Research ArticlePain

Inhibition of somatosensory mechanotransduction by annexin A6

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Science Signaling  19 Jun 2018:
Vol. 11, Issue 535, eaao2060
DOI: 10.1126/scisignal.aao2060
  • Fig. 1 Sensitivity of Anxa6 KO mice to noxious mechanical stimuli.

    (A) Response to stimulation of the paw with von Frey hairs in WT and Anxa6 KO mice. (B) Tail withdrawal threshold of WT and Anxa6 KO mice in response to the application of a blunt probe. n = 9 WT and 8 KO mice. **P < 0.01 Student’s unpaired t test.

  • Fig. 2 Mechanotransduction in Anxa6 KO sensory neurons.

    Mechanotransduction was assessed in cultured sensory neurons from WT and Anxa6 KO DRGs. (A) Membrane capacitances of recorded WT and Anxa6 KO neurons with RA, IA, or SA mechano-gated current and in nonresponsive (NR) neurons. Cm, membrane capacitance. (B) Distribution of the recorded neurons based on the MA current displayed. (C) Minimal stimulation intensity required to elicit a >40-pA MA current in WT and Anxa6 KO neurons. (D) Maximum current density (Imax/Cm) recorded in WT and Anxa6 KO neurons. (E to H) Current density plotted against the displacement of the mechanical probe for RA (E), IA (F), and SA (G) currents and the merged transient RA/IA currents (H). (A, C, and D) *P < 0.05, WT compared to Anxa6 by two-way analysis of variance (ANOVA) followed by Fisher’s least significant difference (LSD) multiple comparisons test. (B) *P < 0.05 by χ2 test. (C) Two-way ANOVA followed by Sidak P = 0.13, 0.80, and 0.99 for RA, IA, and SA, respectively. (D) Two-way ANOVA followed by Sidak P = 0.046, 0.45, and 0.059 for RA, IA, and SA, respectively. (E to H) Two-way ANOVA P = 0.002, 0.0007, 0.0007, and <0.0001 for RA, IA, SA, and RA/IA, respectively. n values are given in (B) and represent the number of neurons.

  • Fig. 3 Mechanotransduction in sensory neurons overexpressing ANXA6.

    Sensory neurons from WT mice were cultured and transfected with a human ANXA6-pIRES2-tdTomato construct or the corresponding pIRES2-tdTomato empty vector (EV). Only neurons expressing tdTomato were selected for mechano-clamp experiments. (A) Membrane capacitance of control and ANXA6-overexpressing neurons. (B) Distribution of the recorded neurons based on the MA current displayed. (C) Minimal stimulation intensity required to elicit a >40-pA MA current in control and ANXA6-overexpressing neurons. (D) Maximum current density (Imax/Cm) recorded in control and Anxa6-overexpressing neurons. (E to H) Current density plotted against the displacement of the mechanical probe for RA (E), IA (F), and SA (g) currents and merged transient RA/IA currents (H). (A, C, and D) *P < 0.05, **P < 0.01, EV compared to ANXA6 by two-way ANOVA followed by Fisher’s LSD multiple comparisons test. (B) χ2 contingency test P = 0.7452. (E to H) Two-way ANOVA P = 0.97, 0.0004, <0.0001, and <0.0001 for SA, RA, IA, and RA/IA, respectively. n values are given in (B) and represent the number of neurons.

  • Fig. 4 Piezo2 MA current modulation by ANXA6 coexpression.

    ND-C cells were cotransfected with hPIEZO2-IRES-eGFP and/or hANXA6-pIRES2-tdTomato or their respective EVs IRES-eGFP and pIRES2-tdTomato according to the indicated combinations, and mechanotransduction was assessed by the mechano-clamp technique. (A) Membrane capacitance of cells transfected as indicated. (B) Minimal stimulation intensity required to elicit a >40-pA MA current in cells transfected as indicated. (C) Maximum recoded MA current density in cells transfected as indicated. (D) Current density plotted against the displacement of the mechanical probe in cells transfected as indicated. (A to D) *P < 0.05, **P < 0.01, ***P < 0.001 compared to PIEZO2 + EV by one-way ANOVA followed by Fisher’s LSD test (A to C) or two-way ANOVA followed by Fisher’s LSD multiple comparisons test (D). n = 4 EV + EV, 6 EV + ANXA6, 10 PIEZO2 + EV, and 5 PIEZO2 + ANXA6 cells.

  • Fig. 5 Effect of virally mediated overexpression of ANXA6 in sensory neurons on noxious mechanical sensitivity and mechanical hypersensitivity in the MIA model of osteoarthritis pain.

    WT mice received one intrathecal injection of AAV-TT viruses containing either a hANXA6-IRES-GFP construct or the IRES-GFP control. Pain thresholds were then monitored for 12 weeks, after which osteoarthritis was induced by a unilateral injection of MIA into the knee joint, and mechanical hypersensitivity was assessed for another 3 weeks. (A) Noxious mechanical sensitivity of mice injected with the indicated viruses and submitted to the paw pressure test. (B) Sensitivity to noxious heat of mice injected with the indicated viruses. (C) Mechanical hypersensitivity after unilateral osteoarthritis (OA) induction in mice injected with the indicated viruses. (D) Weight-bearing asymmetry as evaluated by the weight ratio between ipsilateral and contralateral rear paws in mice injected with the indicated viruses. Data are shown as mean ± SEM; two-way repeated-measures ANOVA with Dunnett’s multiple comparison test; **P < 0.01, ****P < 0.001 compared to 0 within AAV-TT-IRES-GFP; +P < 0.05, ++P < 0.01 compared to 0 within AAV-TT-ANXA6-IRES-GFP; n = 12 mice per group.

  • Table 1 NMB-1–interacting candidates shared between DRG and ND-C cells.

    Mouse Uniprot numbers are given. Total matches refer to combined numbers of positive peptide matches for each candidate.

    GeneUniprotNameMolecular weight Total matches
    Anxa6P14824Annexin A6,
    calcium-dependent
    phospholipid binding
    75,83836
    FlnaQ8BTM8Filamin-A, actin binding274,14228
    Myh9A2VCK1Myosin-9, actin
    filament binding
    226,23222
    VimP20152Vimentin, structural
    constituent of cytoskeleton
    53,65521
    Anxa5P48036Annexin A5,
    calcium-dependent
    phospholipid binding
    35,7308
    HnrpuO88568Heterogenous nuclear
    ribonucleoprotein U, core
    promoter binding activity
    87,8638
    Snd1Q78PY7Staphylococcal nuclease
    domain-containing protein 1
    nuclease domain-containing
    protein 1, cadherin binding
    involved in cell-cell adhesion
    102,0257
    NonoQ99K48Non-POU domain-containing
    octamer-binding protein,
    chromatin binding
    54,5066
    L1camQ6PGJ3Neural cell adhesion
    molecule L1, PDZ domain
    binding
    140,9695
    Pdia6Q3THH1Protein disulfide-isomerase
    A6, protein disulfide
    isomerase activity, platelet
    activation
    48,6275
    Anxa4Q9R0V2Annexin A4,
    calcium-dependent
    phospholipid binding
    24,2114
    Cpsf6Q6NVF9Cleavage and
    polyadenylation specific
    factor 6, 68 kDa (predicted),
    isoform CRA_b, mRNA
    binding
    59,1164
    Matr3Q8K310Matrin-3, nucleotide binding94,5723
    Sfxn3Q91V61Sideroflexin-3, tricarboxylate
    secondary active
    transmembrane transporter
    activity
    31,6363

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/535/eaao2060/DC1

    Fig. S1. Isolation of NMB-1 peptide-binding partners.

    Fig. S2. Interaction between NMB-1 and annexin A6.

    Fig. S3. Representative traces of MA currents in vitro.

    Fig. S4. ATP6V1B2 does not confer mechanosensitivity to HEK or ND-C cells but inhibits Piezo2 current.

    Fig. S5. Transduction of DRG neurons using AAV-TT serotype.

    Table S1. Top NMB-1–binding candidates in ND-C cells.

    Data file S1. NMB-1–binding candidates in DRG neurons.

    Data file S2. NMB-1–binding candidates from differentiated ND-C cells.

  • Supplementary Materials for:

    Inhibition of somatosensory mechanotransduction by annexin A6

    Ramin Raouf, Stéphane Lolignier, Jane E. Sexton, Queensta Millet, Sonia Santana-Varela, Anna Biller, Alice M. Fuller, Vanessa Pereira, Jyoti S. Choudhary, Mark O. Collins, Stephen E. Moss, Richard Lewis, Julie Tordo, Els Henckaerts, Michael Linden, John N. Wood*

    *Corresponding author. Email: j.wood{at}ucl.ac.uk

    This PDF file includes:

    • Fig. S1. Isolation of NMB-1 peptide-binding partners.
    • Fig. S2. Interaction between NMB-1 and annexin A6.
    • Fig. S3. Representative traces of MA currents in vitro.
    • Fig. S4. ATP6V1B2 does not confer mechanosensitivity to HEK or ND-C cells but inhibits Piezo2 current.
    • Fig. S5. Transduction of DRG neurons using AAV-TT serotype.
    • Table S1. Top NMB-1–binding candidates in ND-C cells.
    • Legends for data files S1 and S2

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). NMB-1–binding candidates in DRG neurons.
    • Data file S2 (Microsoft Excel format). NMB-1–binding candidates from differentiated ND-C cells.

    [Download Data files S1 and S2]


    © 2018 American Association for the Advancement of Science

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