Pharmacological Characterization of Purified Full-Length Dopamine Transporter from Drosophila melanogaster
Round 1
Reviewer 1 Report
Pugh et al., provided careful examination of purified Drosophila melanogaster (dDAT) function and compared the results with human DAT (hDAT) and human norepinephrine transporter expressed in COS-7 cells. Purified human DAT (hDAT) stable enough and thus suitable for biophysical assays is not available. The next best option for structure/function studies and identifying molecular mechanism of hDAT, is utilizing the hDAT homologues such as, the dopamine transporter expressed in Drosophila melanogaster (dDAT). Since dDAT displays high sequence homology with the solute carrier 6 transporters, 45% homology for human DAT, NET and SERT, respectively and approximately 80% similarity in transmembrane domains, purified dDAT is a suitable model system. Nevertheless, dDAT’s vs. hDAT’s pharmacological profile remains unknown. The authors characterized purified, full length dDAT in detergent micelles using radioligand binding with the scintillation proximity assay. Since DAT activity is linked to Na and Cl conductance, the authors carefully investigated the consequences of Na and Cl binding on 3H-nisoxetine affinity. In addition, the binding profiles of dDAT substrates and antagonist were determined. The comparison with other transporter systems such as human DAT and human norepinephrine transporter were done in COS-7 cell model system. Overall, this study employed rigorous and complementary approaches a provided detailed pharmacological profile for purified, full length dDAT. The results provide powerful platform for studying pharmacology and substrate selectivity of DAT, but also other members of the Sodium Symporter family. This study significantly advances our understandings of DAT’s molecular mechanisms and addresses important knowledge gaps in the transporter field.
This is a nicely written manuscript. Rigorous experimental design is employed. Figures are clear and data presented in this study support the conclusion. My recommendation: Accept in present form.
Author Response
We cordially thank the reviewer for the generous decision.
Reviewer 2 Report
Pugh et al. purified full length DAT from Drosophila and characterized the affinity and pharmacological properties of several DAT ligands using radioligand binding and scintillation proximity assay. They observed salt dependent concentration effects on the affinity of the ligands. Besides, they were the first to determine the affinity of dopamine to dDAT. They also compared the affinity obtained from using purified dDAT and dDAT in cells and observed some differences in affinity values. Having the ability to generate purified dDAT paves the way for further biophysical studies for this protein and can inspire similar efforts for other membrane bound receptors. Overall, the manuscript was well-written and the experimental design was good. I recommend the publication of the manuscript in its present form.
---more detailed review according to the suggested guidelines
1. What is the main question addressed by the research?
The authors purified full length dDAT to investigate the binding profiles of several ligands using radioligand binding assays. This has not been performed for dDAT previously, and represents a way forward to conduct biophysical studies on purified receptors as an orthogonal method to validate ligand binding. The authors also examined the effect of ion concentrations on the binding affinity of ligands and compared the binding affinities of these ligands with that obtained in cellular assays.
2. Do you consider the topic original or relevant in the field? Does it
address a specific gap in the field?
The research topic is relevant to the field because of potential differences in binding affinities and pharmacological functions from using different heterologous cellular systems to study the receptor in question that could arise due to a variety of factors – overexpression, different gene expression levels, etc. Having the ability to purify receptors and study them using biophysical techniques would provide a more objective way to study the binding and pharmacological properties of ligands.
3. What does it add to the subject area compared with other published
material?
Thus far, the pharmacological profile of dDAT was elucidated in cell lines stably transfected with dDAT. The authors established a pharmacological profile for purified full length dDAT that has not been performed before. In doing so allows comparison with pharmacological profiles with human DAT and NET. Their approach provides a way to study the pharmacology and ligand selectivity of DAT and potentially other receptors from the NSS family.
4. What specific improvements should the authors consider regarding the
methodology? What further controls should be considered?
Experimental methodology was satisfactory to address the research questions. No further controls needed.
5. Are the conclusions consistent with the evidence and arguments presented
and do they address the main question posed?
Yes.
6. Are the references appropriate?
Yes.
7. Please include any additional comments on the tables and figures.
I suggest the authors present the data in Table 1 consistently. For instance, present all the data as mean +/- sem for clarity and consistency. Please do not use mean [sem interval]. Thank you.
Author Response
We thank the reviewer for the very positive reply. It is so rewarding to find reviewers that are equally satisfied with the results as we are.
Minor point:
“I suggest the authors present the data in Table 1 consistently. For instance, present all the data as mean +/- sem for clarity and consistency. Please do not use mean [sem interval]. Thank you. “
Answer:
We agree that the [SEM interval] is somewhat annoying and appears inconsistent in Table 1 when related to the referenced data. We also agree that Ki values are expressed in many ways in the literature and is commonly shown as means +/- SEM. However, the variability within observations is equally distributed around the pIC50 values (See e.g. Elkins et al. 2013, J Pharmacol Toxicol Methods; https://doi.org/10.1016/j.vascn.2013.04.007). When we change to IC50, that error becomes asymmetric and we have to plot an interval. Thus, if we show the data as +/- SEM, then we must display the pKi-values (e.g. the pKi for nisoxetine would be 8.509 +/- 0.005 M instead of 31 [30; 32] nM). The pKi (or pIC50) values was often reported 2-3 decades ago, and is still used occasionally, specially in screening and SAR studies. I just find it harder as reader to relate affinities to a p-value. Because of this, all affinities in all our publications are shown with [sem interval].
However, we did choose to show the Kd for [3H]nisoxetine as mean +/- SEM because the affinity was determined as a saturation binding experiment. Naturally, this was a mistake as the log distribution is irrespective of the data plotted on a linear or log scale. For consistencies to our own data, we have therefore changed that into mean [SEM interval].
We hope the reviewer accepts our respectful reluctance in changing all data to mean +/- sem.
