Chloride ions evoke taste sensations by binding to the extracellular ligand-binding domain of sweet/umami taste receptors

  1. Nanako Atsumi
  2. Keiko Yasumatsu
  3. Yuriko Takashina
  4. Chiaki Ito
  5. Norihisa Yasui
  6. Robert F Margolskee
  7. Atsuko Yamashita

  • Curated by eLife

    eLife logo

    eLife assessment

    This fundamental study presents solid evidence for T1r (sweet /umami) taste receptors as chloride (Cl-) receptors, based on a combination of state-of-the-art techniques to demonstrate that T1r receptors from Medaka fish bind chloride and that this binding induces a conformational change in the heteromeric receptor. This conformational change leads to low-concentration chloride-specific action potential firing in nerves from neurons containing these receptors in mice, results that represent an important advance in our understanding of the logic of taste perception.

  • Curated by Biophysics Colab

    Biophysics Colab logo

    Endorsement statement (17 November 2022)

    The preprint by Atsumi et al. describes how chloride binding to sweet- and umami-sensing proteins (T1R taste receptors) can evoke taste sensation. The authors use an elegant combination of structural, biophysical and electrophysiological approaches to locate a chloride binding site in the ligand-binding domain of medaka fish T1r2a/3 receptors. They convincingly show that low mM concentrations of chloride induce conformational changes and, using single fiber recordings, establish that mouse chorda tympani nerves are activated by chloride in a T1R-dependent manner. This suggests that chloride binding to sweet receptors could mediate the commonly reported sweet taste sensation following ingestion of low concentrations of table salt. The findings will be of broad relevance to those studying taste sensation and ligand recognition in GPCRs.

    (This endorsement by Biophysics Colab refers to version 2 of this preprint, which has been revised in response to peer review of version 1.)

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Abstract

Salt taste sensation is multifaceted: NaCl at low or high concentrations is preferably or aversively perceived through distinct pathways. Cl − is thought to participate in taste sensation through an unknown mechanism. Here, we describe Cl − ion binding and the response of taste receptor type 1 (T1r), a receptor family composing sweet/umami receptors. The T1r2a/T1r3 heterodimer from the medaka fish, currently the sole T1r amenable to structural analyses, exhibited a specific Cl − binding in the vicinity of the amino-acid-binding site in the ligand-binding domain (LBD) of T1r3, which is likely conserved across species, including human T1r3. The Cl − binding induced a conformational change in T1r2a/T1r3LBD at sub- to low-mM concentrations, similar to canonical taste substances. Furthermore, oral Cl − application to mice increased impulse frequencies of taste nerves connected to T1r-expressing taste cells and promoted their behavioral preferences attenuated by a T1r-specific blocker or T1r3 knock-out. These results suggest that the Cl − evokes taste sensations by binding to T1r, thereby serving as another preferred salt taste pathway at a low concentration.

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  1. Version published to 10.7554/elife.84291 on eLife
  2. eLife

    eLife assessment

    This fundamental study presents solid evidence for T1r (sweet /umami) taste receptors as chloride (Cl-) receptors, based on a combination of state-of-the-art techniques to demonstrate that T1r receptors from Medaka fish bind chloride and that this binding induces a conformational change in the heteromeric receptor. This conformational change leads to low-concentration chloride-specific action potential firing in nerves from neurons containing these receptors in mice, results that represent an important advance in our understanding of the logic of taste perception.

  3. eLife

    Reviewer #1 (Public Review):

    Taste perception is a complicated phenomenon. There are many well-established signaling pathways for the transduction of major taste modalities, however, there are also several taste phenomena that are not well understood. Among these is the mechanism for the perception of low concentrations of sodium chloride as sweet. In the present manuscript by Atsumi et al., the authors present solid evidence that identifies the T1r (sweet /umami) taste receptors as chloride (Cl-) receptors.

    The authors make use of an array of modern experimental approaches to demonstrate that T1r receptors from Medaka fish, which are formed by a heterodimer of T1r2a/T1r3 proteins, are able to bind chloride and that this binding induces a conformational change in the heteromeric receptor. The authors demonstrate binding both by solving the x-ray structure of the T1r2a/T1r3 ligand binding domain with Cl- and demonstrating a chloride-induced conformational change by measuring FRET. This conformational change leads to low concentration, chloride-specific action potential firing in nerves from neurons that contain these receptors in mice.

    The authors suggest that their results solve the standing question of how and why is the low concentration of NaCl perceived as sweet. In general, the results presented here support the conclusions and represent an important advance in our understanding of the logic of taste perception.

  4. Version published to 10.1101/2022.02.23.481615v3 on bioRxiv
  5. Biophysics Colab

    Endorsement statement (17 November 2022)

    The preprint by Atsumi et al. describes how chloride binding to sweet- and umami-sensing proteins (T1R taste receptors) can evoke taste sensation. The authors use an elegant combination of structural, biophysical and electrophysiological approaches to locate a chloride binding site in the ligand-binding domain of medaka fish T1r2a/3 receptors. They convincingly show that low mM concentrations of chloride induce conformational changes and, using single fiber recordings, establish that mouse chorda tympani nerves are activated by chloride in a T1R-dependent manner. This suggests that chloride binding to sweet receptors could mediate the commonly reported sweet taste sensation following ingestion of low concentrations of table salt. The findings will be of broad relevance to those studying taste sensation and ligand recognition in GPCRs.

    (This endorsement by Biophysics Colab refers to version 2 of this preprint, which has been revised in response to peer review of version 1.)

  6. Biophysics Colab

    Authors' response (18 October 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.

    The authors appreciate the positive assessment of the study, as well as the valuable comments and suggestions from the reviewers described below. Considering the referees' remarks, we performed additional control experiments and obtained evidence strengthening the T1r-mediated chloride sensing, as described below.

    RECOMMENDATIONS

    Revisions essential for endorsement:

    1. 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 used the X-ray diffraction data from the Br–-substituted crystal, as well as the long-wavelength data from the Cl–-bound crystal, to draw the anomalous difference-Fourier maps pinpointing the Br–/Cl– positions. The structure models used for phase calculation were obtained by molecular replacement as described in the "Crystallography" section in the Materials and Methods. To show the certainty of the molecular replacement solutions, we added the R-factors for the models in the last sentence of the section. Since the resolutions for these anomalous data were limited and the structural comparison between the Br–-bound and the Cl–-bound forms is not the main subject of the study, no further extensive structural refinement was performed on these data.

    1. 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.

    The authors thank the reviewers' insightful comments. We addressed this point and the results were shown in Figure 4D in the revised manuscript (Figure 3C in the original manuscript) and the third paragraph in the "Taste response to Cl– through T1rs in mouse" section in Results (p. 16, the next paragraph to the Figure 4 in the manuscript). As shown in Figure 4D, solely l-Gln or sucrose application in the absence of chloride (shown as l-Gln or sucrose) induced nerve responses. When those were applied in the presence of 10 mM chloride (shown as l-Gln+NMDG-Cl or sucrose+NMDG-Cl), the responses were increased to the similar levels as the summation of the response of the independent application of each substance. These results suggested that the chloride binding is not required for the receptor activation by sugars or amino acids, and that the binding of the two can occur simultaneously but does not cause synergistic responses.

    1. 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.

    The authors thank the reviewers' important suggestions.

    We performed a FRET assay for T1r2a/T1r3(T105A) mutant, and the results have been added to Figure 3E. In this experiment, we used 10 mM chloride, not ~300 mM, for both the T105A mutant and the wild-type LBD proteins and compared the results of the two. We confirmed that the extent of Cl–-dependent conformational change for the mutant was significantly reduced, as judged by the FRET index change. However, we also performed the same experiment using solely l-Gln as a titrant as the reviewers' suggested, and found that the amino acid-dependent change of the mutant was also significantly reduced. Therefore, although the former result itself agrees with our hypothesis, we are aware that the possibility of the entire protein deactivation during preparation cannot be excluded. Therefore, we presented the result with a notion about the study's limitations, as shown in the third paragraph in the section "Cl–-binding properties in T1r2a/T1r3LBD" in the Results (p. 12, the next paragraph to Table 1 in the manuscript).

    Regarding the single fiber recordings, we performed the NMDG-gluconate application and confirmed that it did not induce significant responses at least up to 10 mM, as shown in Figure 4B. In addition, we described the method and results of our reversibility confirmation test for gurmarin inhibition in the section "Single fiber recording from mouse chorda tympani (CT) nerve" in Methods (the last paragraph of the section, p.25).

    Additional suggestions for the authors to consider:

    1. 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?

    We agree with the significance of the information pointed out by the reviewers, and several points are currently under investigation in the field. However, we decided to keep the current contents in the Introduction due to the length limitation imposed by the submitted journal.

    1. 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.

    The authors thank the reviewers' suggestions. We attempted the Ca2+-imaging in the early stage of the study, but it failed due to the instability of the cellular responses under the Cl–-depleted conditions. In contrast, nerve recordings are durable under a wide range of conditions. We described the situation in the first paragraph in the section "Taste response to Cl– through T1rs in mouse" in the Results. To verify that the nerve responses were attributed to T1rs, we confirmed that the chloride-dependent responses were attenuated by gurmarin, a T1r-specific blocker, and in T1r3-knockout mice, which were added in the revised manuscript.

    Furthermore, we additionally performed a mouse behavioral assay and confirmed the preference for the solution containing chloride relative to H2O, which was again abolished by gurmarin. The results supported our discussion that the chloride is detected through a taste signal transduction pathway mediated by T1rs, as described in the last paragraph of the same section, and shown in Figure 4E, F.

    The authors thank the reviewers' interesting and thoughtful pointing about the polymorphism, which is worth to be addressed in future studies.

    1. 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 authors appreciate the reviewers' helpful suggestion. We have constructed the full-length heterodimer models of T1rs from several species. We hope we will utilize the knowledge derived from them in our future studies.

    1. 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 thank this comment. We added representative recordings of the sucrose data in Figure 4A.

    1. 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.

    As described above, we added the nerve recording data using T1r3-KO mice and proved that the chloride-derived responses were attributed to T1rs.

    We agree with the reviewers' pointing that a halide-specificity to T1rs is an interesting issue to be addressed in future studies.

    1. 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.

    The discussion regarding the chloride-dependent regulation of ligand-binding in other class C GPCRs as well as structurally related receptors (ANPRs) was described in the last paragraph of the Discussion. The relationship between the amino acid-binding and the chloride-binding was addressed in the third paragraph in the "Taste response to Cl– through T1rs in mouse" section in Results (p. 16, the next paragraph to the Figure 4 in the manuscript).

    (This is a response to peer review conducted by Biophysics Colab on version 1 of this preprint.)

  7. Version published to 10.1101/2022.02.23.481615v2 on bioRxiv
  8. Biophysics Colab

    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:

    1. 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).

    2. 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.

    3. 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:

    1. 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?

    2. 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.

    3. 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.

    4. 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.

    5. 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.

    6. 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.)

  9. Version published to 10.1101/2022.02.23.481615v1 on bioRxiv