Research ArticleIon Channels

Local Ca2+ signals couple activation of TRPV1 and ANO1 sensory ion channels

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Science Signaling  28 Apr 2020:
Vol. 13, Issue 629, eaaw7963
DOI: 10.1126/scisignal.aaw7963
  • Fig. 1 Optical interrogation of Cl channel activity in DRG neurons.

    (A) Schematic of the dual-imaging protocol. DRG neurons are transfected with EYFP (H148Q/I152L); NaI (I) is added to the extracellular solutions. Upon stimulation of GPCRs such as the bradykinin receptor B2R, Ca2+ is released from the endoplasmic reticulum (ER) through the IP3R. This rise in intracellular Ca2+ is detected by fura-2 (depicted as brightening of cytosol in the middle and right panels). Activation of ANO1 channels by released Ca2+ results in I influx, which, in turn, induces quenching of the EYFP-QL fluorescence (right). (B) Schematic of expected EYFP (H148Q/I152L) quenching in response to ANO1-mediated I influx [30 mM extracellular NaI; based on (19)]. (C) Schematic of the optimized protocol developed in this study. The concentration of NaI is reduced in steps to develop a protocol with minimal agonist-independent quenching and sufficient dynamic range. (D) Representative traces of EYFP (H148Q/I152L) fluorescence quenching produced by 5 (black trace), 10 (red trace), and 30 mM (gray trace) extracellular NaI and subsequently added GABA (100 μM). (E) Scatter plots summarizing experiments similar to those shown in (D); 5 mM, n = 5 neurons; 10 mM, n = 5 neurons; and 30 mM, n = 7 neurons. Quenching produced after standard bath solution application (black circles), NaI application (red triangles), and NaI and GABA (100 μM) application (gray triangles). *P < 0.05 and ***P < 0.001 (paired t test); n.s, not significant.

  • Fig. 2 All-optical demonstration of coupling of CaCC activation to IP3R-mediated Ca2+ release.

    (A and B) Representative traces showing Ca2+ rises and concurrent EYFP-QL fluorescence quenching in small-diameter DRG neurons transfected with EYFP-QL and loaded with fura-2 AM in response to BK (250 nM) application in the absence (A) or presence (B) of ANO1 inhibitors, T16A-inhA01 (50 μM) or Ani9 (500 nM). (C) Scatter plots summarizing experiments similar to those shown in (A) BK (n = 12 neurons) and (B) T16A-inhA01 (n = 6 neurons) and Ani9 (n = 7 neurons). (D) Representative traces showing fura-2 and EYFP-QL responses to BK application in DRG neurons when Ca2+ was omitted from the extracellular solutions. (E) Comparison between the data for BK application with (n = 12 neurons) and without extracellular Ca2+ (n = 5 neurons). In (C) and (E), *P < 0.05, **P < 0.01, and ***P < 0.001 (paired or unpaired t test, as appropriate).

  • Fig. 3 ER Ca2+ release is required for the TRPV1-mediated activation of CaCC.

    (A) Representative traces showing a Ca2+ rise and a concurrent EYFP-QL fluorescence quenching in a small-diameter DRG neuron transfected with EYFP-QL and loaded with fura-2 AM in response to capsaicin (1 μM) application. (B) Scatter plots summarizing experiments similar to those shown in (A); n = 9 neurons. (C) Comparisons for the fura-2 ratiometric Ca2+ measurement and EYFP-QL fluorescence quenching in response to capsaicin and BK application. (D) Summary of fura-2 Ca2+ imaging experiments in which capsaicin was applied either under control conditions (n = 19 neurons), in the presence of thapsigargin (TG; 1 μM; n = 12 neurons), with no extracellular Ca2+ (n = 9 neurons), or in the presence of cyclopiazonic acid (CPA; 1 μM; n = 16 neurons). (E) Representative traces showing a response to capsaicin in triple imaging experiments, where fura-2 Ca2+ levels (black trace), EYFP-QL fluorescence quenching (red trace), and ER-Ca2+ levels using red-CEPIA (gray trace) were simultaneously monitored in EYFP-QL and red-CEPIA cotransfected DRG neurons. (F) Scatter plots summarizing experiments similar to those shown in (E), n = 8 neurons. In (B) to (F), *P < 0.05, **P < 0.01, and ***P < 0.001 [paired t test and Wilcoxon signed-rank test (B) and (F), unpaired t test (C), and one-way ANOVA (D)].

  • Fig. 4 TG disrupts capsaicin-induced currents in small-diameter DRG neurons.

    (A) Representative whole-cell patch-clamp recordings from DRG neurons held at −60 mV during application of capsaicin (1 μM) in the absence (black trace) or presence (red trace) of the ANO1 inhibitor Ani9 (500 nM). (B) Responses to capsaicin (1 μM) in the presence of TG (1 μM). Capsaicin-induced currents that were smaller (black trace) or of comparable amplitude (red trace) to those obtained under control conditions are shown. (C) Scatter plots summarizing areas under the curve for capsaicin responses in experiments similar to these shown in (A) and (B); control (n = 18 neurons), Ani9 (n = 11 neurons); TG (n = 16 neurons). In (C), *P < 0.05 (Kruskal-Wallis ANOVA with Mann-Whitney test).

  • Fig. 5 ER-localized TRPV1 channels are not engaged during extracellular TRPV1 stimulation in small-diameter DRG neurons.

    (A) Representative traces showing Ca2+ responses in small-diameter DRG neurons loaded with fluo-4 AM in response to capsaicin (1 μM) in the presence of ruthenium red (RR; 10 μM) and after the RR washout. (B) Scatter plots summarizing experiments similar to those shown in (A); RR + CAP (n = 42 neurons); CAP (n = 38 neurons). (C) Representative trace showing fluorescence changes in small-diameter DRG neurons transfected with green-CEPIA in response to capsaicin (1 μM) in the presence of RR (10 μM) and after the RR washout. (D) Scatter plots summarizing experiments similar to those shown in (C); n = 6 neurons. (E) Representative traces showing a Ca2+ response in a small-diameter DRG neuron loaded with fura-2 AM in response to capsaicin (1 μM) and K2 (30 μM). (F) Scatter plots summarizing experiments similar to those shown in (E); CAP, n = 19 neurons; K2, n = 19 neurons; K2 (Ca2+ free), n = 8 neurons. (G) Representative trace showing fluorescence changes in a small-diameter DRG neuron transfected with red-CEPIA in response to K2 (30 μM). (H) Scatter plots summarizing experiments similar to those shown in (G); K2 CEPIA, responding neurons (n = 8 neurons); NR K2 CEPIA, non-responding neurons (n = 4 neurons). In (B), (D), (F), and (H), *P < 0.05, **P < 0.01, and ***P < 0.001 [Mann-Whitney test (B), paired t test (D), Kruskal-Wallis ANOVA with Mann-Whitney test (F), and unpaired t test (H)].

  • Fig. 6 ANO1, TRPV1, and IP3R1s are in close proximity in small-diameter DRG neurons as tested with proximity ligation assay.

    (A to C) PLA images for TRPV1-ANO1 (A), ANO1-IP3R1 (B), and TRPV1-IP3R1 (C) pairs in DRG cultures. Left, brightfield images; middle, PLA puncta detection; right, merged images of brightfield and PLA puncta. Scale bars, 20 μm. (D) Scatter plots summarizing number of PLA puncta per cell in experiments similar to these shown in (A) to (C); TRPV1-ANO1 (n = 41 neurons), ANO1-IP3R1 (n = 16 neurons), and TRPV1-IP3R1 (n = 16 neurons); black outlined symbols depict negative control where only one primary antibody (against ANO1) was used in conjunction with both PLA secondary probes (fig. S6, A and B). In (D), ***P < 0.001 (Kruskal-Wallis ANOVA with Mann-Whitney test).

  • Fig. 7 ANO1, TRPV1, and IP3R1s are found in close proximity in small-diameter DRG neurons as tested with STORM.

    (A to C) Right panels display representative STORM images from DRG neurons, double-labeled for either TRPV1 and ANO1 (A), ANO1 and IP3R1 (B), or TRPV1 and IP3R1 (C) using dye pairs of Alexa Fluor 405/Alexa Fluor 647 (green centroids) and Cy3/Alexa Fluor 647 (red centroids); scale is indicated by the white bars; 5 μm (left), 1 μm (middle), and 0.2 μm (right) for (A) to (C). (D to F) Cluster distributions representing double-labeled TRPV1-ANO1, n = 6 neurons (D); ANO1-IP3R1, n = 9 neurons (E); or TRPV1-IP3R1, n = 6 neurons (F). Summary data for cluster percentages, localization number per cluster, and probability distribution statistics are given in tables S1 to S3.

  • Fig. 8 Imaging of ANO1-TRPV1-IP3R1 complexes using three-color STORM.

    (A and B) Representative STORM images for triple-labeled ANO1 (blue centroids), TRPV1 (green centroids), and IP3R1 (red centroids) in DRG neurons; scale is indicated by the white bars; 5 μm (left, A), 1 μm (middle, A), 0.2 μm (right, A), and 0.2 μm (for all images, B). (C) Cluster distributions representing triple-labeled ANO1-TRPV1-IP3R1 combinations of proteins, protein pairs, or single proteins; n = 12 neurons. Summary data for cluster percentages, localization number per cluster, and probability distribution statistics are given in tables S1 to S3. (D) Schematic depicting possible ANO1-TRPV1-IP3R1 functional coupling in DRG neurons. Our data suggest that a sizable fraction of ANO1, TRPV1, and IP3R1s are in close proximity at the ER-PM junctions and form complexes in small-diameter DRG neurons. TRPV1 activation not only leads to Na+ and Ca2+ influx (middle) but can also activate IP3R Ca2+ release (right), presumably through PLC activation. Release of Ca2+ from the ER maximizes ANO1 activation (right), which ultimately causes Cl efflux and depolarization of the neuron.

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/13/629/eaaw7963/DC1

    Fig. S1. Depolarization-induced I influx is a Ca2+-independent phenomenon.

    Fig. S2. ER Ca2+ store depletion severely reduces Ca2+ transients and CaCC activation in response to capsaicin.

    Fig. S3. Capsaicin applied in extracellular Ca2+-free conditions and acute TG-induced ER Ca2+ leak can induce a degree of CaCC activation.

    Fig. S4. The cell-impermeable TRPV1 agonist K2 evokes cytosolic Ca2+ transients in the absence of extracellular Ca2+.

    Fig. S5. Capsaicin induces PLC activation in DRG neurons.

    Fig. S6. Controls for PLA.

    Fig. S7. CD71 and IP3R1 are not found in close proximity as confirmed by PLA and STORM analysis.

    Fig. S8. Decay rates of STORM fluorescent dye labels after repeated laser activation cycles.

    Table S1. Mean percentages of STORM cluster distributions detected by DBSCAN.

    Table S2. Mean cluster radius sizes of STORM-derived clusters detected by DBSCAN.

    Table S3. Mean number of localizations per cluster from STORM-derived clusters detected by DBSCAN.

  • This PDF file includes:

    • Fig. S1. Depolarization-induced I influx is a Ca2+-independent phenomenon.
    • Fig. S2. ER Ca2+ store depletion severely reduces Ca2+ transients and CaCC activation in response to capsaicin.
    • Fig. S3. Capsaicin applied in extracellular Ca2+-free conditions and acute TG-induced ER Ca2+ leak can induce a degree of CaCC activation.
    • Fig. S4. The cell-impermeable TRPV1 agonist K2 evokes cytosolic Ca2+ transients in the absence of extracellular Ca2+.
    • Fig. S5. Capsaicin induces PLC activation in DRG neurons.
    • Fig. S6. Controls for PLA.
    • Fig. S7. CD71 and IP3R1 are not found in close proximity as confirmed by PLA and STORM analysis.
    • Fig. S8. Decay rates of STORM fluorescent dye labels after repeated laser activation cycles.
    • Table S1. Mean percentages of STORM cluster distributions detected by DBSCAN.
    • Table S2. Mean cluster radius sizes of STORM-derived clusters detected by DBSCAN.
    • Table S3. Mean number of localizations per cluster from STORM-derived clusters detected by DBSCAN.

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