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. 2014 Jun 26:10:43.
doi: 10.1186/1744-8069-10-43.

Contribution of TRPC3 to store-operated calcium entry and inflammatory transductions in primary nociceptors

Hazim Alkhani, Ariel R Ase, Rebecca Grant, Dajan O'Donnell, Klaus Groschner, Philippe Séguéla  1
Affiliations

Contribution of TRPC3 to store-operated calcium entry and inflammatory transductions in primary nociceptors

Hazim Alkhani et al. Mol Pain. .

Abstract

Background: Prolonged intracellular calcium elevation contributes to sensitization of nociceptors and chronic pain in inflammatory conditions. The underlying molecular mechanisms remain unknown but store-operated calcium entry (SOCE) components participate in calcium homeostasis, potentially playing a significant role in chronic pain pathologies. Most G protein-coupled receptors activated by inflammatory mediators trigger calcium-dependent signaling pathways and stimulate SOCE in primary afferents. The aim of the present study was to investigate the role of TRPC3, a calcium-permeable non-selective cation channel coupled to phospholipase C and highly expressed in DRG, as a link between activation of pro-inflammatory metabotropic receptors and SOCE in nociceptive pathways.

Results: Using in situ hybridization, we determined that TRPC3 and TRPC1 constitute the major TRPC subunits expressed in adult rat DRG. TRPC3 was found localized exclusively in small and medium diameter sensory neurons. Heterologous overexpression of TRPC3 channel subunits in cultured primary DRG neurons evoked a significant increase of Gd3+-sensitive SOCE following thapsigargin-induced calcium store depletion. Conversely, using the same calcium add-back protocol, knockdown of endogenous TRPC3 with shRNA-mediated interference or pharmacological inhibition with the selective TRPC3 antagonist Pyr10 induced a substantial decrease of SOCE, indicating a significant role of TRPC3 in SOCE in DRG nociceptors. Activation of P2Y2 purinoceptors or PAR2 protease receptors triggered a strong increase in intracellular calcium in conditions of TRPC3 overexpression. Additionally, knockdown of native TRPC3 or its selective pharmacological blockade suppressed UTP- or PAR2 agonist-evoked calcium responses as well as sensitization of DRG neurons. These data show a robust link between activation of pro-inflammatory receptors and calcium homeostasis through TRPC3-containing channels operating both in receptor- and store-operated mode.

Conclusions: Our findings highlight a major contribution of TRPC3 to neuronal calcium homeostasis in somatosensory pathways based on the unique ability of these cation channels to engage in both SOCE and receptor-operated calcium influx. This is the first evidence for TRPC3 as a SOCE component in DRG neurons. The flexible role of TRPC3 in calcium signaling as well as its functional coupling to pro-inflammatory metabotropic receptors involved in peripheral sensitization makes it a potential target for therapeutic strategies in chronic pain conditions.

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Figures

Figure 1
Figure 1
TRPC family gene expression in sensory ganglia. (A) TRPC mRNA detection using a RT-PCR screen against TRPC1-7 in adult rat DRG. TRPC1 and TRPC3 are the major subunits expressed in DRG, along with low levels of TRPC6 and little or no signal for TRPC 2, 4, 5 and 7. (B) In situ expression profile of all TRPC family members at the whole DRG and spinal cord level. TRPC1 and TRPC3 are expressed at high levels in DRGs, with minimal detection in the spinal cord. Low but significant TRPC6 expression is also observed at the DRG level. (C) Emulsion staining with cellular resolution clearly shows that TRPC3 mRNA is localized in a subpopulation of small and medium diameter neurons (arrows) in DRG and trigeminal ganglia (TGG). Representative negative small-diameter and large-diameter neurons are also indicated (stars). Scale bar = 50 μm.
Figure 2
Figure 2
Contribution of TRPC channels to SOCE in DRG neurons. All traces are representative calcium responses recorded using the Fura-2 ratiometric method. (A) Thapsigargin (Thaps, 1 μM, 7 min) evokes a biphasic SOCE response in cultured rat DRG neurons (control). The first phase reflects the release of calcium from ER stores while the second phase reflects the influx of extracellular calcium, upon calcium re-addition to the perfusion solution (calcium add-back protocol), via Orai and/or TRPC channels. The addition of the Orai blocker Gd3+ (1 μM, 7 min) resulted in a significant decrease of calcium influx by 50%, suggesting a possible role for other channels including TRPCs in the remaining response (n = 8-19, P < 0.05). (B) SKF96365 (30 μM), a general SOCE and TRPC blocker, completely blocks SOCE (n = 14-23, P < 0.0005). (C) Effect of SKF96365 on TRPC3 activity. Activation of TRPC3 with the DAG analog OAG (50 μM, 3 min), is significantly inhibited by SKF96365 (approximately 75%), indicating a major role for TRPC3 in the TRPC component of the thapsigargin-induced SOCE response observed in DRG neurons (n = 15-18, P < 0.01).
Figure 3
Figure 3
TRPC3 participates in SOCE in DRG neurons. (A) TRPC3 shRNA-mediated knockdown was validated in a functional assay to determine the inhibitory effect of the shRNA on OAG-evoked calcium entry in DRG neurons (n = 9-11, P < 0.0001). (B) shRNA-mediated knockdown of TRPC3 in the SOCE assay clearly shows a strong contribution of this channel to the overall calcium influx. A decrease of approximately 42% is observed (n = 13-27, P < 0.05). (C) Heterologous overexpression of TRPC3 (OE) in DRG neurons produced a drastic increase in SOCE-mediated calcium influx of approximately 85% (n = 16-19, P < 0.01). (D) The inhibition of TRPC3 activity by the selective blocker Pyr10 (10 μM, 15 min) also resulted in a strong decrease of SOCE activity, with calcium influx dropping approximately 50% (n = 34-39, P < 0.01). The cumulative inhibition of both Orai and TRPC3 with Gd3+ and Pyr10, respectively, almost completely abolished SOCE response in DRG (n = 18-21, P < 0.001).
Figure 4
Figure 4
Functional coupling between TRPC3 and UTP/P2Y2 signaling in DRG neurons. (A) The addition of 100 μM UTP for 7 minutes to the calcium-free perfusion solution activates P2Y2 receptors, which initiate both SOCE and ROCE responses. The addition of Gd3+ removes the Orai component of the UTP-evoked response, which accounts for approximately 35% of the overall calcium influx (n = 17-19, P < 0.05). (B) The blockade of TRPC3 specifically with Pyr10 (10 μM, 15 min) resulted in a drastic decrease of UTP-evoked calcium entry, resulting in 60% inhibition (n = 25-28, P < 0.001). (C) shRNA-mediated knockdown of TRPC3 induced in similar decrease of P2Y2-mediated calcium entry (n = 34-46, P < 0.0001). (D) Heterologous overexpression of TRPC3 (OE) resulted in 90% increase of calcium influx in the UTP response, indicating a strong link between TRPC3 activity and P2Y2 transduction (n = 24-45, P < 0.001).
Figure 5
Figure 5
TRPC3 is linked to proteases/PAR2 transduction in DRG neurons. (A) Addition of 100 μM AC55541 to the calcium-free perfusion solution activates PAR2 receptors, which initiate both SOCE and ROCE responses. Treatment with Gd3+ removed the Orai component, which produced a small but non-significant decrease in calcium influx (n = 22-24, P > 0.05). (B) The selective inhibition of TRPC3 with Pyr10 (10 μM, 15 min) resulted in a drastic decrease (66%) of AC55541-evoked calcium entry (n = 19-29, P < 0.001). (C) shRNA-mediated knockdown of TRPC3 induced a similar decrease in PAR2-mediated calcium entry of approximately 66% (n = 24-73, P < 0.0001). (D) Heterologous overexpression of TRPC3 (OE) increased by 135% the calcium influx following PAR2 activation, indicating a strong link between TRPC3 activity and PAR2 function (n = 18-26, P < 0.0001).
Figure 6
Figure 6
TRPC3 is involved in peripheral sensitization induced by PAR2 and P2Y2 receptors. Intrinsic excitability of a representative DRG nociceptor recorded sequentially under current-clamp conditions (RMP = -67 mV). (A) Firing of action potentials was consistently triggered by current pulses (200 pA; 50 ms duration) injected at fixed time intervals (n = 25). No firing was detected in conditions of subthreshold current injection (50 pA; 50 ms duration) (B) unless the cells were first sensitized by application of both PAR2 agonist AC55541 (100 μM) and P2Y2 agonist UTP (100 μM) (n = 14/25) (C). (D) Treatment with the specific TRPC3 antagonist Pyr10 (10 μM) suppressed this sensitization effect in most neurons (n = 6/9). (E) At the end of each recording, cells were tested for viability and displayed normal firing activity after Pyr10 washout (n = 6).
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