Transition metal ion FRET uncovers K+ regulation of a neurotransmitter/sodium symporter

Billesbølle, Christian B.; Mortensen, Jonas S.; Sohail, Azmat; Schmidt, Solveig G.; Shi, Lei; Sitte, Harald H.; Gether, Ulrik; Løland, Claus J.

doi:10.1038/ncomms12755

Cited by 45 publications

(92 citation statements)

References 45 publications

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“…Furthermore, the ability of Na + to close the cytoplasmic pathway and open the extracellular pathway was evident whether K + or NMDG + (N-methyl-D-glucamine) was used as the control cation ( Fig. 1B), contrary to a recent suggestion that K + , rather than Na + , was responsible for conformational change (32). We do not understand the reason for this discrepancy.…”

Section: Resultscontrasting

confidence: 65%

“…We do not understand the reason for this discrepancy. However, the previous study used a LeuT mutant in which two residues were mutated to histidine to coordinate a Ni 2+ ion, and another mutated to cysteine to attach a fluorophore so that conformational changes could be measured, in detergent, by transition metal FRET (32). In contrast, our constructs contained only one mutation, to cysteine, and reactivity was measured in E. coli membranes in the absence of detergent.…”

Section: Resultsmentioning

confidence: 99%

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Structural elements required for coupling ion and substrate transport in the neurotransmitter transporter homolog LeuT

Zhang

Tavoulari

Sinning

et al. 2018

Preprint

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The coupled transport of ions and substrates allows transporters to accumulate substrates using the energy of transmembrane ion gradients and electrical potentials. During transport, conformational changes that switch accessibility of substrate and ion binding sites from one side of the membrane to the other must be controlled so as to prevent uncoupled movement of ions or substrates. In the Neurotransmitter:Sodium Symporter (NSS) family, Na + stabilizes the transporter in an outward-open state, thus decreasing the likelihood of uncoupled Na + transport. Substrate binding, in a step essential for coupled transport, must overcome the effect of Na + , allowing intracellular substrate and Na + release from an inward-open state. However, the specific elements of the protein that mediate this conformational response to substrate binding are unknown. Previously, we showed that in the prokaryotic NSS transporter LeuT, the effect of Na + on conformation requires the Na2 site, where it influences conformation by fostering interaction between two domains of the protein (JBC 291: 1456(JBC 291: , 2016. Here, we used cysteine accessibility to measure conformational changes of LeuT in E. coli membranes. We identified a conserved tyrosine residue in the substrate binding site required for substrate to convert LeuT to inward-open states by establishing an interaction between the two transporter domains. We further identify additional required interactions between the two transporter domains in the extracellular pathway. Together with our previous work on the conformational effect of Na + , these results identify mechanistic components underlying ion-substrate coupling in NSS transporters. Significance StatementMembrane transport proteins are responsible for moving substrates such as nutrients, vitamins, drugs and signaling molecules across cellular membranes. A subset of these proteins, the ion-coupled transporters, use a transmembrane ion gradient to drive energetically unfavorable substrate movement from a lower concentration on one side of the membrane to a higher concentration on the other side. They do this by coupling the movement of substrate and ions in the same or the opposite direction. Coupled transport requires conformational changes that occur exclusively or predominantly when specific conditions of ion and substrate binding are met. This work identifies, for a family of Na + -coupled neurotransmitter transporters, how the rules controlling these conformational changes are encoded in the transporter structure.

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Section: Resultscontrasting

confidence: 65%

Section: Resultsmentioning

confidence: 99%

Structural elements required for coupling ion and substrate transport in the neurotransmitter transporter homolog LeuT

Zhang

Tavoulari

Sinning

et al. 2018

Preprint

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“…The role of K + ions in the function of the related SERT transporter is well-established67, and we have recently described significant effects that the presence of a K + ion in the Na2 site can have on the dynamics of the extracellular end of the cognate LeuT and its transport cycle(see refs 72 and 73). While it is tempting to speculate that the ability of K + ions to enter the intracellular region of DAT could facilitate Na + /Na2 release from the intermediate state, a more complete evaluation of the impact that K + ions may have on various elements of NSS mechanisms will require future considerations based on quantitative free energy-based computations.…”

Section: Discussionmentioning

confidence: 95%

A Markov State-based Quantitative Kinetic Model of Sodium Release from the Dopamine Transporter

Razavi

Khelashvili

Weinstein

2017

Sci Rep

126

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The dopamine transporter (DAT) belongs to the neurotransmitter:sodium symporter (NSS) family of membrane proteins that are responsible for reuptake of neurotransmitters from the synaptic cleft to terminate a neuronal signal and enable subsequent neurotransmitter release from the presynaptic neuron. The release of one sodium ion from the crystallographically determined sodium binding site Na2 had been identified as an initial step in the transport cycle which prepares the transporter for substrate translocation by stabilizing an inward-open conformation. We have constructed Markov State Models (MSMs) from extensive molecular dynamics simulations of human DAT (hDAT) to explore the mechanism of this sodium release. Our results quantify the release process triggered by hydration of the Na2 site that occurs concomitantly with a conformational transition from an outward-facing to an inward-facing state of the transporter. The kinetics of the release process are computed from the MSM, and transition path theory is used to identify the most probable sodium release pathways. An intermediate state is discovered on the sodium release pathway, and the results reveal the importance of various modes of interaction of the N-terminus of hDAT in controlling the pathways of release.

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“…Interestingly, this requires a negatively charged Glu-290, the carboxylate of which corresponds to bound Cl − in the eukaryotic transporters (37). A recent study indicates that intracellular K + precludes Na + rebinding to the inward facing conformation of LeuT (38). Our data are consistent with this sequence of events and together with other studies (3, 4, 39) contribute to the emerging concept that antiport of K + or of H + is a key feature in the transport cycle of the LeuT superfamily.…”

Section: Discussionmentioning

confidence: 99%

Electrogenic Binding of Intracellular Cations Defines a Kinetic Decision Point in the Transport Cycle of the Human Serotonin Transporter

Hasenhuetl

Freissmuth

Sandtner

2016

Journal of Biological Chemistry

151

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The plasmalemmal monoamine transporters clear the extracellular space from their cognate substrates and sustain cellular monoamine stores even during neuronal activity. In some instances, however, the transporters enter a substrate-exchange mode, which results in release of intracellular substrate. Understanding what determines the switch between these two transport modes demands time-resolved measurements of intracellular (co-)substrate binding and release. Here, we report an electrophysiological investigation of intracellular solute-binding to the human serotonin transporter (SERT) expressed in HEK-293 cells. We measured currents induced by rapid application of serotonin employing varying intracellular (co-)substrate concentrations and interpreted the data using kinetic modeling. Our measurements revealed that the induction of the substrate-exchange mode depends on both voltage and intracellular Na+ concentrations because intracellular Na+ release occurs before serotonin release and is highly electrogenic. This voltage dependence was blunted by electrogenic binding of intracellular K+ and, notably, also H+. In addition, our data suggest that Cl− is bound to SERT during the entire catalytic cycle. Our experiments, therefore, document an essential role of electrogenic binding of K+ or of H+ to the inward-facing conformation of SERT in (i) cancelling out the electrogenic nature of intracellular Na+ release and (ii) in selecting the forward-transport over the substrate-exchange mode. Finally, the kinetics of intracellular Na+ release and K+ (or H+) binding result in a voltage-independent rate-limiting step where SERT may return to the outward-facing state in a KCl- or HCl-bound form.

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Transition metal ion FRET uncovers K+ regulation of a neurotransmitter/sodium symporter

Cited by 45 publications

References 45 publications

Structural elements required for coupling ion and substrate transport in the neurotransmitter transporter homolog LeuT

Structural elements required for coupling ion and substrate transport in the neurotransmitter transporter homolog LeuT

A Markov State-based Quantitative Kinetic Model of Sodium Release from the Dopamine Transporter

Electrogenic Binding of Intracellular Cations Defines a Kinetic Decision Point in the Transport Cycle of the Human Serotonin Transporter

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