Exploring the K⁺ binding site and its coupling to transport in the neurotransmitter:sodium symporter LeuT

Reviewer #1 (Public Review):

This manuscript tackles an important question, namely how K+ affects substrate transport in the SLC6 family. K+ effects have previously been reported for DAT and SERT, but the prototypical SLC6-fold transporter LeuT was not known to be sensitive to the K+ concentration. In this manuscript, the authors demonstrate convincingly that K+ inhibits Na+ binding, and Na+-dependent amino acid binding at high concentrations, and that K+ inside of vesicles containing LeuT increases the transport rate. However, outside K+ apparently had very little effect. Uptake data are supplemented with binding data, using the scintillation proximity assay, and transition metal FRET, allowing the observation of the distribution of distinct conformational states of the transporter.
Overall, the data are of high quality. I was initially concerned about the use of solutions of very high ionic strength (the Km for K+ is in the 200 mM range), however, the authors performed good controls with lower ionic strength solutions, suggesting that the K+ effect is specific and not caused by artifacts from the high salt concentrations.

The major issue I have with this manuscript is with the interpretation of the experimental data. Granted that the K+ effect seems to be complex. However, it seems counterintuitive that K+ competes with Na+ for the same binding site, while at the same time accelerating the transport rate. Even if K+ prevents rebinding of Na+ on the inside of vesicles, it would be expected that K+ then stabilizes this Na+-free conformation, resulting in a slowing of the transport rate. However, the opposite is found. I feel that it would be useful to perform some kinetic modeling of the transport cycle to identify a mechanism that would allow K+ to act as a competitive inhibitor of Na+ binding and rate-accelerator at the same time.

This ties into the second point: It is not mentioned in the manuscript what the configuration of the vesicles is after LeuT reconstitution. Are they right-side out? Is LeuT distributed evenly in inside-out and right-side out orientation? Is the distribution known? If yes, how does it affect the interpretation of the uptake data with and without K+ gradient?

Finally, mutations were only made to the Na1 cation binding site. These mutations have an effect mostly to be expected, if K+ would bind to this site. However, indirect effects of mutations can never be excluded, and the authors acknowledge this in the discussion section. It would be interesting to see the effect of K+ on a couple of mutants that are far away from Na+/substrate binding sites. This could be another piece of evidence to exclude indirect effects, if the K+ affinity is less affected.

Reviewer #2 (Public Review):

To characterize the relationship between Na+ and K+ binding to LeuT, the effect of K+ on Na+- dependent [3 H] leucine binding was studied using a scintillation proximity assay. In the presence of K+ the apparent affinity for sodium was reduced but the maximal binding capacity for this ion was unchanged, consistent with a competitive mechanism of inhibition between Na+ and K+.

To obtain a more direct readout of K+ binding to LeuT, tmFRET was used. This method relies on the distance-dependent quenching of a cysteine-conjugated fluorophore (FRET donor) by a transition metal (FRET acceptor). This method is a conformational readout for both ion- and ligand-binding. Along with the effect of K+ on Na+-dependent [3 H] leucine binding, the findings support the existence of a specific K+ binding site in LeuT and that K+ binding to this site induces an outward closed conformation.

It was previously shown that in liposomes inlaid with LeuT by reconstitution, intra-vesicular K+ increases the concentrative capacity of [ 3 H] alanine. To obtain insights into the mechanistic basis of this phenomenon, purified LeuT was reconstituted into liposomes containing a variety of cations, including Na+ and K+ followed by measurements of [ 3 H] alanine uptake driven by a Na+ gradient. The ionic composition of the external medium was manipulated to determine if the stimulation of [3 H] alanine uptake by K+ was due to an outward directed potassium gradient serving as a driving force for sodium-dependent substrate transport by moving in the direction opposite to that of sodium and the substrate. Remarkably it was found that it is the intra-liposomal K+ per se that increases the transport rate of alanine and not a K+ gradient, suggesting that binding of K+ to the intra-cellular face of the transporter could prevent the rebinding of sodium and the substrate thereby reducing their efflux from the cell. These conclusions assume that the measured radioactive transport is via right-side-out liposomes rather than from their inverted counterparts (in case of a random orientation of the transporters in the proteoliposomes). Even though this assumption is likely to be correct, it should be tested.

Since K+- and Na+-binding are competitive and K+ excludes substrate binding, the Authors chose to focus on the Na1 site where the carboxyl group of the substrate serves as one of the groups which coordinate the sodium ion. This was done by the introduction of conservative mutations of the amino acid residues forming the Na1 site. The potassium interaction in these mutants was monitored by sodium dependent radioactive leucine binding. Moreover, the effect the effect of Na+ with and without substrate as well as that of potassium on the conformational equilibria was measured by tmFRET measurements on the mutants introduced in the construct enabling the measurements. The results suggest that K+-binding to LeuT modulates substrate transport and that the K+ affinity and selectivity for LeuT is sensitive to mutations in the Na1 site, pointing toward the Na1 site as a candidate site for facilitating the interaction between K+ in some NSS members.

The data presented in this manuscript are of very high quality. They are a detailed extension of results by the same group (Billesbolle et. al, Ref. 16 from the list) providing more detailed information on the importance of the Na1 site for potassium interaction. Clearly this begs for the identification of the binding site in a potassium bound LeuT structure in the future. Presumably LeuT was studied here because it appears that it is relatively easy to determine structures of many conformational states. Furthermore, convincing evidence showed that the stimulatory effect of K+ on transport is not because of energization of substrate accumulation but is rather due to the binding of this cation to a specific site.