Tyrosine phosphorylation tunes chemical and thermal sensitivity of TRPV2 ion channel
- State Key Laboratory of Virology, College of Life Sciences, Department of Anesthesiology, Zhongnan Hospital of Wuhan University, Frontier Science Center for Immunology and Metabolism, Wuhan University, China
- Institute of Biology Paris Seine, Neuroscience Paris Seine, CNRS UMR8246, INSERM U1130, Sorbonne Universite, France
- The Key Laboratory of Neural and Vascular Biology, Ministry of Education Hebei Medical University, China
Figures
Figure 1 with 5 supplements
Transient receptor potential vanilloid 2 (TRPV2) activities are enhanced in the presence of Mg2+.
(A) Mg2+ potentiates 2-aminoethyl diphenylborinate (2-APB) responses in a representative rat bone marrow-derived macrophage (BMDM) cell. The cell was exposed to 0.3 mM 2-APB without or with 5 mM Mg2+…
Figure 1—figure supplement 1
Expression of transient receptor potential vanilloid 2 (TRPV2) in bone marrow-derived macrophages (BMDMs).
(A) Relative mRNA expression levels of different Trpv and Trpc channels in BMDMs were assessed by qPCR. (B) Expression of TRPV2 protein in BMDMs. (C) Immunoblot analysis (with anti-TRPV2 or …
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Figure 1—figure supplement 1—source data 1
Uncropped, unedited blots for Figure 1—figure supplement 1B.
- https://cdn.elifesciences.org/articles/78301/elife-78301-fig1-figsupp1-data1-v2.zip
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Figure 1—figure supplement 1—source data 2
Uncropped, unedited blots for Figure 1—figure supplement 1C.
- https://cdn.elifesciences.org/articles/78301/elife-78301-fig1-figsupp1-data2-v2.zip
Figure 1—figure supplement 2
Mg2+ potentiates transient receptor potential vanilloid 2 (TRPV2) currents expressed in various cell lines.
(A–C) Representative whole-cell recordings in CHO, Hela, or ND7/23 that expressed TRPV2 showing the response to 0.3 mM 2-aminoethyl diphenylborinate (2-APB), 0.3 mM 2-APB plus 5 mM Mg2+ and 3 mM …
Figure 1—figure supplement 3
Effect of Mg2+ on 2-aminoethyl diphenylborinate (2-APB)-evoked transient receptor potential vanilloid 2 (TRPV2) currents.
(A) Representative whole-cell recordings in bone marrow-derived macrophages (BMDMs) showing the responses to 0.3 mM 2-APB, 0.3 mM 2-APB plus 5 mM Mg2+ and 3 mM 2-APB. The dotted line indicates zero …
Figure 1—figure supplement 4
Effects of various divalent cations on 2-aminoethyl diphenylborinate (2-APB)-evoked transient receptor potential vanilloid 2 (TRPV2) currents.
(A–H) Representative whole-cell currents in TRPV2-expressing HEK293T cells induced by 0.3 mM 2-APB, the combination of 0.3 mM 2-APB and various divalent cations Mn2+ (A), Ca2+ (B), Ba2+ (C), Zn2+ (D)…
Figure 1—figure supplement 5
Effect of intracellular Mg2+ on dose responses to 2-aminoethyl diphenylborinate (2-APB) for activation of transient receptor potential vanilloid 2 (TRPV2).
(A–B) Representative whole-cell currents at –60 mV evoked by increasing concentrations of 2-APB in TRPV2-expressing HEK293T cells. Note the pipette solution contained 0 mM Mg2+ (A) or 5 mM Mg2+ (B). …
Figure 2
Mg2+ has an indirect effect on transient receptor potential vanilloid 2 (TRPV2) channels.
(A) High concentrations of Mg2+ have no direct effect on TRPV2 channels from the extracellular side. Representative whole-cell currents at –60 mV in a TRPV2-expressing HEK293T cells consecutively …
Figure 3 with 2 supplements
Tyrosine phosphokinase JAK1 upregulates channel activity via phosphorylation of transient receptor potential vanilloid 2 (TRPV2).
(A) Representative whole-cell recordings from TRPV2-expressing HEK293T cells showing the responses to 3 mM 2-aminoethyl diphenylborinate (2-APB) before and after the treatment by 0.3 mM 2-APB plus 5 …
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Figure 3—source data 1
Uncropped, unedited blots for Figure 3B.
- https://cdn.elifesciences.org/articles/78301/elife-78301-fig3-data1-v2.zip
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Figure 3—source data 2
Uncropped, unedited blots and gels for Figure 3F.
- https://cdn.elifesciences.org/articles/78301/elife-78301-fig3-data2-v2.zip
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Figure 3—source data 3
Uncropped, unedited blots for Figure 3H.
- https://cdn.elifesciences.org/articles/78301/elife-78301-fig3-data3-v2.zip
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Figure 3—source data 4
Uncropped, unedited blots for Figure 3I.
- https://cdn.elifesciences.org/articles/78301/elife-78301-fig3-data4-v2.zip
Figure 3—figure supplement 1
Effects of Mg2+ on transient receptor potential vanilloid 2 (TRPV2) responses with or without addition of adenosine triphosphate (ATP) in the pipette solutions.
(A–B) Representative whole-cell currents in a TRPV2-expressing HEK293T cell elicited with 0.3 mM 2-aminoethyl diphenylborinate (2-APB), 0.3 mM 2-APB plus 5 mM Mg2+ and 3 mM 2-APB. Please note the …
Figure 3—figure supplement 2
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of the phosphorylation of transient receptor potential vanilloid 2 (TRPV2).
LC-MS/MS analysis showing the phosphorylation of TRPV2 in HEK293T cells after treatment by 0.3 mM 2-aminoethyl diphenylborinate (2-APB) plus 5 mM Mg2+, followed by immunoprecipitation (with …
Figure 4 with 2 supplements
JAK1 has three phosphorylation sites on the transient receptor potential vanilloid 2 (TRPV2) channel.
(A) Representative whole-cell currents at –60 mV elicited with 0.3 mM 2-APB, 0.3 mM 2-APB plus 5 mM Mg2+ and 3 mM 2-APB in HEK293T cells that expressed TRPV2(Y335F). Bars represent duration of …
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Figure 4—source data 1
Uncropped, unedited blots and gels for Figure 4E.
- https://cdn.elifesciences.org/articles/78301/elife-78301-fig4-data1-v2.zip
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Figure 4—source data 2
Uncropped, unedited blots for Figure 4K.
- https://cdn.elifesciences.org/articles/78301/elife-78301-fig4-data2-v2.zip
Figure 4—figure supplement 1
Partial amino acid sequence alignment of transient receptor potential vanilloid 2 (TRPV2) channels.
Multiple alignments of TRPV2 amino acid sequences surrounding Y335, Y471, and Y525 from rat, mouse, human, rabbit, macaque, cattle, dog, horse, and whale. The residues of Y335, Y471, and Y525 are …
Figure 4—figure supplement 2
Inside-out recordings showing the effects of Mg2+ on 2-aminoethyl diphenylborinate (2-APB)-evoked currents.
(A–C) Representative currents at +60 mV recorded in an inside-out patch excised from transient receptor potential vanilloid 2 (TRPV2)(wild-type)-expressing cells (A), the TRPV2(wild-type)-expressing …
Figure 5 with 1 supplement
Increasing the phosphorylation level of transient receptor potential vanilloid 2 (TRPV2) by inhibition of dephosphorylase activity enhances the channel sensitivity to its stimuli.
(A) Whole-cell recordings from TRPV2-expressing HEK293T cell were consecutively challenged with 0.3 mM 2-aminoethyl diphenylborinate (2-APB), 0.3 mM 2-APB plus 5 mM Mg2+ and 3 mM 2-APB. The cells …
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Figure 5—source data 1
Uncropped, unedited blots for Figure 5C.
- https://cdn.elifesciences.org/articles/78301/elife-78301-fig5-data1-v2.zip
Figure 5—figure supplement 1
Inhibition of protein tyrosine phosphatase (PTP) activity by inhibitors enhanced the transient receptor potential vanilloid 2 (TRPV2) sensitivity to 2-aminoethyl diphenylborinate (2-APB) and heat in TRPV2-expressing HEK293T cells.
(A–B) Representative whole-cell recordings from TRPV2-expressing HEK293T cells pretreated with PTP inhibitor 1 (A) and PTP inhibitor 2 (B). The cells were exposed to increasing concentrations of …
Figure 6 with 1 supplement
Protein tyrosine phosphatase non-receptor type 1 (PTPN1) is a phosphatase that mediates the dephosphorylation of transient receptor potential vanilloid 2 (TRPV2).
(A) Immunoblot analysis (with anti-PTPN1 or anti-β-action) of HEK293T cells transfected for 48 hr with PTPN1-targeting shRNA (shPTPN1#1 and shPTPN1#2) or shControl to test knockdown efficiency of …
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Figure 6—source data 1
Uncropped, unedited blots for Figure 6A.
- https://cdn.elifesciences.org/articles/78301/elife-78301-fig6-data1-v2.zip
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Figure 6—source data 2
Uncropped, unedited blots for Figure 6B.
- https://cdn.elifesciences.org/articles/78301/elife-78301-fig6-data2-v2.zip
Figure 6—figure supplement 1
Tyrosine phosphorylation sets the agonist and heat sensitivity of transient receptor potential vanilloid 2 (TRPV2).
This study demonstrates that JAK1 phosphokinase mediates Mg2+-dependent phosphorylation of TRPV2 at Y335, Y471, and Y525 residues. And, increasing tyrosine phosphorylation of TRPV2 lowers its …
Author response image 1
Determination of the temperature threshold for activation of TRPV2.
(A) Representative current traces for TPRV2-expressing HEK293T cells in response to a family of temperature pulses ranging from 42 oC to 57 oC. (B) Current vs. temperature relations at −60 mV …
Author response image 2
Mg2+-induced sensitization of TRPV2 is reversible.
(A) Representative whole-cell of TRPV2 currents evoked by 0.3 mM 2-APB, 0.3 mM 2-APB plus 5 mM Mg2+, subsequently repeated applications of 0.3 mM 2-APB and 3 mM 2-APB. (B) Time courses of peak …
Author response image 3
Expression of TRPV2 in DRG neurons.
Relative mRNA expression levels of different Trpv channels in BMDMs were assessed by qPCR.
Author response image 4
Effect of Mg2+-dependent phosphorylation on human TRPV2.
(A) Whole-cell currents at -60 mV in a hTRPV2-expressing HEK293T cell treated with 1.5 mM 2-APB, 1.5 mM 2-APB plus 5 mM Mg2+, and 3 mM 2-APB. (B) Relative cationic permeabilities (PX/PNa) of human …
Author response image 5
Effect of several phosphatase inhibitors for Mg2+-dependent enhancement of TRPV2.
Whole-cell recordings from TRPV2-expressing HEK293T cell were consecutively challenged with 0.3 mM 2-APB, 0.3 mM 2-APB plus 5 mM Mg2+, and 3 mM 2-APB. The cells were treated with LB-100
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Anti-Phosphotyrosine antibody (Rabbit monoclonal) | Abcam | Cat#ab179530; RRID: AB_828379 | WB (1:1000) |
| Antibody | Anti-Phospho-(Ser/Thr)Phe antibody (Rabbit polyclonal) | Abcam | Cat#ab17464; RRID: AB_443891 | WB (1:1000) |
| Antibody | Anti-Flag antibody (Rabbit polyclonal) | Proteintech | Cat#20543–1-AP; RRID: AB_11232216 | WB (1:3000) |
| Antibody | Anti-TRPV2 antibody (Rabbit polyclonal) | Alomone Labs | Cat#ACC-032; RRID: AB_2040266 | WB (1:500), IP (1:200) |
| Antibody | Anti-mouse IgG (H+L) (Goat polyclonal) | Jackson Immunoresearch | Cat#115-035-003; RRID: AB_10015289 | (5 μg) |
| Antibody | Anti-rabbit IgG (H+L) (Goat polyclonal) | Jackson Immunoresearch | Cat#111-005-003; RRID: AB_2337913 | (5 μg) |
| Antibody | Anti-JAK1 antibody (Rabbit monoclonal) | Abcam | Cat#ab133666 | WB (1:1000) |
| Antibody | Anti-PTPN1 antibody (Rabbit monoclonal) | Abcam | Cat#ab244207; RRID: AB_2877148 | WB (1:1000) |
| Antibody | Anti-Flag Affinity Gel (Mouse monoclonal) | Bimake | Cat#B23102; RRID: AB_2728745 | (15 μl) |
| Other | ProteinIso Protein G Resin | TransGen | Cat#DP401 | (30 μl) |
| Chemical compound, drug | 2-APB | Sigma-Aldrich | Cat#D9754, CAS: 524-95-8 | TRPV2 agonist |
| Chemical compound, drug | MgCl2·6H2O | Sigma-Aldrich | Cat#M2393, CAS: 7791-18-6 | |
| Chemical compound, drug | Na2-ATP | Sigma-Aldrich | Cat#A2383; CAS: 34369-07-8 | |
| Chemical compound, drug | EDTA | Biosharp | Cat#BS107; CAS: 60-00-4 | |
| Chemical compound, drug | AMP-PNP | Sigma-Aldrich | Cat#A2647; CAS: 25612-73-1 | |
| Chemical compound, drug | MK-2206 | TargetMol | Cat#T1952; CAS: 1032350-13-2 | Akt inhibitor |
| Chemical compound, drug | Staurosporine | TargetMol | Cat#T6680; CAS: 62996-74-1 | PKC inhibitor |
| Chemical compound, drug | KN-93 Phosphate | TargetMol | Cat#T2606; CAS: 1188890-41-6 | CaMKII inhibitor |
| Chemical compound, drug | D4476 | TargetMol | Cat#T2449; CAS: 301836-43-1 | CK1 inhibitor |
| Chemical compound, drug | U0126-EtOH | TargetMol | Cat#T6223; CAS: 1173097-76-1 | MEK1/2 inhibitor |
| Chemical compound, drug | Ruxolitinib | TargetMol | Cat#T1829; CAS: 941678-49-5 | JAK1 inhibitor |
| Chemical compound, drug | Salubrinal | TargetMol | Cat#T3045; CAS: 405060-95-9 | PP1 inhibitor |
| Chemical compound, drug | LB-100 | MCE | Cat#HY-18597; CAS: 1632032-53-1 | PP2A inhibitor |
| Chemical compound, drug | Cyclosproin A | TargetMol | Cat#T0945; CAS: 59865-13-3 | PP2B inhibitor |
| Chemical compound, drug | Cantharidin | Aladdin | Cat#c111020; CAS: 56-25-7 | PP1 and PP2A inhibitors |
| Chemical compound, drug | CCT007093 | TargetMol | Cat#T1927; CAS:176957-55-4 | PPM1D inhibitor |
| Chemical compound, drug | PTP inhibitor 1 | TargetMol | Cat#T7084; CAS: 2491-38-5 | PTPs inhibitor |
| Chemical compound, drug | PTP inhibitor 2 | TargetMol | Cat#T7541; CAS: 2632-13-5 | PTPs inhibitor |
| Cell lines (species) | Human embryo kidney (HEK) 293T (human) | ATCC | Cat#CRL-3216; RRID: CVCL_0063 | |
| Software, algorithms | QStudio | Developed by Dr Feng Qin from University of New York at Buffalo | ||
| Software, algorithms | Micro-Manager 1.4 | Vale Lab, UCSF | ||
| Software, algorithms | Clampfit | Molecular Devices, Sunnyvale, CA | ||
| Software, algorithms | IGOR | Wavemetrics, Lake Oswego, OR | ||
| Software, algorithms | SigmaPlot | SPSS Science, Chicago, IL | ||
| Software, algorithms | OriginPro | OriginLab Corporation, Northampton, MA | ||
| Software, algorithms | ImageJ | Schneider et al., 2012 |
