT2 and 60 s −1 for ASCT1 (Figure 6), providing a lower limit for the turnover rate, since a slight limitation of these rate constants by solution exchange cannot be fully excluded. In any case, these values provide an estimate of amino acid exchange rates by these SLC1 family subtypes, suggesting that ASCT2 is a marginally faster exchanger than ASCT1. The knowledge of these turnover rates will have substantial implications for our understanding of the impact of ASCTs in physiological, as well as pathophysiological processes related to the metabolic turnover of neutral amino acids, in particular glutamine.
In summary, the work presented here compares the functional and kinetic properties of the neutral amino acid transporters ASCT1 and 2 from the SLC1 family. Both transporters share the high affinity for Na + in the apo-, amino acid-free configuration, suggesting that this Na + binding site (likely the Na3 site) is always occupied under physiological conditions. In contrast, the Na + binding step following amino acid binding has a much lower apparent affinity, in the 15 to 25 mM range. Both transporters show sub-millimolar apparent affinities for serine in the inward-facing conformation, suggesting a lack of sidedness with respect to substrate affinities. Finally, despite overall electroneutral amino acid exchange, both transporters display fast electrogenic charge movement in response to rapid amino acid application, most likely caused by rearrangements of the transport domain during substrate translocation, or Na + binding. From the recovery of these charge movements after amino acid removal, the turnover rate was estimated in the range of >65 s −1 for ASCT2 and only slightly slower for ASCT1. Together these data may be useful to understand the involvement of ASCTs in amino acid homeostasis under physiological conditions, as well as in certain cancer cells, in which ASCT2 is upregulated.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/biom12010113/s1, Figure S1: Apparent affinity for substrate is dependent on [Na + ]; Figure S2: Transient current recovery rates depend on the substratetitle; Figure  Funding: This study was supported by a grant from the National Institutes of Health (R01 GM108911) to Avner Schlessinger and Christof Grewer and the R15 GM135843-01 awarded to Christof Grewer.

Data Availability Statement:
Original data can be found in the supplementary information or will be made available upon request.

Conflicts of Interest:
The authors declare no conflict of interest.