Peer review process
Consolidated peer review report (6 April 2022)
GENERAL ASSESSMENT
The sweet and umami sensor proteins, taste receptors type 1 (T1Rs) are important GPCRs underlying taste sensation. In humans, amino acids bind and activate the T1r1/3 heterodimeric receptors leading to umami taste perception, whereas sugars activate the T1r2/3 receptors leading to sweet taste perception. In this manuscript, Atsumi and colleagues combine structural, biophysical and electrophysiological methods to show that Cl- ions also bind to T1Rs, at low mM concentrations, to evoke taste sensation. The authors (1) identify a putative evolutionarily conserved Cl- binding site in the crystal structures of isolated LBDs from medaka fish T1r2a/3 receptors, (2) show that Cl- ions promote protein stability and induce conformational changes in these mfT1r2a/3 LBDs, independent of orthosteric ligands, and (3) demonstrate that mouse chorda tympani nerves are activated by Cl- ions via a T1R-specific mechanism. Based on these findings, the authors conclude that low concentrations of Cl- may bind to sweet receptors and mediate the commonly reported sweet taste sensation following ingestion of low concentrations of table salt.
The elucidation of the molecular mechanism(s) underlying salt taste sensation is a physiologically relevant question that will appeal to a broad audience. Moreover, the authors use an impressive array of different approaches to broadly cover numerous aspects, ranging from structural biology, to biophysics and physiological recordings. Overall, the identification of the chloride ion binding site is convincing, based on the previously solved structure, as well as the bromide ion substitution and long-wavelength Cl- anomalous difference analysis performed in this work. This analysis is supported by biophysical measurements showing that Cl- substantially stabilizes the wild type complex against thermal denaturation, but does not stabilize a point mutant in the putative Cl- binding site. The single fiber recordings suggest there is physiological relevance to the biophysical and structural findings, although they could be strengthened by additional control experiments. Overall, the possibility of Cl- ions acting as a sweet receptor ligand is enticing and the work will likely motivate additional research on this subject.
RECOMMENDATIONS
Revisions essential for endorsement:
The authors should provide refinement statistics and methodology for both the Cl-- and Br-- bound structures, and some comparison between these two structures (global structural alignment & RMSD should be sufficient).
We would recommend that the authors perform nerve recordings using artificial saliva rather than water as the perfusate. This is a key point because the chloride concentration in saliva is approximately 15 mM. Thus, according to their binding data, most T1rs should have chloride bound at baseline. Perhaps this means that chloride binding is required to allow sucrose or other ligands to cause sufficient conformational changes and receptor activation? If this is the mechanism, it would still be quite interesting, but would change the framing/interpretation as presented in the manuscript. If additional experiments are not feasible, the authors should carefully discuss this point.
Some of the conclusions would be strengthened by additional control experiments, especially for the data obtained using FSEC-TS (Fig. 2C) and single fibre recordings (Fig. 3). For instance, how specific is the T105A mutation in abolishing Cl--dependent conformational changes? Did the authors check how the T105A mutation affects the ability of the LBD to undergo conformational changes in response to (1) L-Gln only and (2) Cl- only? Have the authors tried running these experiments at lower Cl- concentrations? 304 mM Cl- (page 16, line 363) is much higher compared to the effective concentration range claimed by the authors. For the single fibre recordings, have the authors tried applying 10 mM NMDG-gluconate? Having this negative control will provide more confidence in the specificity of Cl--induced impulses. Also, we would recommend a demonstration of reversibility in the gurmarin effect shown in Fig 3A.
Additional suggestions for the authors to consider:
The introduction would benefit from greater focus and clarity to make the work more accessible to readers. Despite the overall focus on T1rs, only a quarter of the introduction revolves around these receptors. Additional information would help the reader to understand the research topic. For example, how many isoforms are there? Are these receptors obligate heterodimers? How similar are the mf T1r2a/3 compared to the human T1r2/3 receptors? If mf T1r2a/3 receptors are activated by amino acids, how useful a proxy are they in understanding sweet-sensing human T1r2/3 receptors? If T1r3 is found in both heterodimers, and amino acids bind to T1r3, how do these receptors discern between sweet and umami taste? What are the mechanisms underlying activation of these receptors? How are these receptors usually studied functionally?
Given the focus on isolated LBDs of (non-human) mfT1r2a/3 receptors, the authors are encouraged to comment on the probability of Cl- binding, and the subsequent conformational rearrangement observed in the isolated LBDs, actually translating to activation of (full-length) human receptors (and ultimately taste stimulation). Since the authors have previously assessed the function of hsT1r2/3 in HEK293 cells using Ca2+ imaging (PMID: 25029362), evaluation of the activation properties of Cl- at full-length receptors and testing the effects of T1r3 mutations on these Cl- effects would help to strengthen the manuscript. Also, there are several reported polymorphisms in the gnomAD database around the Cl- ion binding site (Thr102Met, Gly143Arg, Pro144Ser/Leu), so it would be interesting and helpful to test the effects of these variants that are found in the population. We do not expect the authors to perform these experiments, but in the absence of more conclusive functional data on full-length receptors, the authors should consider discussing these potential caveats in the text.
Given the availability of AlphaFold Multimer and the well-defined stoichiometry of the complex, did the authors attempt to predict a model of the full-length heterodimer? This may be informative with regards to the mechanism of signal transduction to the transmembrane domain.
The nerve recording data would be more convincing if the authors could provide electrical recordings to truly sweet compounds at physiologically relevant concentrations (sucrose and artificial sweeteners). Currently, they only show data for 20 mM L-glutamine, which is not particularly sweet in Fig 3a-b, and then summary data for sucrose in Fig 3b.
The authors may wish to include a comment about whether bromide has the same effect on taste perception as chloride, and point out that gurmarin is a non-selective antagonist. Ideally, the nerve recordings should be done in T1r knockout mice to formally prove the mechanism. Although this may be beyond the scope of this work, a brief mention of this caveat seems warranted.
Finally, the discussion would benefit from additional mention of ligand binding in relevant heterodimeric class C GPCRs, as well as the observation that chloride appears to work via a distinct mechanism despite its binding site being spatially very close to that of Gln.
REVIEWING TEAM
Reviewed by:
Alexander T. Chesler, Principal Scientist, NCCIH, NIH, USA: Ion channel function, regulation and physiology
Han Chow Chua, Assistant Professor, University of Copenhagen, Denmark: Ion channel structure and function
Oliver B. Clarke, Assistant Professor, Columbia University, USA: Protein structural biology
Curated by:
Stephan A. Pless, Professor, University of Copenhagen, Denmark
(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.)