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Bradykinin and nerve growth factor release the capsaicin receptor from PtdIns(4,5)P2-mediated inhibition

Nature volume 411, pages 957–962 (2001)Cite this article

Abstract

Tissue injury generates endogenous factors that heighten our sense of pain by increasing the response of sensory nerve endings to noxious stimuli1,2. Bradykinin and nerve growth factor (NGF) are two such pro-algesic agents that activate G-protein-coupled (BK2) and tyrosine kinase (TrkA) receptors, respectively, to stimulate phospholipase C (PLC) signalling pathways in primary afferent neurons3,4. How these actions produce sensitization to physical or chemical stimuli has not been elucidated at the molecular level. Here, we show that bradykinin- or NGF-mediated potentiation of thermal sensitivity in vivo requires expression of VR1, a heat-activated ion channel on sensory neurons. Diminution of plasma membrane phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) levels through antibody sequestration or PLC-mediated hydrolysis mimics the potentiating effects of bradykinin or NGF at the cellular level. Moreover, recruitment of PLC-γ to TrkA is essential for NGF-mediated potentiation of channel activity, and biochemical studies suggest that VR1 associates with this complex. These studies delineate a biochemical mechanism through which bradykinin and NGF produce hypersensitivity and might explain how the activation of PLC signalling systems regulates other members of the TRP channel family.

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Figure 1: VR1 is essential for the development of bradykinin- or NGF-evoked thermal hypersensitivity in vivo.
Figure 2: Bradykinin (BK) and nerve growth factor (NGF) sensitize VR1 in heterologous expression systems.
Figure 3: Phospholipase C is involved in nerve growth factor (NGF) modulation of VR1 function.
Figure 4: Phosphatidyl-4,5-inositol bisphosphate (PtdIns(4,5)P2) antibody application mimics modulation of VR1 by bradykinin (BK) or nerve growth factor (NGF).
Figure 5: VR1, TrkA and phospholipase Cγ (PLC-γ) form a signalling complex.

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Acknowledgements

We are grateful to W. Neuhausser for providing dissociated DRG neurons, W. Mobley for 2.5 S NGF, D. Shelton for advice regarding NGF administration and J. Poblete and K. Simpson for technical assistance. We thank members of our laboratories for many helpful suggestions and constructive criticism. This work was supported by a NSF predoctoral fellowship (E.P.), NIH predoctoral neuroscience training grant (S.S.), a German Academy of Natural Scientists Leopoldina postdoctoral fellowship (S.-E.J.) and by the NIH (M.V.C., A.I.B. and D.J.).

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Author notes

  1. Huai-hu Chuang, Elizabeth D. Prescott, Shannon Shields and Allan I. Basbaum: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Cellular and Molecular Pharmacology, University of California, San Francisco, 94143-0450, California, USA

    Huai-hu Chuang, Elizabeth D. Prescott, Sven-Eric Jordt & David Julius

  2. Molecular Neurobiology Program, Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, 10016, New York, USA

    Haeyoung Kong & Moses V. Chao

  3. Departments of Anatomy and Physiology and the W. M. Keck Center for Neuroscience, University of California, San Francisco, 94143-0444, California, USA

    Shannon Shields & Allan I. Basbaum

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Corresponding author

Correspondence to David Julius.

Supplementary information

Supplementary Figure 1

(A) In transfected HEK cells, BK (20 nM) elicits a strongly rectifying VR1 basal current that is inhibited by 5 mM capsazepine, as shown by the holding current trace (left) and the ramp traces (right). (B) PIP2 Ab-induced VR1 basal current (-60 mV) is suppressed by 3 mM capsazepine; representative ramp traces from specified time points (* and **) are shown at right. (PDF 26 kb)

Supplementary Figure 2

[3H]-RTX binding to membranes from VR1-transfected HEK293 cells is displaced by TPA. Data are plotted as percentage of specific [3H]-RTX binding. IC50 for TPA = 256 ± 8 nM (average of four independent experiments; each point in duplicate). Note that phorbol-12,13-dibutyrate (PDBu) did not affect [3H]-RTX binding. Data were fit to the Hill equation. (DOC 33 kb)

Supplementary Figure 3

VR1 associates with wild type TrkA and TrkA mutants. HEK cells were transfected with equal amounts of VR1 and TrkA or TrkA mutant cDNAs. Immune complexes (above) were formed and analyzed as described in text. Western blots (below) of 40 mg total soluble HEK protein indicate equal expression levels of VR1 and Trks among samples. (PDF 21 kb)

Supplementary Figure 4

Effects of EGFR activation on VR1 currents. Two-electrode voltage clamp analysis was performed as described in text. Representative traces are shown above, and quantitation of 5 VR1 injected and 8 VR1/EGFR injected oocytes is shown below as SEM, p < 0.005. Note calcium activated chloride current (denoted with star) indicative of EGFR stimulation and consequent PLC activation. (PDF 26 kb)

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Chuang, Hh., Prescott, E., Kong, H. et al. Bradykinin and nerve growth factor release the capsaicin receptor from PtdIns(4,5)P2-mediated inhibition. Nature 411, 957–962 (2001). https://doi.org/10.1038/35082088

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