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. 2017 May 12;7(1):1842.
doi: 10.1038/s41598-017-01999-4.

A-Kinase Anchoring Protein 79/150 Scaffolds Transient Receptor Potential A 1 Phosphorylation and Sensitization by Metabotropic Glutamate Receptor Activation

Allison Doyle Brackley  1 , Ruben Gomez  2 , Kristi A Guerrero  2 , Armen N Akopian  3 , Marc J Glucksman  4 , Junhui Du  4 , Susan M Carlton  5 , Nathaniel A Jeske  6   7   8
Affiliations

A-Kinase Anchoring Protein 79/150 Scaffolds Transient Receptor Potential A 1 Phosphorylation and Sensitization by Metabotropic Glutamate Receptor Activation

Allison Doyle Brackley et al. Sci Rep. .

Abstract

Mechanical pain serves as a base clinical symptom for many of the world's most debilitating syndromes. Ion channels expressed by peripheral sensory neurons largely contribute to mechanical hypersensitivity. Transient Receptor Potential A 1 (TRPA1) is a ligand-gated ion channel that contributes to inflammatory mechanical hypersensitivity, yet little is known as to the post-translational mechanism behind its somatosensitization. Here, we utilize biochemical, electrophysiological, and behavioral measures to demonstrate that metabotropic glutamate receptor-induced sensitization of TRPA1 nociceptors stimulates targeted modification of the receptor. Type 1 mGluR5 activation increases TRPA1 receptor agonist sensitivity in an AKA-dependent manner. As a scaffolding protein for Protein Kinases A and C (PKA and PKC, respectively), AKAP facilitates phosphorylation and sensitization of TRPA1 in ex vivo sensory neuronal preparations. Furthermore, hyperalgesic priming of mechanical hypersensitivity requires both TRPA1 and AKAP. Collectively, these results identify a novel AKAP-mediated biochemical mechanism that increases TRPA1 sensitivity in peripheral sensory neurons, and likely contributes to persistent mechanical hypersensitivity.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
mGluR5, AKAP and TRPA1 Contribute to Mechanical Hyperalgesic Priming Behavior. (A) Male Sprague-Dawley rats were injected intraplantarly (ipl) with vehicle (Veh) or carrageenan (Cg, 5 μl of 1% solution in 50 μl PBS final). Over the next 4 days, rats were then injected with Veh or MTEP (400 μg in 50 μl PBS, ipl). Rats were tested for paw withdrawal threshold (PWT, g) on day 5, then injected with PGE2 (100ng in 50 μl PBS, ipl), and again tested for PWT 0.5, 4 and 24 h post-PGE2. n = 6 rats/treatment paradigm; ***p < 0.005; two-way ANOVA with Bonferroni post-hoc analysis. (B) AKAP WT and KO mice (WT and KO, respectively) were tested for PWT (g) before ipl injections (Pre-Cg). Mice were then injected with vehicle (No Cg) or Cg (5 μl of 1% solution in 10 μl PBS, final, ipl) on Day 1. Mice were tested for baseline PWT on Day 5 (Post-Cg), then injected with PGE2 (50ng in 10 μl, ipl) and tested for PWT at 0.5, 1, 2, and 4 h post-PGE2. n = 6–8 mice/genotype/treatment paradigm, ***p < 0.005; two-way ANOVA with Bonferroni post-hoc analysis. (C) TRPA1 WT and KO mice (WT and KO, respectively) were tested and treated identically as in panel B. n = 6 mice/genotype/treatment paradigm; ***p < 0.005; two-way ANOVA with Bonferroni post-hoc analysis. (D) TRPA1 WT and KO mice (WT and KO, respectively) were pre-injected with vehicle (Veh, ddH2O, 5 μl) or 8-Br-cAMP (8-Br, 10 μM, 5 μl) for 5 min prior to mustard oil (MO, 10 μl of 0.01% solution) injection ipl. Nociceptive behavior including licking, biting, scratching, and/or petting the injected area was quantified in seconds over a 3 × 5 min bin collecting period following MO injection. N = 6 mice/genotype/treatment paradigm; *p < 0.05; two-way ANOVA with Bonferroni post-hoc analysis.
Figure 2
Figure 2
mGluR sensitization of Mechanical Responses in Nociceptors from AKAP WT and KO mice. (A) Discharge rates of receptive fields of WT nociceptors following mustard oil (MO) application in ascending doses (2 minutes each). *p < 0.05 vs. pre-drug baseline, Kruskal-Wallis ANOVA on Ranks with Dunn’s posthoc analysis. (B) Representative traces from (A,B) AKAP WT and (C,D) KO nociceptor nociceptor field activity in response to 10 µM DHPG (A,C) alone or (B,D) applied with 10 µM MO. (C). Collective drug-induced activity discharge rates of receptive fields from experiments conducted in panel B. *p < 0.05, significant vs. pre-drug; +p < 0.05; significant vs KO at same dose; Kruskal-Wallis ANOVA on Ranks with Dunn’s posthoc analysis.
Figure 3
Figure 3
mGluR sensitization of Mustard Oil Responses in Nociceptors from AKAP WT and KO mice. (A) Recorded threshold to activation to a ramp stimulus following exposure to 1 mM or 3 mM DHPG from skin-nerve preparation nociceptor receptive fields of AKAP WT and KO mice and (B). Discharge rates to a suprathreshold mechanical stimulus (250 mN). *p < 0.05; significant vs. pre-drug; +p < 0.05, significant vs. KO at same dose; Kruskal-Wallis ANOVA on Ranks with Dunn’s posthoc analysis.
Figure 4
Figure 4
TRPA1 Phosphorylation by PKA and PKC is Dependent on AKAP Expression. Cultured rat trigeminal neurons were grown on 10-cm plates, and transfected with siRNA directed against AKAP, as illustrated. Cultures were then incubated with 32P-orthophosphate for 4 h, prior to treatment with vehicle 8-Br-cAMP (10 μM, A,B), or vehicle vs. phorbol 12,13-dibutyrate (PDBu, 1 μM, (C,D) for 5 min before harvesting and immunoprecipitation (IP) of TRPA1. Immunoprecipitants and aliquots of general lysates were resolved by SDS-PAGE and Western blot (WB), as depicted. Representative autoradiographic and WB results are shown (A and C). Quantified autoradiographic results were normalized to total TRPA1 IP (B and D). Results are representative of 4–5 independent trials; **p < 0.05; ns = no significance; two-way ANOVA with Bonferroni post-hoc analysis.
Figure 5
Figure 5
Site-Directed Mutagenesis of Potential PKA phosphorylation sites on TRPA1. Cumulative traces from Chinese hamster ovary (CHO) cells transiently transfected with various cDNAs in the following combinations: (A) TRPA1 and empty green fluorescence protein vector (GFP), (B) TRPA1, AKAP and GFP, (C) TRPA1 S87A, AKAP and GFP, (D) TRPA1 S179G, AKAP and GFP, (E) TRPA1 S318A, AKAP and GFP, or (F) TRPA1 S1101G, AKAP and GFP. Real-time Ca2+ accumulation measurements were recorded from GFP-positive cells following vehicle (black) or 8-Br-cAMP (10 μM, red) pre-treatment (5 min) to a mustard oil (MO, 50 μM, 30 sec) challenge. ***p < 0.005; **p < 0.01; *p < 0.05; two-way ANOVA with Bonferroni post-hoc analysis. G. Quantified results from cumulative recordings (n = 12–46); ns = no significance; ***p < 0.005; **p < 0.01; trace significance determined by two-way ANOVA with Bonferroni posthoc analysis and quantified data significance determined by unpaired student’s t test.
Figure 6
Figure 6
Site-Directed Mutagenesis of Potential PKC phosphorylation sites on TRPA1. Cumulative traces from Chinese hamster ovary (CHO) cells transiently transfected with various cDNAs in the following combinations: (A) TRPA1 and empty green fluorescence protein vector (GFP), (B) TRPA1, AKAP and GFP, (C) TRPA1 S119G, AKAP and GFP, (D) TRPA1 T281A, AKAP, and GFP, (E) TRPA1 S441A, AKAP, and GFP, (F) TRPA1 S455G, AKAP, and GFP, (G) TRPA1 T529A, AKAP, and GFP, (H) TRPA1 T536A, AKAP, and GFP. Real-time Ca2+ accumulation measurements were recorded from GFP-positive cells following vehicle (black) or PDBu (1 μM, red) pre-treatment (5 min) to a mustard oil (MO, 50 μM, 30 sec) challenge. ***p < 0.005; *p < 0.05; ns = no significance; one-way ANOVA. (I) Quantified results from cumulative recordings (n = 24–64/cDNA transfection paradigm), ns = no significance; ***p < 0.005; **p < 0.01; trace significance determined by two-way ANOVA with Bonferroni posthoc analysis and quantified data significance determined by unpaired student’s t test.
Figure 7
Figure 7
mGluR5-sensitization of TRPA1 Current is Sensitive to PKA and PKC Inhibition. Mustard oil (MO; 25 μM)-activated current was recorded from DRG sensory neurons. MO was applied for 30 sec. Vehicle (ddH2O), DHPG, H89 (PKA inhibitor) and GFX (PKC inhibitor) were applied at concentrations of 10 μM (each). DRG neurons were incubated for 10 min with drugs marked on X-axis, followed by co-application of MO. *p 
Figure 8
Figure 8
8-Br-cAMP-sensitization of TRPA1 Requires PKA-phosphorylation Site. Mustard oil (MO, 50 μM)-activated Ca2+ mobilization was monitored from DRG sensory neurons harvested from TRPA1 KO mice and transfected with cDNA corresponding to green fluorescent protein (eGFP-N1, GFP) and either pcDNA3 (empty vector, E.V., A), mouse TRPA1 WT (TRPA1, B), mouse TRPA1 S87A (S87A, C), mouse TRPA1 S179G (S179G, D), mouse TRPA1 S318A (S318A, E), or mouse TRPA1 S1101G (S1101G, F). For 5 min prior to recordings, DRG were pre-incubated for 5 min in vehicle (ddH2O) or 8-Br-cAMP (10 μM), then DRG were challenged with MO (30 sec) and Ca2+ accumulation was measured. Cumulative traces are displayed in panels A–F, quantitative measurements from multiple neurons displayed in panel G. *p < 0.05; **p < 0.01; ns = no significance; n = 15–39 MO responsive cells per TRPA1 cDNA transfection; trace significance determined by two-way ANOVA with Bonferroni posthoc analysis and quantified data significance determined by unpaired student’s t test.
Figure 9
Figure 9
mGluR5-sensitization of TRPA1 Requires PKA and PKC-phosphorylation Sites. Mustard oil (MO, 50 μM)-activated Ca2+ mobilization was monitored from DRG sensory neurons harvested from TRPA1 KO mice and transfected with cDNA corresponding to green fluorescent protein (eGFP-N1, GFP) and either mouse TRPA1 WT (TRPA1, A), mouse TRPA1 S87A (S87A, B), mouse TRPA1 S119G (S119G, C), mouse TRPA1 T281A (T281A, D), or mouse TRPA1 T529A (T529A, E). For 5 min prior to recordings, DRG were pre-incubated for 5 min in vehicle (ddH2O) or DHPG (10 μM), then DRG were challenged with MO (30 sec) and Ca2+ accumulation was measured. Cumulative traces are displayed in panels A–E, quantitative measurements from multiple neurons displayed in panel F. **p < 0.01; ns = no significance; n = 21–35 MO responsive cells per TRPA1 cDNA transfection; trace significance determined by two-way ANOVA with Bonferroni posthoc analysis and quantified data significance determined by unpaired student’s t test.
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