Engineering vanilloid-sensitivity into the rat TRPV2 channel

  1. Feng Zhang
  2. Sonya M Hanson
  3. Andres Jara-Oseguera
  4. Dmitriy Krepkiy
  5. Chanhyung Bae
  6. Larry V Pearce
  7. Peter M Blumberg
  8. Simon Newstead
  9. Kenton J Swartz  Is a corresponding author
  1. National Institutes of Health, United States
  2. Memorial Sloan Kettering Cancer Center, United States
  3. National Cancer Institute, United States
  4. University of Oxford, United Kingdom

Abstract

The TRPV1 channel is a detector of noxious stimuli, including heat, acidosis, vanilloid compounds and lipids. The gating mechanisms of the related TRPV2 channel are poorly understood because selective high affinity ligands are not available, and the threshold for heat activation is extremely high (> 50 {degree sign}C). Cryo-EM structures of TRPV1 and TRPV2 reveal that they adopt similar structures, and identify a putative vanilloid binding pocket near the internal side of TRPV1. Here we use biochemical and electrophysiological approaches to investigate the resiniferatoxin (RTx) binding site in TRPV1 and to explore the functional relationships between TRPV1 and TRPV2. Collectively, our results support the interaction of vanilloids with the proposed RTx binding pocket, and demonstrate an allosteric influence of a tarantula toxin on vanilloid binding. Moreover, we show that sensitivity to RTx can be engineered into TRPV2, demonstrating that the gating and permeation properties of this channel are similar to TRPV1.

Article and author information

Author details

  1. Feng Zhang

    Molecular Physiology and Biophysics Section, Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, United States
    Competing interests
    No competing interests declared.
  2. Sonya M Hanson

    Computational Biology Program, Memorial Sloan Kettering Cancer Center, Bethesda, United States
    Competing interests
    No competing interests declared.
  3. Andres Jara-Oseguera

    Molecular Physiology and Biophysics Section, Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, United States
    Competing interests
    No competing interests declared.
  4. Dmitriy Krepkiy

    Molecular Physiology and Biophysics Section, Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, United States
    Competing interests
    No competing interests declared.
  5. Chanhyung Bae

    Molecular Physiology and Biophysics Section, Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, United States
    Competing interests
    No competing interests declared.
  6. Larry V Pearce

    Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, Bethesda, United States
    Competing interests
    No competing interests declared.
  7. Peter M Blumberg

    Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, Bethesda, United States
    Competing interests
    No competing interests declared.
  8. Simon Newstead

    Department of Biochemistry, University of Oxford, Oxford, United Kingdom
    Competing interests
    No competing interests declared.
  9. Kenton J Swartz

    Molecular Physiology and Biophysics Section, Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, United States
    For correspondence
    swartzk@ninds.nih.gov
    Competing interests
    Kenton J Swartz, Reviewing editor, eLife.

Ethics

Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocol (#1253-15) of the National Institute of Neurological Disorders and Stroke.

Copyright

This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

Metrics

  • 2,547
    views
  • 678
    downloads
  • 53
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Feng Zhang
  2. Sonya M Hanson
  3. Andres Jara-Oseguera
  4. Dmitriy Krepkiy
  5. Chanhyung Bae
  6. Larry V Pearce
  7. Peter M Blumberg
  8. Simon Newstead
  9. Kenton J Swartz
(2016)
Engineering vanilloid-sensitivity into the rat TRPV2 channel
eLife 5:e16409.
https://doi.org/10.7554/eLife.16409

Share this article

https://doi.org/10.7554/eLife.16409

Further reading

    1. Structural Biology and Molecular Biophysics
    Jinsai Shang, Douglas J Kojetin
    Research Advance

    Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor transcription factor that regulates gene expression programs in response to ligand binding. Endogenous and synthetic ligands, including covalent antagonist inhibitors GW9662 and T0070907, are thought to compete for the orthosteric pocket in the ligand-binding domain (LBD). However, we previously showed that synthetic PPARγ ligands can cooperatively cobind with and reposition a bound endogenous orthosteric ligand to an alternate site, synergistically regulating PPARγ structure and function (Shang et al., 2018). Here, we reveal the structural mechanism of cobinding between a synthetic covalent antagonist inhibitor with other synthetic ligands. Biochemical and NMR data show that covalent inhibitors weaken—but do not prevent—the binding of other ligands via an allosteric mechanism, rather than direct ligand clashing, by shifting the LBD ensemble toward a transcriptionally repressive conformation, which structurally clashes with orthosteric ligand binding. Crystal structures reveal different cobinding mechanisms including alternate site binding to unexpectedly adopting an orthosteric binding mode by altering the covalent inhibitor binding pose. Our findings highlight the significant flexibility of the PPARγ orthosteric pocket, its ability to accommodate multiple ligands, and demonstrate that GW9662 and T0070907 should not be used as chemical tools to inhibit ligand binding to PPARγ.

    1. Structural Biology and Molecular Biophysics
    Yuanyuan Wang, Fan Xu ... Yongning He
    Research Article

    SCARF1 (scavenger receptor class F member 1, SREC-1 or SR-F1) is a type I transmembrane protein that recognizes multiple endogenous and exogenous ligands such as modified low-density lipoproteins (LDLs) and is important for maintaining homeostasis and immunity. But the structural information and the mechanisms of ligand recognition of SCARF1 are largely unavailable. Here, we solve the crystal structures of the N-terminal fragments of human SCARF1, which show that SCARF1 forms homodimers and its epidermal growth factor (EGF)-like domains adopt a long-curved conformation. Then, we examine the interactions of SCARF1 with lipoproteins and are able to identify a region on SCARF1 for recognizing modified LDLs. The mutagenesis data show that the positively charged residues in the region are crucial for the interaction of SCARF1 with modified LDLs, which is confirmed by making chimeric molecules of SCARF1 and SCARF2. In addition, teichoic acids, a cell wall polymer expressed on the surface of gram-positive bacteria, are able to inhibit the interactions of modified LDLs with SCARF1, suggesting the ligand binding sites of SCARF1 might be shared for some of its scavenging targets. Overall, these results provide mechanistic insights into SCARF1 and its interactions with the ligands, which are important for understanding its physiological roles in homeostasis and the related diseases.