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. 2008 May 7;28(19):4904-17.
doi: 10.1523/JNEUROSCI.0233-08.2008.

Protein kinase A anchoring via AKAP150 is essential for TRPV1 modulation by forskolin and prostaglandin E2 in mouse sensory neurons

Katrin Schnizler  1 , Leonid P Shutov, Michael J Van Kanegan, Michelle A Merrill, Blake Nichols, G Stanley McKnight, Stefan Strack, Johannes W Hell, Yuriy M Usachev
Affiliations

Protein kinase A anchoring via AKAP150 is essential for TRPV1 modulation by forskolin and prostaglandin E2 in mouse sensory neurons

Katrin Schnizler et al. J Neurosci. .

Abstract

Phosphorylation-dependent modulation of the vanilloid receptor TRPV1 is one of the key mechanisms mediating the hyperalgesic effects of inflammatory mediators, such as prostaglandin E(2) (PGE(2)). However, little is known about the molecular organization of the TRPV1 phosphorylation complex and specifically about scaffolding proteins that position the protein kinase A (PKA) holoenzyme proximal to TRPV1 for effective and selective regulation of the receptor. Here, we demonstrate the critical role of the A-kinase anchoring protein AKAP150 in PKA-dependent modulation of TRPV1 function in adult mouse dorsal root ganglion (DRG) neurons. We found that AKAP150 is expressed in approximately 80% of TRPV1-positive DRG neurons and is coimmunoprecipitated with the capsaicin receptor. In functional studies, PKA stimulation with forskolin markedly reduced desensitization of TRPV1. This effect was blocked by the PKA selective inhibitors KT5720 [(9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylicacid hexyl ester] and H89 (N-[2-(p-bromo-cinnamylamino)-ethyl]-5-isoquinoline-sulfon-amide 2HCl), as well as by the AKAP inhibitory peptide Ht31. Similarly, PGE(2) decreased TRPV1 desensitization in a manner sensitive to the PKA inhibitor KT5720. Both the forskolin and PGE(2) effects were strongly impaired in DRG neurons from knock-in mice that express a mutant AKAP150 lacking the PKA-binding domain (Delta36 mice). Protein kinase C-dependent sensitization of TRPV1 remained intact in Delta36 mice. The PGE(2)/PKA signaling defect in DRG neurons from Delta36 mice was rescued by overexpressing the full-length human ortholog of AKAP150 in these cells. In behavioral testing, PGE(2)-induced thermal hyperalgesia was significantly diminished in Delta36 mice. Together, these data suggest that PKA anchoring by AKAP150 is essential for the enhancement of TRPV1 function by activation of the PGE(2)/PKA signaling pathway.

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Figures

Figure 1.
Figure 1.
AKAP150 is associated with TRPV1 in DRG neurons. A, DRG neurons express multiple AKAPs. Western blots of lysates of adult mouse brain, spinal cord, and DRG tissue were probed with antibodies against Yotiao, AKAP250, AKAP150, AKAP15/18, and α-tubulin, respectively (3–5 independent experiments, 12–20 animals). B, AKAP150–TRPV1 complexes were isolated from adult mouse spinal cord (top) and DRG (bottom) membrane fractions. Immunoprecipitations (IP) were performed with an antibody against TRPV1 or control IgG. Immunoprecipitates were separated by SDS-PAGE and probed with antibodies against Yotiao, AKAP250, AKAP150, AKAP15/18, and TRPV1, respectively (spinal cord: 5 independent experiments, 27 animals; DRG: 9 independent experiments, 40 animals). C, Histogram shows size (cross-sectional area of the cell body) distribution of AKAP150-positive DRG neurons relative to all neurons. D, Immunostaining shows subcellular distribution of TRPV1 (left) and AKAP150 (right) in a DRG neuron expressing both proteins. E, Pie chart shows the proportion of DRG neurons expressing AKAP150 (red), TRPV1 (green), both proteins (yellow), or none (white) in culture.
Figure 2.
Figure 2.
Time dependence of TRPV1 recovery from desensitization in a paired-pulse stimulation protocol. A–C, [Ca2+]i transients were evoked by two applications of capsaicin (100 nm for 10 s; indicated by arrowheads), which were separated by 3 min (A), 30 min (B), or 60 min (C). D, Plot of the response ratio (Amplitude2/Amplitude1) as a function of the time interval between two capsaicin applications. Data are presented as mean ± SEM and were obtained from 13–51 cells from 4–14 animals for each time point. Data points were fitted with a single-exponential function (smooth curve) using a nonlinear, least-squares curve fitting algorithm (Origin 7 software).
Figure 3.
Figure 3.
Forskolin reduces TRPV1 desensitization in a PKA- and AKAP-dependent manner. A–F, [Ca2+]i changes in DRG neurons were evoked by two subsequent capsaicin applications (100 nm for 10 s, 3 min between the applications indicated by arrowheads) under control conditions (HH buffer; A) and during treatments with 10 μm forskolin (FSK; B), 10 μm forskolin and 10 μm H89 (C), 10 μm forskolin and 1 μm KT5720 (D), 10 μm forskolin and 50 μm Ht31 (E), and 10 μm forskolin and 50 μm Ht31P (F). Treatments with H89, KT5720, Ht31, and Ht31P started 20 min before the first capsaicin application and were continued throughout the experiments as indicated by the horizontal bars. G, Bar graph summarizes the effects of forskolin (10 μm; FSK), the PKA inhibitors H89 (10 μm) and KT5720 (1 μm), the Epac activator CPTOMe (10 μm), the PLC inhibitor U73122 (1 μm), and AKAP inhibitory peptides (Ht31 or Ht31P at 50 μm) on TRPV1 desensitization in DRG neurons from WT mice. Data were obtained from experiments such as those shown in A–F and are presented as mean ± SEM. *p < 0.05 and ***p < 0.001 relative to control; #p < 0.05 and ##p < 0.01 relative to forskolin treatment alone; one-way ANOVA with Bonferroni's post hoc test.
Figure 4.
Figure 4.
Disruption of PKA binding to AKAP150 inhibits TRPV1 modulation by forskolin. A, Diagram shows primary structure of AKAP150 with binding sites for PKC, calcineurin (CaN), and PKA (modified from Dell'Acqua et al., 2006). In the truncated version of AKAP150 (AKAP150-Δ36), the last 36 amino acids of the C terminus are removed to disrupt PKA binding. B, PKA RIIα associates with AKAP150 in WT but not in Δ36 mice. AKAP150 was immunoprecipitated from Triton X-100 solubilized spinal cord membranes with an anti-AKAP150 antibody, and immunoprecipitates were analyzed by immunoblotting with an antibody against PKA RIIα (4 independent experiments, 24 animals from each strain). C, PKA RIIα coimmunoprecipitates with TRPV1 from adult mouse spinal cord in WT but not AKAP150-Δ36 mice. Immunoprecipitations were performed with an antibody against TRPV1 or nonimmune control antibodies, separated by SDS-PAGE, and probed with antibodies against AKAP150 and the regulatory subunit of PKA RIIα (5 independent experiments, 27 animals from each strain). D, E, [Ca2+]i elevations elicited by two stimulations with capsaicin (100 nm for 10 s, 3 min between the stimulations indicated by arrowheads) were recorded in DRG neurons from Δ36 mice. Between capsaicin applications, cells were superfused with either control HH buffer (D) or HH buffer containing 10 μm forskolin (FSK, horizontal bar) (E). F, Bar graph shows that the ability of forskolin to reduce TRPV1 desensitization was significantly impaired in DRG neurons from Δ36 mice. Response ratios were obtained from experiments such as those shown in D and E and are presented as mean ± SEM. For comparison between DRG neurons from WT (white bars) and Δ36 (gray bars) mice, data for WT/control and WT/forskolin conditions were replotted from Figure 3G. **p < 0.01, one-way ANOVA with Bonferroni's post hoc test.
Figure 5.
Figure 5.
Sensitization of TRPV1 via PKC is not impaired in DRG neurons from Δ36 mice. A, B, [Ca2+]i responses in DRG neurons from WT mice (A) and Δ36 mice (B) were evoked by two applications of capsaicin (arrowheads; 100 nm for 10 s, 3 min apart). Cells were superfused with 500 nm PdBu (horizontal bar) between the capsaicin applications to induce PKC activation. C, [Ca2+]i recording in a DRG neuron (Δ36 mice) that did not initially respond to 100 nm capsaicin (10 s) application but became responsive to capsaicin after PKC stimulation. Similar sensitizing effects of PdBu on capsaicin-induced [Ca2+]i changes were observed in 4 of 121 DRG neurons from WT mice and in 14 of 169 DRG neurons from Δ36 mice.
Figure 6.
Figure 6.
PGE2 reduces desensitization of TRPV1 via AKAP150-anchored PKA. A–D, Representative [Ca2+]i responses evoked by two applications of capsaicin (arrowheads; 100 nm for 10 s, 3 min apart) in DRG neurons treated with 5 μm PGE2 alone (A, B) or with 5 μm PGE2 and 1 μm KT5720 (C, D). [Ca2+]i changes are compared between DRG neurons from WT mice (A, C) and those from Δ36 mice (B, D). Applications of PGE2 (horizontal black bar) and KT5720 (horizontal white bar) are shown under the traces. Incubation with 1 μm KT5720 started 20 min before the first capsaicin application. E, Bar graph summarizes the effects of PGE2 and KT5720 on TRPV1-mediated [Ca2+]i responses in DRG neurons from WT (white bars) and Δ36 (gray bars) mice. Data were obtained from experiments such as those shown in A–D and are presented as mean ± SEM. Data for WT/control and Δ36/control were replotted from Figures 3G and 4E, respectively, to enable comparison of the PGE2 effects in both strains of animals. **p < 0.01, relative to WT/control; #p < 0.05 and ##p < 0.01 relative to Δ36/PGE2; one-way ANOVA with Bonferroni's post hoc test.
Figure 7.
Figure 7.
PGE2 reduces desensitization of capsaicin-evoked currents in DRG neurons from WT but not from Δ36 mice. A–D, Representative patch-clamp recordings (Vhold of −60 mV) of capsaicin-induced (1 μm, 10 s) currents from WT (A, C) and Δ36 (B, D) DRG neurons that were exposed to the control solution (A, B) or solution containing 5 μm PGE2 (C, D; black bars) between two capsaicin applications. E, Bar graph quantifies reduction of desensitization of capsaicin-evoked currents by PGE2 and the effect of the PKA inhibitor KT5720 (1 μm) in DRG neurons from WT and Δ36 mice. Data were obtained from experiments like those described in A–D. Cells were incubated with KT5720 for 3 min before the first capsaicin exposure. **p < 0.01, ***p < 0.001, one-way ANOVA with Bonferroni's post hoc test.
Figure 8.
Figure 8.
AKAP79 rescues the PGE2 effect in DRG neurons from Δ36 mice. A–D, DRG neurons from Δ36 mice were transfected with EYFP (A), AKAP79–EYFP (B), D-AKAP2–EGFP (C), or AKAP15/18–EGFP (D). Images show fluorescence distribution in transfected neurons (λex = 475 nm, λem = 530 nm; scale bars, 10 μm). Transfected DRG neurons were loaded with fura-2 and [Ca2+]i transients were evoked by two subsequent applications of capsaicin (arrowheads; 100 nm for 10 s, 3 min apart). Treatments with 5 μm PGE2 are indicated by horizontal bars under the traces. E, Bar graph summarizes the effects of PGE2 on TRPV1 in DRG neurons from WT and Δ36 mice transfected with either EYFP or plasmids encoding different AKAPs. Data were obtained from experiments such as those shown in A–D and are presented as mean ± SEM. The bars represent 21 cells/15 animals for WT/EYFP/control, 47 cells/15 animals for WT/EYFP/PGE2, 31 cells/7 animals for Δ36/EYFP/PGE2, 18 cells/11 animals for Δ36/AKAP79/PGE2, 14 cells/4 animals for Δ36/D-AKAP1/PGE2, 9 cells/6 animals for Δ36/D-AKAP2/PGE2, 21 cells/10 animals for Δ36/AKAP15/18/PGE2, and 9 cells/5 animals for Δ36/AKAP250/PGE2. **p < 0.01, one-way ANOVA with Bonferroni's post hoc test.
Figure 9.
Figure 9.
PGE2-induced enhancement of thermal sensitivity is impaired in Δ36 mice. A, Representative recording of heat-activated currents (top trace) induced by a transient increase of extracellular temperature (bottom trace). This specific recording was obtained from a WT DRG neuron voltage clamped at −60 mV (also shown in B). B–E, Heat-activated currents were induced as described in A and plotted as a function of extracellular temperature for DRG neurons from WT (B, C) and Δ36 mice (D, E) under control conditions (B, D) or in the presence of 5 μm PGE2 (3 min pretreatment; C, E). Gray vertical arrows indicate threshold temperature for each plot, which was defined as the intersection between the lines (gray dotted lines) approximating the baseline and the clearly increasing temperature-dependent inward current (Sugiura et al., 2002, 2004). F, Bar graph summarizes temperature thresholds of heat-activated currents in DRG neurons from WT (white bars) and Δ36 (gray bars) mice, recorded under control conditions and after treatment with 5 μm PGE2 alone or in combination with either 1 μm KT5720 (PGE2/KT) or 20 μm Ht31 (PGE2/Ht). Ht31 was included in the pipette solution, and KT5720 was applied for 6 min before the recordings. **p < 0.01, ***p < 0.001, ANOVA with Bonferroni's post hoc test. G, PGE2-induced thermal hyperalgesia was studied in WT and Δ36 mice. PGE2 was injected into plantar surface of the hindpaw (100 ng in 10 μl) of WT (n = 16; square) or Δ36 (n = 11; triangle) mice or a mixture of PGE2 plus Ht31 (100 ng + 8 μg in 10 μl) was injected into plantar surface of a WT mouse hindpaw (n = 15; circle). Paw-withdrawal latency to radiant heat was measured at various times after the injection. The baseline latency for each group (time = 0 min) was determined before the injection by averaging the results of three tests separated by a 5 min interval. *p < 0.05, relative to WT/Ht31; #p < 0.05, relative to Δ36; ANOVA with Bonferroni's post hoc test.
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