The dopamine transporter antiports potassium to increase the uptake of dopamine

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1. Version published to 10.1038/s41467-022-30154-5

   May 4, 2022
2. 

   Biophysics Colab

   Apr 19, 2022

   Biophysics Colab

   Consolidated peer review report (13 December 2021)

   GENERAL ASSESSMENT

   The plasma membrane biogenic amine transporters fulfill important roles in neurotransmission by regulating synaptic serotonin, norepinephrine and dopamine. They are targets for drugs used to treat depression anxiety and other psychiatric disorders and also for drugs of abuse. Additionally, these transporters have been the subject of much research from the 1960’s to the present and have come to represent models for how transporters in general move their substrate neurotransmitters across the plasma membrane and how that process is coupled to transmembrane ion gradients.

   This preprint addresses the role of K+ in transport of dopamine (DA) by the Drosophila dopamine transporter (dDAT). In this family, only the serotonin transporter (SERT) had been shown …

   Biophysics Colab

   Consolidated peer review report (13 December 2021)

   GENERAL ASSESSMENT

   The plasma membrane biogenic amine transporters fulfill important roles in neurotransmission by regulating synaptic serotonin, norepinephrine and dopamine. They are targets for drugs used to treat depression anxiety and other psychiatric disorders and also for drugs of abuse. Additionally, these transporters have been the subject of much research from the 1960’s to the present and have come to represent models for how transporters in general move their substrate neurotransmitters across the plasma membrane and how that process is coupled to transmembrane ion gradients.

   This preprint addresses the role of K+ in transport of dopamine (DA) by the Drosophila dopamine transporter (dDAT). In this family, only the serotonin transporter (SERT) had been shown to use the outwardly directed K+ gradient as a driving force for transport. In another transporter family, K+ coupling to transport was shown for excitatory amino acid transporters. The preprint shows interactions between K+ and dDAT in three areas, competition with Na+ for facilitation of inhibitor binding, conformational changes assessed using hydrogen-deuterium exchange and mass spectrometry, and flux studies in proteoliposomes.

   Transporters in this family use an inwardly directed Na+ gradient to drive substrate uptake, and two Na+ binding sites have been detected in high resolution structures of these proteins. This preprint presents strong evidence that K+ acts competitively with Na+ to inhibit the ability of dDAT to bind nisoxetine, an inhibitor of the human norepinephrine transporter (hNET) that also binds avidly to dDAT. It also presents some supporting data with hDAT showing similar behavior with another radioligand specific for the human homologue.

   The effect of K+ on dDAT conformation was assessed using HDX-MS. Compared with the control condition (200 mM Cs+), dDAT in 200 mM K+ exchanged less D+ for H+ in multiple locations throughout the protein, but found no locations where exchange was enhanced by K+. The measurement was repeated comparing 200 mM Na+ with 200 mM K+. Several of the regions that were stabilized by K+ relative to Cs+ (lower exchange) showed enhanced exchange relative to Na+. Other areas that were stabilized by K+ relative to Na+ were also stabilized relative to Cs+ but several regions that were stabilized relative to Cs+ showed similar exchange rates in Na+ and K+. These results could indicate that K+ induces a more inward-open conformation of dDAT relative to Na+.

   The experiments with dDAT reconstituted in proteoliposomes provide the strongest support for the antiport of K+ for dopamine. Successful reconstitution of a biogenic amine transporter has not been convincingly demonstrated and this result represents an important step forward for the field. The data in Fig. 3b and extended data 4a show specific thermodynamic coupling between transmembrane K+ and dopamine gradients. There is some ambiguity in the interpretation of these experiments that will be addressed in the recommendations.

   The single vesicle experiments described in Fig. 4 provide additional analysis of Na+ influx and K+ efflux in response to imposition of gradients and addition of substrate. These experiments show in more detail the ion fluxes across the membranes of individual proteoliposomes. In agreement with former measurements of DAT-mediated uncoupled ion fluxes, they show that dDAT catalyzes Na+ influx and K+ efflux even in the absence of dopamine. Fluxes were blocked by nortriptyline, demonstrating that these were catalyzed by dDAT and not due to leaky liposomes.

   RECOMMENDATIONS

   Revisions essential for endorsement:

   There are six changes in the text that we feel are necessary, and only one experiment, which we feel would not be difficult for the authors to perform.

   Discussion and calculations:

   1. Because the demonstration of coupling between a K+ gradient and DA accumulation is so significant, it is important to show that it represents catalysis – that is, each reconstituted dDAT molecule is capable of transporting multiple molecules of substrate. The figures show only relative amounts (% of maximum, cpm) and no measure of the amount of reconstituted dDAT in the transport assays. Molar ratios of accumulated dopamine and the amount of reconstituted dDAT (which must be known by the authors) can be translated into the number of molecules transported per transporter. The amount of dDAT could be corrected using the data in extended Fig. 4b, to count only functional transporters. Ideally, the DA/dDAT ratio would be much larger than one, to distinguish the uptake from any kind of 1:1 binding phenomenon.
   2. There is an alternative explanation for the ability of a K+ gradient to drive DA accumulation in Fig. 3b. Imposing an outwardly directed K+ gradient under conditions where there is K+ efflux would generate a diffusion potential (negative inside) which itself could act as a driving force for DAT if the transport process involves inward movement of positive charge. The coupling between the K+ gradient and DA accumulation could therefore be direct, as it is in SERT, or indirect, mediated by the diffusion potential. Unless the authors have convincing reasons to exclude the possibility the latter possibility, they should include that interpretation as an alternative explanation of the results that cannot be ruled out.
   3. The dopamine-independent Na+ and K+ leaks suggest that ion gradients imposed by dilution may not be constant with time. Typically, when ion gradients dissipate with time, substrate transport does not show the stable accumulation observed in Fig. 3b and extended data 4a, but rather an overshoot where a rising phase of accumulation is followed by a falling phase as the driving force of the ion gradients wanes. The relatively rapid increase in intravesicular Na+ and loss of intravesicular K+ shown in Fig. 4b seem in conflict with the long period of stable dopamine accumulation in Fig. 3b. How do the authors explain this difference? The reviewers suggest an experiment in the following section to address this issue.
   4. In describing the HDX-MS studies, there should be some explanation for why Cs+ was used as an inert cation while NMDG+ was used for the binding experiments. In addition, the statement that K+ “stabilizes dDAT structural dynamics” is not quite accurate. HDX data suggest that while this is true when compared to Cs+, comparison with Na+ reveals mixed effects on deuterium uptake in presence of K+. The authors have previously shown that there are multiple regions of dDAT displaying EX1 kinetics in presence of Na+ (ref 20). Does this still hold true for K+? Discussion of deuterium uptake kinetics (especially in the areas shown to have displayed on EX2 behavior) would be valuable when comparing the effect of Na+ and K+ on dDAT conformational dynamics. Is there a way the changes in HDX could be explained that would provide some connection with the other observations? For example, could it be argued that the replacement of Na+ with K+ reversed the effect of Na+ at the Na2 site, which is to favor an outward-facing conformation? The statement at the end of the HDX-MS results seems somewhat tepid (“could suggest that K+ induces a more inward facing dDAT state than Na+.”). It would be more helpful for the reader to point out how the HDX-MS results can be interpreted as evidence for a given conformation, perhaps by comparing these results with conformational results obtained by other methods under similar ionic conditions.
   5. The preprint presents 4 phenomena connecting K+ to dDAT function. There is competition between K+ and Na+ for inhibitor binding, differences between the effects of Na+ and K+ in HDX studies, the ability of Na+ gradients (and amplification by internal K+) to drive dopamine accumulation, and the ability of dDAT to catalyze K+ and Na+ fluxes across the membrane. What is the connection between the flux studies and the other results? If the authors have any hypotheses that link either of these with the accumulation and ion flux data, they should express them. Otherwise, the binding and HDX-MS data seem unconnected from the rest of the experiments in the study.
   6. Another laboratory has published a paper on the role of K+ in dopamine, norepinephrine and serotonin transport (DOI: https://doi.org/10.7554/eLife.67996). That paper concluded that intracellular K+ interacted with each of these transporters but was antiported for substrate only in SERT, not in NET or DAT. This paper was not cited, perhaps by oversight, but it is very relevant, and the authors should address the differences in their conclusions and possibly attempt to explain them.

   Experimental work:

   1. Additional evidence that ion gradients are driving accumulation of intravesicular dopamine could be obtained easily by dissipating the ion gradients after accumulation using an ionophore such as nigericin, monensin or gramicidin, any of which will exchange Na+ and K+ across the membrane and remove the energetic driving force responsible for dopamine accumulation. If added, for example, at 10 min into the time course of Fig. 3b, any of these would cause rapid efflux of accumulated dopamine if the ion gradients were maintaining dopamine accumulation.

   Additional suggestions for the authors to consider:

   Discussion:

   1. The preprint only briefly addresses the many ways that the proteoliposome preparation could be heterogeneous. Transporters could be inserted in inside-out or right-side-out orientations, some of the dDAT added to the reconstitution process could be inactive, vesicles could have 0, 1, 2… etc. copies of DAT. Some of the results indicate that the number of vesicles that show changes could be affected by dopamine, others show that the rate of change was affected. The authors might want to expand the very brief mention of heterogeneity on the first page of the discussion (page 7) to include these other issues and how they might influence the results.
   2. Extended Data Figure 6a shows slow uptake of dopamine in the absence of internal potassium, but the plateau level is the same as in its presence. This would suggest a kinetic effect rather than a thermodynamic effect. On the other hand, this is not observed in Figure 3b. Please explain.
   3. It might be useful to know the sequence identity and similarity between dDAT and hDAT.
   4. In Extended Data Figure 4a it looks like the counts observed with internal sodium represent uncorrected non-specific binding. Correct?

   Experimental:

   1. As stated above, the mechanism by which K+ stimulates DA accumulation could be direct antiport or it could be through generation of a membrane potential. One way to address this issue would be to dissipate any potential by adding a proton ionophore such as FCCP or 2,4-dinitrophenol with a pH buffer in both the medium and the proteoliposomes lumen. If the protonophore inhibited DA accumulation, it would suggest that the potential was the driving force and not direct coupling to K+ as the preprint infers.
   2. In SERT, a pH difference (acid inside) stimulated 5-HT accumulation in the absence of K+, suggesting that protons could replace K+ ions. That would be interesting to test in this system.
   3. One way to show that the ion fluxes in proteoliposomes were catalyzed by dDAT and not a property of the membrane would be to drive ion flux using a H+ diffusion potential. Imposing a pH difference (alkaline inside) in the presence of a proton ionophore should stimulate outward K+ flux. Adding nortriptyline should eliminate the contribution of dDAT to this flux.
   4. It would be informative to know if Cl- was required for the processes studied here, which would strengthen the argument that transport by dDAT was involved. For example, it would be interesting to know if Cl- was required for DA-stimulated K+ efflux and Na+ influx, and the accumulation of DA in response to internal K+.

   REVIEWING TEAM

   Reviewed by:

   Suraj Adhikary, Scientist, Janssen Pharmaceuticals, USA: Membrane protein structural biology and biophysics.

   Baruch Kanner, Professor, The Hebrew University of Jerusalem, Israel: The molecular mechanism of sodium-coupled neurotransmitter transport.

   Christopher Mulligan, Lecturer, University of Kent, UK: Molecular mechanisms of secondary-active transporters.

   Gary Rudnick, Professor, Yale University School of Medicine, USA: Mechanisms of membrane transport proteins.

   Curated by:

   Gary Rudnick, Professor, Yale University School of Medicine, USA

   (This consolidated report is a result of peer review conducted by Biophysics Colab on version 1 of this preprint. Minor corrections and presentational issues have been omitted for brevity.)

   Read the original source
3. 

   Version published to 10.21203/rs.3.rs-1043992/v1 on Research Square

   Nov 3, 2021