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. 2021 Oct 4;220(10):e202002083.
doi: 10.1083/jcb.202002083. Epub 2021 Aug 25.

Depolarization induces nociceptor sensitization by CaV1.2-mediated PKA-II activation

Jörg Isensee  1 , Marianne van Cann  1 , Patrick Despang  2 , Dioneia Araldi  3 , Katharina Moeller  1 , Jonas Petersen  4 , Achim Schmidtko  4 , Jan Matthes  2 , Jon D Levine  3 , Tim Hucho  1
Affiliations

Depolarization induces nociceptor sensitization by CaV1.2-mediated PKA-II activation

Jörg Isensee et al. J Cell Biol. .

Abstract

Depolarization drives neuronal plasticity. However, whether depolarization drives sensitization of peripheral nociceptive neurons remains elusive. By high-content screening (HCS) microscopy, we revealed that depolarization of cultured sensory neurons rapidly activates protein kinase A type II (PKA-II) in nociceptors by calcium influx through CaV1.2 channels. This effect was modulated by calpains but insensitive to inhibitors of cAMP formation, including opioids. In turn, PKA-II phosphorylated Ser1928 in the distal C terminus of CaV1.2, thereby increasing channel gating, whereas dephosphorylation of Ser1928 involved the phosphatase calcineurin. Patch-clamp and behavioral experiments confirmed that depolarization leads to calcium- and PKA-dependent sensitization of calcium currents ex vivo and local peripheral hyperalgesia in the skin in vivo. Our data suggest a local activity-driven feed-forward mechanism that selectively translates strong depolarization into further activity and thereby facilitates hypersensitivity of nociceptor terminals by a mechanism inaccessible to opioids.

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Figures

Figure 1.
Figure 1.
Depolarization activates PKA-II in nociceptive neurons. (A) HCS microscopy images of rat DRG neurons stimulated with solvent (Ctrl) or KCl (40 mM) for 1 min. Cultures were immunolabeled with UCHL1 to identify the neurons and pRII to quantify PKA-II signaling activity. Red frames mark enlarged image sections; green or red encircled neurons in the left panel indicate the mask to selected or rejected objects, respectively (see Materials and methods). Scale bars, 100 µm. (B) Time course of pRII intensity in DRG neurons stimulated with KCl (40 mM). (C) Dose–response curve of pRII intensity in DRG neurons exposed to KCl (0–80 mM, EC50 = 10 mM) for 3 min. (D) Time course of pRII intensity in DRG neurons treated with veratridine (VT; 100 µM) to open VGSCs in comparison to the KCl (40 mM) response. (E) Size and RIIβ intensity distributions of control and KCl-stimulated DRG neurons used to determine the gating thresholds for the following subgroup analysis. PDE, phosphodiesterase. (F) Cell density plots showing single-cell data of pRII intensities versus the neuronal areas of KCl (40 mM)–stimulated sensory neurons. Dashed lines indicate gating thresholds used to calculate the percentage of cells in the respective quadrant. Combined data of n = 4 experiments with a total of >3,000 neurons per condition. (G) Quantification of responding smaller (<1,400 µm2) and larger (>1,400 µm2) neurons in n = 4 replicate experiments with a total of >3,000 neurons per condition. (H) Cell density plots showing single-cell data of pRII versus RIIβ intensities in the same neurons shown in F. (I) Quantification of responding RIIβ− and RIIβ+ neurons. HCS data in B–D are means ± SEM; n = 3–4 independent experiments; >2,000 neurons/condition; two-way ANOVA with Bonferroni’s test; **, P < 0.01; ***, P < 0.001 indicate significance levels between baseline and stimulated conditions at the respective time point.
Figure S1.
Figure S1.
KCl induces pRII-increase predominantly in nociceptive neurons. (A) Dose–response curve of pRII intensity in mouse DRG neurons exposed to KCl (0–80 mM, EC50 = 10 mM) for 3 min. Data are means ± SEM; n = 4 independent experiments; >2,000 neurons/condition; two-way ANOVA with Bonferroni’s test; *, P < 0.05; ***, P < 0.001 indicate significance levels between baseline and stimulated conditions. (B) Cell density plots of cultured rat DRG neurons labeled for pRII and subgroup markers RIIβ, NaV1.8, CGRP, and NF200. The cells were stimulated with KCl (40 mM, lower panel) or buffer (Ctrl, upper panel) for 3 min to activate PKA-II. Dashed lines indicate gating thresholds used to calculate the percentage of cells in the respective quadrant. Single-cell data of >10,000 neurons per plot from one experiment with pooled cells from n = 3 rats are shown. (C) Marker intensity distributions used to determine the gating thresholds for the subgroup analysis. (D) Quantification pRII intensity and responding pRII(+) neurons in marker-negative and marker-positive neurons from the experiment shown in B and C. Each data point represents the mean of a single culture well.
Figure 2.
Figure 2.
Calcium influx through CaV1.2 induces PKA-II activity during depolarization. (A) Expression pattern of VGCC α subunits in overnight cultures of rat and mouse DRG determined by RNA-seq (Isensee et al., 2014b). TPM, transcripts per kilobase million. (B) Expression pattern of VGCC α subunits in mouse DRG neuron subgroups determined by single-cell RNA-seq (Zeisel et al., 2018). (C) Time course of pRII intensity in KCl-depolarized rat sensory neurons after pretreatment (10 min) with the NMDA receptor antagonist D-AP5 (10 µM), the Cav3.1-3.3 blocker TTA-P2 (1 µM), and a combination of the Cav2.1/2.2 blocker ω-agatoxin IVA (100 nM), ω-conotoxin MVIIC (200 nM), and ω-conotoxin GVIA (1 µM). (D) Inhibitory effect of verapamil (20 or 200 µM, 10-min pretreatment) on the pRII increase induced by KCl depolarization. (E) Dose–response curve showing the effect of verapamil (0–200 µM; IC50 = 16 µM) on pRII signals induced by KCl depolarization (3 min). (F) Inhibitory effect of diltiazem (100 µM, 10 min) on the KCl-induced pRII increase. (G) Dose–response curve showing the inhibition of KCl-induced pRII signals by diltiazem (0–200 µM; IC50 = 37 µM). (H) Reinforcing effect of the CaV1 agonist (S)-(-)-Bay K 8644 (2 µM, 10 min) on pRII signals induced by a low dose of KCl (10 mM). (I) Dose–response curve showing the reinforcing effect of Bay K 8644 (0–5 µM; EC50 = 80 nM) on pRII signals induced by KCl (10 mM, 3 min). (J) Chelation of extracellular calcium with EGTA (2.5 mM, 30-min prestimulation) abolished the pRII response to KCl-depolarization. (K) Effect of the cell-permeable calcium chelator BAPTA-AM (100 µM, 60 min) on pRII signals induced by KCl depolarization (compound effect: F1,28 = 10.9, P < 0.003). Values in C–K represent means ± SEM; n = 3–4 experiments; >2,000 neurons/condition; two-way ANOVA with Bonferroni’s test; §, P < 0.05; §§, P < 0.01; §§§, P < 0.001 indicate significance levels between KCl-induced pRII signals in the absence or presence of an agonist/antagonist.
Figure S2.
Figure S2.
The increase of pRII intensity induced by KCl is independent of NaV, TRPA1, TRPV1, or ionomycin-induced calcium influx. (A) Time course of pRII intensity in lidocaine-pretreated (2, 20, and 200 µM; 10 min) rat DRG neurons stimulated with KCl (40 mM). (B) Dose–response curve of pRII intensities in DRG neurons of WT, TRPA1, TRPV1, and TRPA1/V1 double-knockout mice exposed to KCl (0–80 mM, EC50 = 10 mM) for 3 min. (C) Calcium imaging (FLIPR Calcium 5 dye) showing calcium influx evoked by ionomycin (2 or 5 µM) followed by KCl (40 mM). Values in are means ± SEM; n = 4; >1,000 neurons/condition. (D) Time course of pRII intensities in DRG neurons stimulated with ionomycin (2 or 5 µM) versus KCl (40 mM). Data in A, B, and D are means ± SEM; n = 3–4 independent experiments; >2,000 neurons/condition; two-way ANOVA with Bonferroni’s test; *, P < 0.05; **, P < 0.01; ***, P < 0.001 indicate significance levels between baseline and stimulated conditions. Calcium imaging data in C are means ± SEM; n = 4 independent experiments with a total of >500 analyzed neurons/condition.
Figure 3.
Figure 3.
Deletion of CaV1.2 in NaV1.8+ nociceptors reduces PKA-II activity. (A) Expression pattern of NaV1.8 (Scn10a), CaV1.2 (Cacna1c), and RIIβ (Prkar2b) in subgroups of DRG neuron determined by single-cell RNA-seq (Zeisel et al., 2018). (B) Conditional mouse model to delete CaV1.2 in NaV1.8-expressing DRG neurons. (C and D) Distribution of UCHL1 and RIIβ expression levels in DRG neurons of cKO mice and respective controls lacking Cre recombinase (Ctrl). (E) Total numbers of viable DRG neurons after overnight culture determined by HCS microscopy (n = 3 females and 2 males per genotype). (F) Single-cell data and mean intensities obtained using a CaV1.2-specific antibody (clone N263/31) indicating down-regulation of CaV1.2 (n = 10 cultures from three mice per genotype, >6,000 neurons per genotype, Student’s t test). The primary antibody was omitted in respective controls (w/o AB). (G) Basal genotype difference of pRII intensity in all solvent stimulated RIIβ+ control neurons (n = 3 females and 2 males per genotype, >15,000 neurons/condition, Student’s t test). (H) Dose-dependent induction of pRII intensity by KCl (0–80 mM) in cKO and Ctrl mice (n = 3 females and 2 males per genotype; genotype effect: F4,62 = 5.6, extra-sum-of-squares F test). (I) Dose-dependent induction of pRII intensity by 10 mM KCl after 10 min preincubation with (S)-(-)-Bay K 8644 (0–5 µM) in cKO and Ctrl mice. (n = 3 females and 2 males per genotype; genotype effect: F4,62 = 13.9, extra-sum-of-squares F test). (J) Single-cell data of selected condition shown in I. Values in G–I are means ± SEM; *, P < 0.05; ***, P < 0.001.
Figure S3.
Figure S3.
CaV1-expression in neurons of cKO mice or after AAV-mediated knock down of CaV1.2. (A) Single-cell data and mean intensities obtained from Ctrl and cKO mice using a CaV1-specific antibody (clone L57/46; n = 10 cultures from three mice per genotype, >6,000 neurons per genotype, Student’s t test). (B and C) Single-cell data and mean intensities obtained using a CaV1-specific antibody (clone L57/46) at 8 div (B, n = 4, >4,000 neurons per condition) and 15 div (C, n = 4, >1,500 neurons per condition) after transduction with AAV-PHP.S expressing either a scrambled (Scr) or Cacna1c-specific shRNA, indicating down-regulation of CaV1 in GFP+ neurons. The primary antibody was omitted in respective controls (w/o AB).
Figure 4.
Figure 4.
Adenoviral knockdown of CaV1.2 reduces PKA-II activity. (A) Representative HCS microscopy images of mouse DRG neurons transduced with AAV-PHP.S-U6-shRNA:Scramble-CAG-GFP (Scr) or AAV-PHP.S-U6-shRNA:Cacna1c-CAG-GFP (Cac) to knock down CaV1.2. Neurons were transduced after overnight culture (1 div) and fixed 1 wk (8 div) or 2 wk (15 div) later. Cultures were immunolabeled for the neuronal marker UCHL1 and pRII to quantify PKA-II signaling activity. The expression of GFP indicated efficient transduction. Nuclei were stained with Hoechst 34580. Scale bar, 100 µm. (B and C) GFP expression levels in individual neurons (left) and mean numbers of GFP+ neurons after 8 div (n = 6, >6,000 neurons per condition, Student’s t test) as well as after 15 div (n = 6, total of >1,000 neurons per condition, Student’s t test). (D and E) Single-cell data of all analyzed neurons (left) and mean intensities in GFP− and GFP+ neurons (right) obtained using a CaV1.2-specific antibody (clone N263/31) at 8 div (D; n = 4, >4,000 neurons) and 15 div (E, n = 3, >1200 neurons) indicating down-regulation of CaV1.2 in GFP+ neurons. The primary antibody was omitted in respective controls (w/o AB). (F and G) Effect of AAV-mediated CaV1.2 knockdown on pRII intensity levels induced by 3-min stimulation with KCl at 8 div (F; n = 6, >6,000 neurons, two-way ANOVA with Bonferroni’s test) and 15 div (G, n = 6, >1,000 neurons per condition). (H) Cell density plots showing single-cell data of pRII intensities versus GFP expression after 15 div as shown in G. Values in B–G are means ± SEM; *, P < 0.05; ***, P < 0.001.
Figure S4.
Figure S4.
The KCl-induced increase of pRII intensity cannot be altered by inhibition of adenylycyclases or phosphodiesterases. (A) Expression pattern of AC isoforms in adult rat DRGs determined by RNA-seq (Isensee et al., 2014b). (B–I) Effect of the AC inhibitors NB001, ST034307, SQ22536, and NKY80 (100 µM each, 30-min pretreatment) on the pRII increase induced by KCl (40 mM) or 5-HT (250 nM). (J and K) Effect of the phosphodiesterase inhibitor IBMX (100 µM, 30-min pretreatment) on the pRII increase induced by 5-HT (250 nM) or KCl (40 mM). The data shown are not normalized to the baseline difference due to IBMX treatment alone. Data in A are means ± SD, n = 6 replicates. Values in B–K represent means ± SEM; n = 3–4 experiments; >2,000 neurons/condition; two-way ANOVA with Bonferroni’s test; §§, P < 0.01; §§§, P < 0.001 indicate significance levels between 5-HT-induced pRII signals in the absence or presence of an inhibitor.
Figure 5.
Figure 5.
KCl-induced PKA-II activity is cAMP independent but modulated by calpains. (A) The pRII increase induced by KCl (40 mM) was not inhibited by fentanyl (Fent; 10 µM), oxycodone (Oxy; 10 µM), or [Leu5]-enkephalin (Enk; 10 µM) in rat sensory neurons. (B and C) Effect of the phosphodiesterase inhibitor IBMX (100 µM, 30-min pretreatment) on the pRII increase induced by 5-HT (250 nM) or KCl (40 mM). Fig. S4, J and K show not-normalized data indicating basal elevation of pRII intensity by IBMX. (D and E) The cAMP antagonist Rp-cAMPS-pAB (10 µM) has no effect on the induction of pRII intensity by KCl (40 mM). (F) Effect of the calpain inhibitor MDL28170 (100 µM, 30 min) on the pRII increase by KCl (40 mM). (G) Time course of pERK1/2 intensity in DRG neurons treated with veratridine (VT; 100 µM) to open VGSCs in comparison to the KCl (40 mM) response. (H) Inhibitory effect of verapamil (VP; 200 µM, 10 min pretreatment) on ERK1/2 phosphorylation induced by depolarization (40 mM KCl). (I) The pERK1/2 response to a low dose of KCl (10 mM) is reinforced by (S)-(-)-Bay K 8644 (2 µM, 10 min). (J) Chelation of extracellular calcium with EGTA (2.5 mM, 30 min) prevents the pERK1/2 increase. (K) The CaMKII inhibitor AIP (1 µM, 30 min) does not inhibit the induction of pERK1/2 by depolarization. (L) Pretreatment with the PKA inhibitor H89 (25 µM, 30 min) reduces the pERK1/2 response to KCl. Values represent means ± SEM; n = 3–4 experiments; >2,000 neurons/condition; two-way ANOVA with Bonferroni’s test; §, P < 0.05; §§, P < 0.01; §§§, P < 0.001 indicate significance levels between KCl-induced pRII signals in the absence or presence of an inhibitor.
Figure S5.
Figure S5.
Neither CaMKII nor MEK inhibitors modulate KCl-induced pRII increases. (A and B) Effect of the CaMKII inhibitor KN93 (compound effect: F1,42 = 16.3, P < 0.0003) and its inactive analogue KN-92 (compound effect: F1,42 = 17, P < 0.0002; 10 µM each, 30-min pretreatment) on the pRII increase induced by KCl (40 mM). (C) Exposure to AIP (1 µM, 30 min) is not inhibiting the pRII response to KCl (40 mM). (D) Exposure to the ERK1/2 inhibitor U0126 (1 µM, 30 min) is not inhibiting the pRII response to KCl (40 mM). (E) Dose–response of KCl (0–80 mM) in the absence or presence of U0126 (1 µM, 30 min). (F) The MEK inhibitor U0126 (1 µM, 30 min) prevents the pERK1/2 increase after KCl depolarization. Values represent means ± SEM; n = 3–4 experiments; >2,000 neurons/condition; two-way ANOVA with Bonferroni’s test; §, P < 0.05; §§§, P < 0.001 indicate significance levels between KCl-induced pRII signals in the absence or presence of an inhibitor.
Figure 6.
Figure 6.
PKA-dependent phosphorylation of Ser1928 regulates CaV1.2 gating. (A) Representative images of rat DRG neurons stimulated with solvent (Ctrl) or KCl (40 mM) for 1 min. Cultures were labeled for UCHL1 to identify the neurons, phospho-Ser1928 of CaV1.2 (pCaV1.2), and CaV1 channels (CaV1, clone L57/46). Green or red encircled objects indicate automatically selected or rejected objects, respectively. Scale bar, 100 µm. (B) Enlarged section demonstrating the modified image analysis to quantify in nuclear (orange) and cytoplasmic (blue) regions of neurons (green). (C) Cell density plots of single-cell data of pCaV1.2/CaV1-labeled neurons stimulated with solvent control (Ctrl) or KCl (40 mM) for 1 min. The Spearman’s rank correlation coefficient (ρ) and its P value are shown. (D) Depolarization (40 mM KCl) induces CaV1.2 phosphorylation, which is inhibited by verapamil (VP; 200 µM, 10 min). (E) The CaV1 intensity is unchanged after depolarization. (F and G) KCl-induced increase of pCaV1.2 intensity in cytoplasmic versus nuclear regions. (H) Effect of the PKA inhibitor H89 on the pCaV1.2 increase induced by depolarization. (I) Effect of the calcineurin inhibitor FK506 on the of pCaV1.2 increase induced by depolarization. (J) Inhibitory effect of the PKA inhibitor H89 (25 µM, 30 min) on the pRII increase induced by depolarization. (K) Dose–response of KCl (0–80 mM) in the absence or presence of H89 (25 µM, 30 min). Data in D–J represent means ± SEM; n = 3–4 experiments; >2,000 neurons/condition; two-way ANOVA with Bonferroni’s test; **, P < 0.01; ***, P < 0.001 indicate significance levels between baseline and stimulated conditions; §, P < 0.05; §§, P < 0.01; §§§, P < 0.001 indicate significance levels between the absence and presence of an agonist/antagonist.
Figure 7.
Figure 7.
Repetitive depolarization induces calcium and PKA-dependent sensitization of calcium currents in small diameter rat DRG neurons. (A) Normalized I-V curves, V0.5act, and representative traces for calcium currents (ICa) before and 60 s after applying a train of 10 1-s-long depolarizations to 0 mV. (B) Normalized I-V curves, V0.5act, and representative traces for barium currents (IBa) before and 60 s after applying a train of 10 1-s-long depolarizations to 0 mV. (C) Normalized I-V curves, V0.5act, and representative traces for calcium currents (ICa) before and 60 s after applying a train of 10 1-s-long depolarizations to 0 mV. DRG neurons were preincubated with H89 (25 µM, 1 h). Numbers of analyzed neurons are given in parentheses. Data represent means ± SEM of at least three animals. Currents underlying the I-V curves were elicited from −40 to +50 mV in 10-mV increments with 5 mM Ca2+ or Ba2+ as charge carrier. The holding potential was −90 mV. Paired t test; *, P < 0.05.
Figure 8.
Figure 8.
Depolarization induces hyperalgesia in vivo. (A and C) Evaluation of mechanical nociceptive thresholds (Randall–Selitto test) 5, 15, 45, 60, 120, 180, and 1,440 min after intradermal injection of KCl (5 µl of 80 mM KCl solution in dH2O) in the dorsum of the hindpaw of male rats. A biphasic mechanical hyperalgesia was observed in the ipsilateral paw (A, percentage reduction from baseline: F(1,10)=186.0, **, P < 0.01, ***, P < 0.001, ****, P < 0.0001; C, nociceptive threshold: F(1,10)=47.75, **, P < 0.01, ***, P < 0.001, ****, P < 0.0001, when the ipsilateral and contralateral paws were compared in all time points; two-way repeated-measures ANOVA followed by Sidak's test). (B and D) Vehicle (5 µl, dH2O + 1% ethanol) or verapamil (10 µg diluted in 5 µl dH2O + 1% ethanol) was injected intradermally in the dorsum of the hindpaw. 30 min later, KCl (5 µl of 80 mM KCl solution in dH2O) was injected at the same site and mechanical nociceptive threshold evaluated 5, 15, 45, 60, 120, 180, 360, and 1,440 min later. In the group that received verapamil, hyperalgesia was inhibited (B, percentage reduction in nociceptive baseline: F(1,10) = 222.0, ****, P < 0.0001; D, nociceptive threshold: F(1,10) = 22.38, *, P = 0.02, **, P < 0.01, ***, P < 0.001, ****, P < 0.0001, when the vehicle- and verapamil-treated groups are compared at all time points after the injection of KCl; two-way repeated-measures ANOVA followed by Sidak's test). Data represent means ± SEM, n = 6 rats per group.
Figure 9.
Figure 9.
Model of CaV1.2 regulation in DRG neurons. Depolarization of DRG neurons results in CaV1.2 and calcium-dependent activation of PKA-II. The induction of PKA-II activity is not sensitive to opioids or inhibitors of calcium-stimulated ACs, phosphodiesterases (PDEs), and cAMP antagonists such as Rp-cAMPS, indicating a cAMP-independent activation mechanism (red shadow). In addition, inhibition of CaMKII or ERK1/2 is not affecting the induction of PKA-II activity after depolarization, excluding that PKA-II is downstream of these kinases. Depolarization sensitizes CaV1.2 by PKA-dependent phosphorylation of Ser1928. Dephosphorylation of CaV1.2 by the phosphatase calcineurin (CaN) inactivates the channel. CsA, cyclosporine A; MOR, µ opioid receptor.
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References

    1. Agarwal, N., Offermanns S., and Kuner R.. 2004. Conditional gene deletion in primary nociceptive neurons of trigeminal ganglia and dorsal root ganglia. Genesis. 38:122–129. 10.1002/gene.20010 - DOI - PubMed
    1. Alexander, S.P., Benson H.E., Faccenda E., Pawson A.J., Sharman J.L., Spedding M., Peters J.A., and Harmar A.J.. CGTP Collaborators . 2013. The Concise Guide to PHARMACOLOGY 2013/14: G protein-coupled receptors. Br. J. Pharmacol. 170:1459–1581. 10.1111/bph.12445 - DOI - PMC - PubMed
    1. Aley, K.O., and Levine J.D.. 1999. Role of protein kinase A in the maintenance of inflammatory pain. J. Neurosci. 19:2181–2186. 10.1523/JNEUROSCI.19-06-02181.1999 - DOI - PMC - PubMed
    1. Araldi, D., Khomula E.V., Ferrari L.F., and Levine J.D.. 2018. Fentanyl Induces Rapid Onset Hyperalgesic Priming: Type I at Peripheral and Type II at Central Nociceptor Terminals. J. Neurosci. 38:2226–2245. 10.1523/JNEUROSCI.3476-17.2018 - DOI - PMC - PubMed
    1. Ataman, B., Boulting G.L., Harmin D.A., Yang M.G., Baker-Salisbury M., Yap E.L., Malik A.N., Mei K., Rubin A.A., Spiegel I., et al. . 2016. Evolution of Osteocrin as an activity-regulated factor in the primate brain. Nature. 539:242–247. 10.1038/nature20111 - DOI - PMC - PubMed

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