Article activity feed

  1. Evaluation Summary:

    The TRPV2 channel plays essential roles in many cell types in the body, including macrophages and cardiomyocytes, but its physiological mechanisms of activation and regulation remain largely unknown. Mo and collaborators describe a novel regulatory mechanism of TRPV2 channels in which phosphorylation at three different tyrosine residues by JAK1 sensitizes channels to activation by 2-APB and heat, whereas de-phosphorylation by PTPN1 reverses sensitization. Changes in the dynamics of TRPV2 channel phosphorylation could have important physiological consequences in many cell types expressing these channels. The data are of significant importance for the scientific community interested in function und relevance of transient receptor potential ion channels.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)

  2. Reviewer #1 (Public Review):

    The TRPV2 channels are expressed in multiple cell types and have been shown to be essential for the development and function of the heart and activation of phagocytosis by macrophages. TRPV2 channels are directly activated by heating, but this requires extreme temperatures outside the physiologically relevant range (> 50{degree sign}C). Although some mechanisms such as oxidative modification of methionine residues have been found to enable channel activation by heat at physiological temperatures, the endogenous mechanisms of TRPV2 channel activation remain largely unknown. Here Xiaoyi Mo and collaborators describe a novel mechanism of TRPV2 channel regulation by phosphorylation/dephosphorylation that could tune TRPV2 channel sensitivity to heat and other stimuli allowing its activation in rat bone marrow macrophages and potentially other cell types under physiological conditions. Using patch-clamp electrophysiology, the authors find that extracellular magnesium co-applied with the chemical agonist 2-APB slowly sensitizes responses to sub-saturating concentrations of 2-APB and heat in rat bone marrow-derived macrophages and DRG neurons that endogenously express TRPV2, as well as in heterologous expression systems. The authors find no effect when other divalent cations are applied from the extracellular side or when large concentrations of a divalent ion chelator are included in the intracellular solution, suggesting that the inward flow of magnesium in a whole cell is required to observe sensitization of the channels. The authors also find no sensitization when cytosolic ATP is substituted by a non-hydrolyzable analog, suggesting that a kinase might be responsible for the observed effect of magnesium on channel sensitization. By testing multiple pharmacological kinase inhibitors, the authors find that only a JAK1 kinase inhibitor ablates the sensitizing effect of magnesium. Using biochemical assays and mass-spectrometry, the authors provide further evidence that the presence of magnesium enhances TRPV2 channel phosphorylation, and show that siRNA-dependent knockdown of JAK1 kinase reduces magnesium-dependent sensitization in patch-clamp experiments and biochemical assays. Using site-directed mutagenesis, the authors identify three tyrosine residues responsible for the observed sensitizing effect. Finally, using pharmacological phosphatase inhibitors, siRNA-dependent knockdown of different phosphatases, and biochemical assays of phosphorylation, the authors find that protein tyrosine phosphatase non-receptor type 1 (PTPN1) is capable of de-phosphorylating TRPV2 channels to reverse sensitization caused by JAK1. The data presented are of high quality, and most conclusions are supported by the evidence provided. The findings are relevant, as they identify a novel regulatory mechanism that could enable TRPV2 channel activity under physiological conditions.

    One concern in the data is that application of 2-APB alone is much shorter than when co-applied with extracellular magnesium in all experiments in the manuscript (e.g. Figs. 1A, C, E, Fig. 1 Suppl. 1 A-C). Because the sensitization caused by 2-APB and magnesium takes > 100 s to develop, it is unclear from the data whether a longer stimulation with 2-APB alone would have some sensitizing effect. In general, the authors do not appear to account for the length of stimulation with 2-APB and magnesium in experiments where this is an important factor, such as the dose-response relations for magnesium or 2-APB (Fig. 1G and H), the comparison between different divalent cations (Fig. 1 Suppl. 2 A-H), the effects of kinase or phosphatase inhibitors (Figs. 2E and 5B), the effect of magnesium applied alone at different concentrations (Fig. 2A) or the effects of the various tyrosine mutants (Fig. 4A, G, H and I).

    Normalization of the current vs temperature relations and the quantitation of the threshold is not appropriate, because the apparent threshold depends on how data is normalized and different temperatures are used to normalize the data sets being compared (Figs. 1K, 5K, 6G, and Fig. 5 Suppl. 1G).

    The data provided to support that channels containing substitutions E609Q and E614Q have reduced permeability to divalent cations and magnesium specifically is not adequate. First, the calcium imaging data is largely qualitative and provides no information about magnesium permeability. It is also unclear why the authors utilized CBD instead of 2-APB. Direct measurement of the reversal potential with or without extracellular magnesium would be required.

    Regarding the in-vitro kinase assays (Fig. 3F), it is unclear why a smear is apparent on the third lane, and also limited information is provided regarding the mass-spectrometry results, making it hard for the general reader to assess these results.

    An important concern pertains to the phagocytosis assay. Because the authors are utilizing primary cells for this, it is unclear whether the effects of JAK1 inhibition on phagocytosis are specific to TRPV2 channel activity. These cells do express other types of ion channels, and the observed changes are not very robust. Importantly, the authors did not stimulate cells with magnesium and 2-APB, so it is unclear what other signal would be responsible for triggering TRPV2 channel activity and phagocytosis.

    Finally, the authors provide very limited discussion about how TRPV2 channel phosphorylation/de-phosphorylation could be modulated physiologically. To observe robust sensitization, the authors need to raise the extracellular magnesium concentration > 10 mM. It is unclear whether the resulting increases in cytosolic magnesium under these conditions could occur physiologically, or whether there are other signaling pathways regulating the function of JAK1 of PTPN1.

  3. Reviewer #2 (Public Review):

    How TRPV2 is exactly integrated into signaling pathways of an organism is a relatively open question. There is no compelling evidence for a specific endogenous TRPV2 ligand, and its thermosensitive threshold at 52 degrees Celsius is nearly outside the practical range, where it could help an organism avoid noxious temperatures. These findings have prompted speculation that cell signaling pathways play an important part in the physiological role of the channel. The research article, "Tyrosine phosphorylation tunes chemical and thermal sensitivity of TRPV2 ion channel", by Mo et al. makes a significant contribution to our understanding of how TRPV2 function is integrated with cell signaling and its physiological role. By leveraging a newly discovered TRPV2 response to rapid recovery of cellular Mg2+ levels from low initial concentrations, the authors conduct a compelling study of how phosphorylation of TRPV2 at three amino acids determine the functional response of the channel in the physiology of bone-marrow-derived macrophages (BMDM). The research follows a logical progression of experiments to identify the key amino acids that are targeted for phosphorylation in this pathway and is a fine example of a clearly conceptualized and actualized study. In terms of the interpretation of the results, the importance of the phosphorylation events in TRPV2 in relation to physiological function is well supported by the evidence. However, some caution should be given to interpreting the Mg2+ dependent mechanism of TRPV2 modulation as having physiological relevance. The Mg2+ experiments with TRPV2 proceeded from an initial state with very low intracellular Mg2+ concentrations to relatively high levels (0.1-100 mM), and no experiments were conducted that explored Mg2+ concentration changes from nominal initial levels (1-5 mM).

  4. Reviewer #3 (Public Review):

    Mo et al., present novel and, in most parts conclusive data showing that the function of TRPV2 is tightly regulated by phosphorylation of specific tyrosine residues.

    In the hands of the authors, in vitro electrophysiological experiments revealed a remarkable sensitizing effect of high concentrations of extracellular Mg on ion-currents activated by 2-APB or high heat on rat TRPV2. This effect is reproducible in different cell types and did not seem to differ between recombinant channels and TRPV2 endogenously expressed in macrophages and neurons. While it remains a little unclear why, or with what hypothesis the authors conducted these initial experiments, they present a fair amount of evidence indicating that this Mg-induced effect is due to an indirect intracellular effect rather than a direct interaction with Mg on specific extracellular or intracellular sites of TRPV2. While Mg applied alone completely failed to activate TRPV2 examined in the whole-cell mode as well as in inside-out cell-free patches, it potentiated currents evoked by 2-APB or heat. In consecutive experiments, the authors present some evidence that Mg permeates through the pore of TRPV2 to increase phosphorylation of TRPV2. Using selective inhibitors of different kinases, mass spectrometry and shRNA-mediated knockdown, the authors present very convincing evidence that JAK1 is the main kinase responsible for this Mg-induced effect. Of note, inhibition of JAK1 has also reduced phagocytosis of BMDM cells. Given that TRPV2 is known to be important for phagocytosis, this finding indeed indicates that the degree of phosphorylation of TRPV2 is crucial for the function of macrophages - e.g. giving this in vitro study an exciting physiological relevance. Using site-directed mutagenesis, the tyrosine residues Y335, Y471 and Y525 were found to the key sites for JAK1-induced phosphorylation. Finally, the authors very elegantly demonstrate that the degree of TRPV2-phosphorylation also seems to be finely tuned by the phosphatase PTPN1.

    In most parts of this well-written study, the conclusions drawn by the authors are supported by conclusive data generated by state-of-art in vitro techniques. The findings are truly novel, and as very little is still known about the precise functional properties of TRPV2, the data presented here may have a substantial impact on future research on these ion channels. Saying this, some aspects might be worth clarifying or at least discussing.

    1. The underlying hypothesis leading the authors to challenge TRPV2 with high concentrations of extracellular Mg should be (better) stated. There are many ways to explore the role of protein phosphorylation in the activity of ion channels, the authors applied many elegant techniques in this study. The starting point with extracellular Mg is somewhat odd, giving the slight impression that the authors did not have protein phosphorylation in mind when they started this study.

    2. A key event of basically all functional data presented in this study is the likely permeation of Mg through the pore of TRPV2. However, ion permeability of TRPV2 is not very well explored in previous reports. Thus, permeation of Mg through TRPV2 should be convincingly demonstrated.

    3. It is easy to accept that probably many signalling pathways in different cell types expressing TRPV2 may regulate phosphorylation of TRPV2 via JAK1/PTPN1. Excessive concentrations of is artificial, well suitable for this mechanistic in vitro study. However, it may be possible to at least speculate which are the endogenously relevant pathways involving JAK1 and PTPN1.