To characterize the relationship between Na⁺ and K⁺ binding to LeuT, we investigated the effect of K⁺ on Na⁺-dependent [³H]leucine binding using the scintillation proximity assay (SPA) (Quick and Javitch, 2007). Accordingly, we performed a Na⁺ titration with 100 nM [³H]leucine (Figure 1A). Choline (Ch⁺), seemingly inert to LeuT (Billesbølle et al., 2016), was used as counter ion to maintain the ionic strength. Na⁺ promoted the binding of [³H]leucine to LeuT with an EC₅₀ for Na⁺ of 7.7 mM, in line with previous studies (Zhao et al., 2011; Shi et al., 2008). In the presence of K⁺, however, the EC₅₀ right-shifted while the B_max remained unchanged, consistent with a competitive mechanism of inhibition between Na⁺ and K⁺ as suggested previously (Billesbølle et al., 2016).

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Excel file containing data for (A) Na⁺-mediated [³H]leucine binding to purified LeuT in the absence of K⁺, and (B) K⁺-dependent displacement of Na⁺-mediated [³H]leucine binding.

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To determine whether the inhibition of [³H]leucine binding by K⁺ was saturable, we titrated in K⁺ in the presence of Na⁺ and [³H]leucine. Displacement of [³H]leucine binding by K⁺ is concentration-dependent, with an IC₅₀ for K⁺ of 235 [225; 243] mM (mean [s.e.m. interval]) (Figure 1B). To ensure that the displacement of [³H]leucine by K⁺ was not caused by artifacts originating from the high total salt concentrations (1.6 M), we repeated the displacement assay with a total ionic strength of 208 mM salt using either Ch⁺ or N-methyl-D-glucamine (NMDG⁺), both of which are inert to LeuT, as counter ions. Again, K⁺ dose-dependently displaced [³H]leucine binding with an inhibition constant not significantly different from that measured in the high salt conditions (Figure 1—figure supplement 1). Although the approach is indirect with respect to K⁺, this saturable, ionic strength-independent displacement of [³H]leucine binding is indicative of a specific, low affinity K⁺ binding site in LeuT.

To obtain a more direct readout of K⁺ binding to LeuT, we turned to tmFRET. This method relies on the distance-dependent quenching of a cysteine-conjugated fluorophore (FRET donor) by a transition metal (FRET acceptor), here Ni²⁺, coordinated to an engineered α-helical His-X₃-His site (Taraska et al., 2009a). In LeuT, we have inserted the His-X₃-His site in extracellular loop (EL)4a (A313H-A317H) and a cysteine at the top of TM10 (K398C) that is labeled with fluorescein-5-maleimide (F5M). The distance between these FRET probes changes upon opening and closing of the extracellular gate in LeuT (Figure 2A and Figure 2—figure supplement 1A). Importantly, this construct, LeuT A313H-A317H-K398F5M (from here and on named LeuT^tmFRET), retains WT activity with respect to ligand binding affinities (Billesbølle et al., 2016). Accordingly, changes in tmFRET intensity is a conformational readout for both ion and ligand binding.

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Excel file containing data for Figure 2B and C providing transition metal ion FRET (tmFRET) efficiencies obtained for by the added ions in 10 mM Ni^2+.

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To determine the Ni²⁺ concentrations required to saturate the His-X₃-His site, we first recorded the FRET efficiency as a function of increasing Ni²⁺ for LeuT incubated with NMDG⁺, K⁺, or Na⁺± leucine (Figure 2—figure supplement 1B). To ensure close to saturating conditions for both Na⁺ and K⁺ while preserving the ionic strength, we applied 800 mM of the ions. The FRET efficiencies at saturating Ni²⁺ concentrations reflect the average distance between the FRET probes and showed that K⁺ uniquely stabilizes a high FRET state, suggesting a shift in the conformational equilibrium of the transporter toward an outward-closed state by K⁺ (Figure 2—figure supplement 1A and B). The Ni²⁺ affinity for the His-X₃-His site is increased ~3-fold when Na⁺ or K⁺ is added to LeuT relative to NMDG⁺. This indicates that both Na⁺ and K⁺ bind and stabilize LeuT, including the His-X₃-His motif, whereas NMDG⁺ does not.

We proceeded by measuring the FRET efficiency only at saturating Ni²⁺ concentrations (10 mM) to obtain FRET efficiencies independent of potential differences in Ni²⁺ affinities. In addition to the conditions above, we measured FRET efficiencies for LeuT incubated in Na⁺ with alanine and in Rb⁺ (Figure 2B). Interestingly, applying Rb⁺, a cation often seen to be able to substitute for K⁺ in biological systems, also stabilized a more outward-closed state relative to that in NMDG⁺. The FRET efficiency in the Na⁺/leucine-bound state was decreased relative to that in the Na⁺-bound state, suggesting a, on average, more open-to-out state when adding leucine. We speculate that LeuT adopts a more conformationally restricted equilibrium upon binding of leucine relative to that with Na⁺ alone, and that this is reflected in a lower FRET efficiency (Figure 2—figure supplement 1A). This is also in line with the observation that alanine gives rise to a higher FRET efficiency than leucine (Figure 2B) as alanine binding allows a higher degree of conformational freedom in the transporter (Zhao et al., 2011; Calugareanu et al., 2022).

The large difference in tmFRET efficiencies between the apo state with NMDG⁺ and the K⁺-bound state of LeuT^tmFRET allowed us to use the K⁺-induced change in conformational dynamics as a proxy for K⁺ binding. To estimate the affinity for K⁺ to apo state LeuT, we recorded the FRET efficiencies for LeuT^tmFRET incubated with Ni²⁺ in increasing concentrations of K⁺ (Figure 2C). We observed an increase in FRET as a function of K⁺ yielding an EC₅₀ of 183 [176; 189] mM, in line with the affinity determined by K⁺-dependent displacement of Na⁺ and [³H]leucine (Figure 1B). This EC₅₀ is also in line with the affinity from the Schild analysis performed previously (Billesbølle et al., 2016). While Na⁺ did not impose major conformational changes, we found that also Rb⁺ induced a conformational response resembling that of K⁺, suggesting that Rb⁺ can indeed substitute for K⁺ although with a lower apparent affinity (Figure 2—figure supplement 1C). Of note, the high salt concentrations did not affect the intrinsic fluorescence properties of the fluorophore, validating that the responses to titration of the ions were direct results of conformational changes (Figure 2—figure supplement 1D). Along with the effect of K⁺ on Na⁺-dependent [³H]leucine binding, this finding supports the existence of a specific K⁺ binding site in LeuT, and that K⁺ binding to this site induces an outward-closed conformation.

We have previously shown for LeuT reconstituted into liposomes that intra-vesicular K⁺ increases the concentrative capacity of [³H]alanine, probably by decreasing its efflux (Billesbølle et al., 2016). To expand on these findings and to characterize how substrate transport was affected by the cations in the intra-vesicular buffer, we reconstituted purified LeuT into liposomes containing either Na⁺, NMDG⁺, Cs⁺, Rb⁺, or K⁺ (Figure 3A). Under each of these conditions, we measured time resolved [³H]alanine uptake driven by a Na⁺ gradient (Figure 3B). With equimolar intra- and extra-vesicular Na⁺ concentrations, no [³H]alanine transport was observed, indicating that the established Na⁺ gradient did drive [³H]alanine uptake (Figure 3B). Interestingly, proteoliposomes containing K⁺ displayed a 2.5-fold increase in concentrative capacity compared to those containing Cs⁺ or NMDG⁺ (Figure 3—figure supplement 1; Table 1). To ensure that variations in the amount of active LeuT in the proteoliposomes did not affect the uptake capacity, we correlated it to the relative number of active LeuT under each condition (Figure 3—figure supplement 1A). The concentrative capacity with Rb⁺ was similar to that with K⁺, indicating that Rb⁺ can functionally substitute for K⁺. These data suggest that K⁺, and Rb⁺, are not obligate for LeuT transport, but add a concentrative potential for accumulation of alanine in addition to that obtained solely by the Na⁺ gradient.

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Word file containing data table for rate constants for time-dependent [³H]alanine transport by LeuT into PLs.

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Word file containing data table for V_max and K_m of [³H]alanine transport by LeuT into PLs.

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Word file containing data table for V_max and K_m of [³H]alanine transport as an effect by various K⁺ concentrations.

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Excel file containing data for [³H]alanine uptake by LeuT reconstituted into PLs.

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Next, we investigated how the identity of the intra-vesicular cation affected alanine uptake rate. The K_m for [³H]alanine in vesicles containing K⁺ was 1.8±0.4 µM, and it was not significantly different upon substitution of intra-vesicular K⁺ with NMDG⁺, Cs⁺, or Rb⁺ (Figure 3C and Figure 3—source data 2). The V_max for [³H]alanine uptake was not significantly different between NMDG⁺- and Cs⁺-containing proteoliposomes. In contrast, the V_max increased 2.5- to 3-fold when NMDG⁺ was substituted with Rb⁺ or K⁺. The increased uptake rate could originate from increases in the rates of certain steps in the transport cycle, from a decrease in [³H]alanine efflux, or a combination of both.

To gain further insight into the relationship between substrate transport and intra-vesicular K⁺, we investigated how the K⁺ concentration affected K_m and transport velocity for [³H]alanine. Accordingly, LeuT-containing proteoliposomes were prepared in a range of intra-vesicular K⁺ concentrations. We observed that the V_max for [³H]alanine transport increased with increasing intra-vesicular K⁺ concentration, whereas the estimated K_m values for alanine were not significantly different (Figure 3—figure supplement 1B and Figure 3—source data 3), suggesting that the effect of K⁺ on V_max increases with increasing occupancy of the K⁺ binding site. To assess how the concentration of extra-vesicular Na⁺ affected the substrate uptake, we measured [³H]alanine uptake at initial velocity conditions in increasing concentrations of extra-vesicular Na⁺. We found that the half-saturating extra-vesicular Na⁺ concentration was ∼25 mM for vesicles containing 200 mM intra-vesicular K⁺, which is in line with the higher affinity for Na⁺ to LeuT compared to K⁺ (Figure 3—figure supplement 1C).

The increase in [³H]alanine transport rate by K⁺ could be due to an imposed driving force by the outward-directed gradient. If so, dissipation of the K⁺ gradient would decrease the [³H]alanine transport rate. To allow for the addition of K⁺ on the extracellular side also, we lowered the inward-directed Na⁺ gradient to 50 mM. The lowered Na⁺ gradient still resulted in an ~3.5-fold increased transport capacity with intra-vesicular K⁺ relative to NMDG⁺ (Figure 3D). However, dissipation of the K⁺ gradient by application of equal amounts of K⁺ on both sides did not change the transport capacity. Addition of K⁺ only on the outside did also not change the transport capacity relative to that with the Na⁺ gradient only (Figure 3D), and only having an outward-directed K⁺ gradient could not drive [³H]alanine transport (Figure 3E). These results suggest that it is the intra-vesicular K⁺ per se that increases the transport rate of alanine and not a K⁺ gradient.

It is difficult to control the directionality of proteins when they are reconstituted into lipid vesicles. They will be inserted in both orientations. Outside-out and inside-out. In the case of LeuT, it is the imposed Na⁺ gradient which determines the directionality of transport. Uptake through the inside-out transporters will probably also happen. Note that the inside-out LeuT will not have the K⁺ binding site exposed to the intra-vesicular environment. Accordingly, a propensity of transporters will likely not be influenced by the added K⁺ and will tend to mask the contribution of K⁺ on the transport mode from the right-side out LeuT. To investigate LeuT directionality in our reconstituted samples, we performed thrombin cleavage of accessible C-terminals on intact and perforated vesicles, respectively. The result suggests that the proportion of LeuT inserted as outside-out is larger than the proportion with an inside-out directionality (Figure 3—figure supplement 1D).

With the elucidation of the impact of K⁺ on LeuT substrate transport, we next sought to identify the binding site for K⁺ in LeuT. Since K⁺ and Na⁺ binding are competitive and K⁺ excludes substrate binding, we chose to focus on the Na1 site (Figure 4). Accordingly, we introduced the following conservative mutations of the amino acid residues in the Na1 site: A22S, A22V, N27Q, T254S, and N286Q. The aim was to keep LeuT functional but perturb K⁺ binding. Since it has been shown that H⁺ can substitute for the K⁺ antiport in SERT (Keyes and Rudnick, 1982), we also mutated the adjacent Glu290, which has been proposed to facilitate H⁺ antiport in LeuT (Zomot et al., 2007; Forrest et al., 2007; Kantcheva et al., 2013; Malinauskaite et al., 2016). Thus, by substituting it with glutamine (LeuT E290Q), we aimed to replicate the protonated state of Glu290. If the mechanism was similar to that in SERT but facilitated through this non-conserved residue, it should prevent K⁺ binding.

Figure 4

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Excel file containing data for Na⁺-mediated [³H]leucine binding for Na1 site mutations and how that is inhibited by K⁺.

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All mutants were expressed in E. coli and purified in DDM. They all bound leucine and alanine (Table 1). The LeuT mutants A22V, A22S, and T254S retained WT-like substrate affinities whereas the mutation E290Q decreased the affinity one order of magnitude and the mutations N27Q and N286Q decreased the affinities about two orders of magnitude. To estimate their Na⁺ affinities, we measured the Na⁺-dependent [³H]leucine binding in a next to saturating [³H]leucine concentration (10× K_d), thereby taking the differences in substrate affinity for the individual mutants into account (Figure 4B and Table 2). Mutation of A22V and N27Q increased the Na⁺ affinity by ~3-fold relative to WT. The T254S mutant caused a 2-fold decrease whereas the A22S and N286Q mutants both decreased the apparent Na⁺ affinity by ~7-fold. The E290Q mutant retained close to WT Na⁺ affinity.

To assess if the mutations in the Na1 site affected the ability to bind K⁺ and test if the competitive mechanism of inhibition was preserved, we repeated the Na⁺-dependent [³H]leucine binding experiments in the presence of 800 mM K⁺. For LeuT WT, the added K⁺ caused an ~6-fold increase in the EC₅₀ for Na⁺-dependent [³H]leucine binding. Interestingly, mutation E290Q and A22V resulted in increased antagonism by K⁺ relative to WT, causing a 15-fold and 35-fold change, respectively (Figure 5 and Table 2). For the remaining mutants, the antagonism by K⁺ was either less (A22S, N27Q, and N286Q) or similar (T254S) (Figure 5 and Table 2). The observation that the inhibition of Na⁺-dependent ligand binding by K⁺ is retained for LeuT E290Q suggests that binding of K⁺ is not dependent on a negative charge at Glu290 and can occur in parallel with H⁺ antiport via Glu290 in LeuT. Also, the fact that the effect of K⁺ on the T254S mutation was indifferent to WT suggests that the serine residue can completely substitute for threonine in this position.

Figure 5

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Excel file containing data for Na⁺-mediated [³H]leucine binding for the Na1 site mutants and in the presence of K⁺.

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Next, we assessed the impact of the mutations in the Na1 site on K⁺ affinity by measuring the potency by which K⁺ inhibits [³H]leucine binding. Again, [³H]leucine was added in a near-saturating concentration (10× K_d) and Na⁺ at its determined EC₅₀. We observed a marked (~10-fold) decrease in K⁺ sensitivity for A22S, N27Q, and N286Q, suggesting perturbed K⁺ binding by these mutants (Figure 4C, Figure 5). Note that the same N27Q mutant had an increased EC₅₀ for Na⁺. In contrast, the potency for K⁺ in inhibiting [³H]leucine binding was almost 5-fold increased by the A22V mutation (Figure 4C, Figure 5, and Table 2). The T254S mutation did not alter the K⁺ sensitivity relative to LeuT WT (Figure 4C, Figure 5, and Table 2). The cognate position to Thr254 in SERT is also a serine residue, which could indicate that the threonine to serine substitution is tolerated in terms of K⁺ sensitivity between NSSs from different species.

Taken together, the Na1 site mutations showed a marked and differentiated response to the effects of both Na⁺ and K⁺. For some mutants, the affinities for both Na⁺ and K⁺ were decreased (A22S and N286Q) or increased (A22V). For one, the effects were differentiated so that the Na⁺ affinity was increased, while the K⁺ affinity was decreased (N27Q).

With coordinates from TM1, -7 and the bound substrate, the Na1 site is likely a central mediator of conformational changes. To investigate how the conformational equilibria in the transporters were affected by the Na1 site mutations, we introduced them into the LeuT^tmFRET background and probed their response to Na⁺, K⁺, and substrate binding with respect to changes in tmFRET. We first investigated the conformational equilibria for N286Q^tmFRET in NMDG⁺ (apo state), in Na⁺ with and without leucine, and in K⁺. Surprisingly, even though the EC₅₀ and IC₅₀ values for Na⁺ and K⁺, respectively, were markedly increased (decreased affinities), the conformational equilibria of N286Q resembled those of LeuT WT (Figure 6A). This suggests that the substantial decreases in ion affinities and selectivity, imposed by the asparagine to glutamine substitution, do not result from conformational biases, but likely from a direct mutual modulation of their binding site. However, we failed to observe any [³H]alanine transport activity by reconstituted LeuT N286Q, likely as a result of its low substrate affinity.

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Excel file containing data for transition metal ion FRET (tmFRET) efficiencies for all mutants in N-methyl-D-glucamine (NMDG⁺), Na⁺, leucine, or K⁺.

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The N27Q^tmFRET showed a markedly lower tmFRET efficiency in the apo state compared to LeuT^tmFRET (Figure 6B), suggesting that the mutation biases the conformational equilibrium toward a more outward-open conformation. Addition of Na⁺ and leucine restored the conformational equilibrium toward significantly more outward-closed states, but not to the same extent as found for the LeuT^tmFRET construct. However, we hardly observed any change in the tmFRET efficiency with K⁺ in the N27Q mutant. This is in line with its marked decrease in K⁺ affinity. The result could either suggest that Asn27 is important for the binding of K⁺, or that the mutation causes a conformational bias which makes this mutant rarely visit the K⁺-selective state. We were unable to measure any [³H]alanine transport activity by the LeuT N27Q mutant when reconstituted into K⁺-containing vesicles.

Mutant A22V^tmFRET displayed similar tmFRET efficiency as LeuT^tmFRET in NMDG⁺ and Na⁺, but the tmFRET efficiency induced by K⁺ was not significantly different from that induced by Na⁺ (Figure 6C). This indicates that the conformational equilibrium of A22V^tmFRET apo-form is not changed by the mutation. In addition, although both Na⁺ and K⁺ ions bind with a higher affinity to LeuT A22V than to WT (Figure 4), the tmFRET data suggest that K⁺ no longer promotes the conformational shift toward the outward-closed conformation. To further evaluate this, we reconstituted the mutant into liposomes and measured its ability to transport alanine (Figure 6—figure supplement 1A). With equimolar Na⁺ on each side of the lipid bilayer, we observed a specific [³H]alanine signal, which could either reflect transport driven by the [³H]alanine gradient alone or simply binding of [³H]alanine to LeuT. In the presence of a Na⁺ gradient, we observed a minor, but significant increase in specific [³H]alanine counts. However, the substitution to intra-vesicular K⁺ did not affect the [³H]alanine activity. Further investigations must clarify whether the changes in observed [³H]alanine activity constitute a transport or a binding event.

The LeuT A22S mutant displayed a decrease in both Na⁺ and K⁺ affinity (Figure 4). When inserting it into the LeuT^tmFRET background (A22S^tmFRET), the pattern in tmFRET efficiencies were largely unaltered from LeuT^tmFRET, although with a minor reduction in responses upon addition of ligands (Figure 6D). This suggests that the mutation only has minor effect on the overall conformational equilibrium independent of the added ligands. When inserted into liposomes, LeuT A22S retained the ability to transport alanine in the presence of a Na⁺ gradient. As for LeuT WT, intra-vesicular K⁺ increased the concentrative capacity for [³H]alanine transport. However, the increase was 3-fold higher than what we observed for LeuT WT (Figure 6—figure supplement 1B). The WT-like conformational equilibrium could suggest that the decreased Na⁺ and K⁺ affinities are due to a direct perturbation of the ion binding site by the A22S mutation.

Finally, we characterized the tmFRET response for T254S^tmFRET. The mutant exhibited a slightly higher tmFRET efficiency in the apo state (NMDG⁺), but its conformational response to Na⁺, substrate, and K⁺ binding was similar to LeuT^tmFRET (Figure 6E). Reconstituted into liposomes, LeuT T254S transported [³H]alanine and retained a WT-like increase in concentrative capacity with intra-vesicular K⁺ (Figure 6—figure supplement 1C). As the substitution is the only apparent difference between the Na1 site in LeuT relative to the human SERT, DAT, and NET, it could suggest a similar functionality of the Na1 site by the four transporters.

In all, although we are unable to discern between direct and indirect effects imposed by the mutants, our results do reflect both concerted and opposed consequences of Na⁺ and K⁺ binding, conformation, and substrate transport.

Unless otherwise stated, reagents were purchased from Merck (Life Sciences).

DNA encoding LeuT from A. aeolicus, fused C-terminally to a thrombin protease cleavage site and an octahistidine tag, was cloned into a pET16b vector. Single mutations in the Na1 site (A22V, A22S, N27Q, T254S, N286Q, E290Q) were inserted into LeuT constructs with pre-existing pairs of mutations (A313H-A317H-K398C; K398C) and without (WT). Full gene sequences were verified by DNA sequencing (Eurofins Genomics).

Expression and purification of LeuT variants containing the tmFRET pair mutations were performed essentially as described previously (Billesbølle et al., 2016). In brief, E. coli C41(DE3) cells were transformed with pET16b plasmid encoding the desired LeuT Na1 site variants and single colonies were cultivated at 37°C in Lysogeny Broth. Expression was induced at OD₆₀₀∼0.6 upon induction with β-D-1-thiogalactopyranoside (IPTG). Harvested cells were disrupted using a cell disruptor (CF1, ConstantSystems) and isolated crude membranes were solubilized with 1.5% (wt/vol) DDM (anagrade, Anatrace). Solubilized LeuT was incubated with Ni²⁺-NTA resin (Thermo Fisher Scientific) and 40 mM imidazole, batch-washed and labeled overnight with F5M (Thermo Fisher Scientific). Following wash of the resin with 15 successive column volumes of buffer (20 mM Tris-HCl [pH 7.4], 20% [vol/vol] glycerol, 200 mM KCl, 0.05% [wt/vol] DDM, 100 µM tris(2-carboxyethyl)phosphine [TCEP] [hydrochloride solution]) containing 90 mM imidazole, immobilized LeuT was eluted and frozen in buffer with 340 mM imidazole. Labeling efficiency and specificity were examined by absorbance measurements (280 and 490 nm) and sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, respectively. For LeuT variants on WT background (devoid of tmFRET mutations), the F5M labeling step was skipped and the resin washing procedure was performed with 3 and 5 column volumes of buffer containing 60 and 90 mM imidazole, respectively. For reconstitution in liposomes, LeuT was dialyzed overnight in buffer (20 mM Tris-HCl [pH 7.4], 20% [vol/vol] glycerol, 200 mM KCl, 0.05% [wt/vol] DDM) to remove imidazole. The protein was subsequently concentrated to >2 mg/ml.

Pharmacological studies were conducted on purified LeuT variants by virtue of the SPA. Leucine affinities for WT LeuT and mutants displaying WT substrate affinities (A22V, A22S) were determined by saturation binding of [³H]leucine (25 Ci/mmol) (PerkinElmer), with unspecific binding corrected for by the addition of 100 µM unlabeled leucine. Alanine and leucine affinities for the remaining LeuT variants were determined by the ability of increasing concentrations of unlabeled alanine or leucine, respectively, to competitively displace a fixed concentration of [³H]leucine. Assayed in a 96-well plate (Corning), LeuT was mixed with Yttrium Silicate Copper (YSi-Cu) His-tag SPA beads (PerkinElmer) and [³H]leucine in binding buffer (200 mM NaCl, 20 mM Tris [pH 8], 20% [vol/vol] glycerol, 0.05% [wt/vol] DDM, and 100 µM TCEP). The following conditions were applied for the mutants tested: WT, K398C, A22V, A22S: 0.3 µg/ml protein, 1.25 mg/ml YSi-Cu His-tag SPA beads, 120 nM [³H]leucine (25 Ci/mmol); N27Q, N286Q: 3 µg/ml protein, 1.6 mg/ml YSi-Cu His-tag SPA beads, 1200 nM [³H]leucine (4 Ci/mmol); E290Q: 1.5 µg/ml protein, 1.6 mg/ml YSi-Cu His-tag SPA beads, 200 nM [³H]leucine (20 Ci/mmol). The Na⁺ dependency on [³H]leucine binding was determined by mixing LeuT, YSi-Cu His-tag SPA beads (in equivalent concentrations as above) and a fixed concentration of [³H]leucine (10× K_d, as determined in 200 mM NaCl) in binding buffer supplemented with increasing concentrations of Na⁺. The specific activity of [³H]leucine was kept approximately inversely proportional to the concentration of [³H]leucine and protein used. This experiment was repeated in the presence of 200 and 800 mM K⁺. The K⁺-dependent inhibition of Na⁺-mediated [³H]leucine binding was assayed by subjecting LeuT mutants (double the concentration as above) to increasing concentrations of K⁺ (0–1600 mM) in the presence of Na⁺ equivalent to the EC₅₀ determined with 10× K_d of [³H]leucine. The unspecific binding of [³H]leucine was determined upon addition of 100 (WT, A22V, A22S) or 300 µM (N27Q, N286Q) of unlabeled leucine. Ionic strengths were preserved by substituting Na⁺ and K⁺ for Ch⁺. For competition binding and ion-dependent [³H]leucine binding experiments, ligand depletion was avoided by maintaining ≥20-fold molar excess of [³H]leucine relative to LeuT. Sealed plates were incubated for ~16 hr at 4°C with gentle agitation and counts per minute (c.p.m.) were recorded on a 2450 MicroBeta² microplate counter (PerkinElmer) in ‘SPA’ mode.

Saturation binding data were corrected for unspecific binding, normalized to B_max and fitted to a one-site binding regression from which K_d values were obtained using GraphPad Prism 9.0. For competition binding, data points were normalized to a control (without competing unlabeled leucine or alanine) and fitted to a single-site log(inhibitor)-response model. Derived IC₅₀ values (inhibitor concentration that reduces binding of radioligand by 50%) were converted to inhibition constants K_i by the Cheng-Prusoff equation:

where [S] and K_d refer to the concentration and affinity, respectively, of the radioligand. Data points for K⁺ competition binding were corrected for unspecific binding and normalized to a control (absent for K⁺), whereas data for Na⁺-dependent [³H]leucine binding were normalized to the maximum response predicted by the model. Both were fitted to the Hill equation from which IC₅₀ and EC₅₀ values, respectively, were extracted. All experiments were performed at least three independent times in triplicates for specific and triplicates/duplicates for unspecific binding. Data points and Hill slopes are reported as mean ± s.e.m. and K_d, K_i, and EC₅₀ values are reported as [s.e.m. interval]. Statistical analysis was performed with a post hoc Dunnett’s multiple test in a one-way analysis of variance (ANOVA), comparing every mean with that obtained in the absence of competitor.

LeuT Na1 site mutants examined with tmFRET were encoded with a set of FRET pair mutations (A313H-A317H-K398C/K398C) designed and characterized previously (Billesbølle et al., 2016). Purified and fluorescein-labeled LeuT variants were centrifuged at 15,000 × g at 4°C for 10 min, and diluted to ~10 nM (adjusted for labeling efficiency) in fluorescence buffer (20 mM Tris-Cl [pH 8], 0.05% [wt/vol] DDM, 100 µM TCEP), supplemented with 800 mM chloride salts of NMDG⁺, Na⁺±50 µM leucine/200 µM alanine, K⁺ or Rb⁺ as specified. Samples were incubated for 30 min at room temperature in the dark. If not otherwise specified, parallel experiments on LeuT constructs with and without the His-X₃-His motif were conducted, here referred to with the suffixes ^tmFRET and ^K398C, respectively. Fluorescence measurements using a single saturating concentration of Ni²⁺ were assayed in a Swartz cuvette (Hellma Analytics) inserted into a FluoroMax-4 spectrophotometer (HORIBA Scientific) temperature controlled to 25°C. Reference-corrected fluorescence intensities (0.1 s integration time) were recorded following the incubation with 10 mM Ni²⁺, using constant excitation and emission wavelengths of 492 and 512 nm, respectively, and 4 nm excitation and emission slit widths. For ion titration experiments, LeuT^tmFRET was incubated in fluorescence buffer containing 750 µM Ni²⁺ and increasing concentrations of Na⁺, K⁺, or Rb⁺ (0–1584 mM), substituted with Ch⁺ to preserve ionic strength. Fluorescence intensities were obtained using a FluoroMax-4 spectrophotometer and the same technical specifications as described above. For Ni²⁺ titration experiments performed in 96-well plates (Corning), LeuT was subjected to increasing concentrations of Ni²⁺ (10^–7 to 10^–2 M) in buffer containing 800 mM of NMDG⁺, Na⁺±50 µM leucine, or K±50 µM leucine. Fluorescence intensities at 512 nm were recorded on a PolarStar Omega plate reader (NMG Labtech) upon excitation at 492 nm.

Fluorescence intensities for LeuT^tmFRET variants (F) were normalized to their equivalents for LeuT^K398C (F_{no site}) without the metal-chelating His-X₃-His site, to correct for the contribution of collisional quenching from free Ni²⁺, dilution, as well as the primary inner filter effect (1 – F/F_{no site}). An increase in 1 – F/F_{no site} implies an enhanced energy transfer between FRET donor (F5M) and acceptor (Ni²⁺) that, when plotted as a function of increasing Ni²⁺, was fitted to a Hill equation from which EC₅₀ and maximal tmFRET values were obtained. Maximal tmFRET efficiencies were converted to distances by the FRET equation:

with E, R, and R₀ being the efficiency of energy transfer, distance between FRET probes, and Förster distance (R₀=12 Å for Ni²⁺/fluorescein; Taraska et al., 2009b), respectively. For ion titration experiments, fluorescence intensities for LeuT^tmFRET (not normalized to LeuT^K398C) at each ion concentration (F) were normalized to fluorescence intensities obtained in the absence (F₀) of Na⁺, K⁺, or Rb⁺ (F₀/F). As for 1 – F/F_{no site}, F₀/F is inversely dependent on the distance between the FRET probes and when plotted against the ion concentration, data points could be fitted to a Hill equation, yielding the Hill slope and EC₅₀ value. Experiments were repeated at least three independent times in triplicates using protein from two separate purifications. Data points and Hill slopes are reported as mean ± s.e.m., whereas EC₅₀ values are mean [s.e.m. interval]. When indicated, data points were subjected to statistical analysis using either post hoc Tukey or Bonferroni multiple comparison test as part of a one-way ANOVA.

E. coli polar lipid extract dissolved in chloroform (Avanti Polar Lipids) was dried under a steam of N₂ for 2 hr. Lipids were re-suspended to 10 mg/ml in reconstitution buffer (200 mM NMDG-Cl, 20 mM Tris/HEPES [pH 7.5]) by vortexing and 2× 10 min bath sonication. The lipid solution was subjected to five freeze-thaw cycles in ethanol dry ice bath. Subsequently, the liposomes were extruded with a mini extruder (Avanti Polar Lipids) 11 times through a Nuclepore Track-Etch Membrane polycarbonate filter of pore size 400 nm (GE Healthcare Life Sciences) and diluted to 4 mg/ml in reconstitution buffer. Liposome destabilization was induced by stepwise addition of 10 µl aliquots of 10% (vol/vol) Triton X-100. Destabilization of liposomes was followed by measuring absorbance at 550 nm. Triton X-100 was added until absorbance of the sample had reached a maximum and started to decrease. LeuT solubilized in DDM was added in a protein to lipid ratio of 1:25 (wt/wt) and the protein-liposome solution was incubated under slow rotation for 30 min at 4°C. Semidry SM-2 Bio-Beads (Bio-Rad Laboratories), equilibrated in reconstitution buffer, were added in aliquots of 8.5 mg beads/mg lipid after 30 min, 60 min, 120 min and after overnight incubation at 4°C with gentle agitation. The beads were filtered out 2 hr after the last addition of bio-beads. The proteoliposome solution was diluted ~20 times in the indicated internal buffer (200 mM salt [NaCl, KCl, NMDG-Cl, CsCl, or RbCl], 20 mM Tris/HEPES [pH 7.5]) and centrifuged for 1 hr at 140,000 × g at 4°C. Pelleted proteoliposomes were re-suspended in the indicated internal buffer to a final concentration of 10 mg lipid/ml. Single-use aliquots of proteoliposomes were flash-frozen in liquid N₂ and stored at –80°C until further use.

Proteoliposomes were thawed and extruded through a Nuclepore Track-Etch Membrane polycarbonate filter with 400 nm pore size (GE Healthcare Life Sciences). Uptake was assayed in a 96-well setup in ultra-low attachment, round bottom plate (Costar) at room temperature (21–23°C). Uptake buffer (20 mM Tris/HEPES [pH 7.5], 200–225 mM salt [NaCl, KCl, or NMDG-Cl]) supplemented with [³H]alanine (Moravek Biochemicals) was added to each well in a volume of 190 µl. Subsequently, 10 µl of proteoliposome solution was added to start the uptake reaction. For time-dependent uptake, the uptake buffer was supplemented with 2 µM [³H]alanine with a specific activity of 1.335 Ci/mmol and proteoliposomes were added at the indicated time points (2–60 min). For concentration-dependent uptake, uptake buffer was supplemented with [³H]alanine with a specific activity of 3.73 Ci/mmol (50–2000 nM) or 1.07 Ci/mmol (3.5–6 µM). The uptake reaction was terminated after 5 min. To assess nonspecific [³H]alanine uptake and binding, proteoliposomes were pre-incubated with 200 µM unlabeled leucine for 15 min, and 100 µM unlabeled leucine was added to the uptake buffer to saturate all transporters with leucine. The uptake reaction was terminated by filtering the samples through a 96-well glass fiber filter (Filtermat B – GF/B, Perkin Elmer) soaked in 1.5% poly(ethyleneimine) solution, using a cell harvester (Tomtec harvester 96 match II). The filter was washed with 1 ml ice-cold wash buffer (200 mM ChCl, 20 mM Tris/HEPES [pH 7.5]) for each of the 96 positions on the filter. Filter plates were dried at 96°C before scintillation sheet (MeltiLex B/HS, Perkin Elmer) was melted on to the filter. Filters were counted on a 2450 MicroBeta² microplate counter (PerkinElmer) in ‘normal’ counting mode.

As for uptake experiments, proteoliposomes were extruded through a Nuclepore Track-Etch Membrane polycarbonate filter with 400 nm pore size (GE Healthcare Life Sciences). Reconstituted LeuT corresponding to 8 µg was treated with thrombin (0.5 unit; Cytiva) and the reaction was quenched upon the addition of 500 µM PMSF after 1, 5, 10, 30, 60, 120, and 180 min. Controls without thrombin and with 1.5% DDM (for 180 min) were included. Following SDS-PAGE, the gel was stained with InstantBlue (abcam) and images were obtained using an ImageQuant 800 (Cystiva).

To assess the amount of active protein in each reconstitution condition, a sample of proteoliposomes from each of the different intra-vesicular buffer conditions was solubilized in buffer (30% glycerol, 1% [wt/vol] DDM, 20 mM Tris/HEPES [pH 7.5], 200 mM NaCl). The samples were left for 3 hr with gentle agitation at 4°C to dissolve proteoliposomes and solubilize LeuT in DDM detergent micelles. The amount of re-solubilized active LeuT was assessed by binding in a saturating [³H]leucine concentration (1 µM) in buffer (200 mM NaCl, 20 mM Tris [pH 8], 0.05% [wt/vol] DDM) by SPA. Measures of maximum binding from each condition were used to normalize the c.p.m. obtained from uptake experiments to ensure that uptake was not affected by variations in protein content in the proteoliposome samples. The highest maximum binding was used as normalization standard.

For time-dependent uptake, specific uptake data, normalized to the amount of active protein (see section above), was fitted to a one-phase association using GraphPad Prism 9.0.

where p is the maximal uptake (the amplitude), k is the rate constant in min^–1, t is time in min, and y is uptake in c.p.m. after normalization. Data from each experiment was subsequently normalized to the value of p from the condition with NMDG⁺. The normalized data sets from each experiment were combined and re-fitted to a one-phase association.

For concentration-dependent uptake, normalized, specific uptake data was fitted to the Michaelis-Menten equation:

where V_max is the maximal uptake rate, K_m is the substrate concentration where half-maximum uptake rate is reached, y is the uptake in c.p.m. after normalization. [s] is the [³H]alanine concentration in µM. Subsequently data was either combined or (if shown in % on the y-axis) normalized to the V_max value determined with intra-vesicular NMDG⁺. The normalized data sets from each experiment were combined and fitted to the Michaelis-Menten equation. All uptake experiments were done in triplicates or duplicates as indicated and repeated three to four times using protein obtained from at least two different purifications and reconstitutions.

For concentration-dependent uptake with different intra-vesicular cations, c.p.m. were converted to d.p.m., and then to pmol of [³H]alanine per min, using the specific activity of [³H]alanine and a counting efficiency of 40% for 2450 MicroBeta² microplate counter (PerkinElmer) in ‘normal’ counting mode.

All newly created materials (e.g. LeuT mutants) are available upon request.

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

We would like to thank Patricia Curran for technical guidance. We also acknowledge the NINDS intramural program for support. Funding sources: Support for this research was provided by the Independent Research Fund Denmark (1030-00036B to CJL), the Lundbeck Foundation (R344-2020-1020 to CJL), the Novo Nordic Foundation (NNF19OC0058496 to CJL), and the Carlsberg Foundation (CF20-0345 to CJL).

1. Preprint posted: April 4, 2023 (view preprint)
2. Sent for peer review: April 4, 2023
3. Preprint posted: June 22, 2023 (view preprint)
4. Preprint posted: December 11, 2023 (view preprint)
5. Version of Record published: January 25, 2024 (version 1)

You can cite all versions using the DOI https://doi.org/10.7554/eLife.87985. This DOI represents all versions, and will always resolve to the latest one.

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