Exploring the K+ binding site and its coupling to transport in the neurotransmitter: sodium symporter LeuT
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Exploring the K+ binding site and its coupling to transport in the neurotransmitter : sodium symporter LeuT. / Schmidt, Solveig G.; Nygaard, Andreas; Mindell, Joseph A.; Loland, Claus J.
In: eLife, Vol. 12, 2024.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Exploring the K+ binding site and its coupling to transport in the neurotransmitter
T2 - sodium symporter LeuT
AU - Schmidt, Solveig G.
AU - Nygaard, Andreas
AU - Mindell, Joseph A.
AU - Loland, Claus J.
PY - 2024
Y1 - 2024
N2 - The neurotransmitter:sodium symporters (NSSs) are secondary active transporters that couple the reuptake of substrate to the symport of one or two sodium ions. One bound Na+ (Na1) contributes to the substrate binding, while the other Na+ (Na2) is thought to be involved in the conformational transition of the NSS. Two NSS members, the serotonin transporter (SERT) and the Drosophila dopamine transporter (dDAT), also couple substrate uptake to the antiport of K+ by a largely undefined mechanism. We have previously shown that the bacterial NSS homologue, LeuT, also binds K+, and could therefore serve as a model protein for the exploration of K+ binding in NSS proteins. Here, we characterize the impact of K+ on substrate affinity and transport as well as on LeuT conformational equilibrium states. Both radioligand binding assays and transition metal ion FRET (tmFRET) yielded similar K+ affinities for LeuT. K+ binding was specific and saturable. LeuT reconstituted into proteoliposomes showed that intra-vesicular K+ dose-dependently increased the transport velocity of [3H]alanine, whereas extra-vesicular K+ had no apparent effect. K+ binding induced a LeuT conformation distinct from the Na+- and substrate-bound conformation. Conservative mutations of the Na1 site residues affected the binding of Na+ and K+ to different degrees. The Na1 site mutation N27Q caused a >10-fold decrease in K+ affinity but at the same time a ~3-fold increase in Na+ affinity. Together, 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 with K+ in some NSSs.
AB - The neurotransmitter:sodium symporters (NSSs) are secondary active transporters that couple the reuptake of substrate to the symport of one or two sodium ions. One bound Na+ (Na1) contributes to the substrate binding, while the other Na+ (Na2) is thought to be involved in the conformational transition of the NSS. Two NSS members, the serotonin transporter (SERT) and the Drosophila dopamine transporter (dDAT), also couple substrate uptake to the antiport of K+ by a largely undefined mechanism. We have previously shown that the bacterial NSS homologue, LeuT, also binds K+, and could therefore serve as a model protein for the exploration of K+ binding in NSS proteins. Here, we characterize the impact of K+ on substrate affinity and transport as well as on LeuT conformational equilibrium states. Both radioligand binding assays and transition metal ion FRET (tmFRET) yielded similar K+ affinities for LeuT. K+ binding was specific and saturable. LeuT reconstituted into proteoliposomes showed that intra-vesicular K+ dose-dependently increased the transport velocity of [3H]alanine, whereas extra-vesicular K+ had no apparent effect. K+ binding induced a LeuT conformation distinct from the Na+- and substrate-bound conformation. Conservative mutations of the Na1 site residues affected the binding of Na+ and K+ to different degrees. The Na1 site mutation N27Q caused a >10-fold decrease in K+ affinity but at the same time a ~3-fold increase in Na+ affinity. Together, 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 with K+ in some NSSs.
KW - Aquifex aeolicus
KW - ion binding site
KW - membrane transport
KW - molecular biophysics
KW - neuroscience
KW - secondary active transport
KW - structural biology
KW - structure-function relationships
KW - transition metal ion FRET
U2 - 10.7554/eLife.87985
DO - 10.7554/eLife.87985
M3 - Journal article
C2 - 38271216
AN - SCOPUS:85183516472
VL - 12
JO - eLife
JF - eLife
SN - 2050-084X
ER -
ID: 382155461