Research ArticleNeuroscience

Peripheral pain is enhanced by insulin-like growth factor 1 through a G protein–mediated stimulation of T-type calcium channels

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Science Signaling  07 Oct 2014:
Vol. 7, Issue 346, pp. ra94
DOI: 10.1126/scisignal.2005283

Abstract

Insulin-like growth factor 1 (IGF-1) is implicated in the nociceptive (pain) sensitivity of primary afferent neurons. We found that the IGF-1 receptor (IGF-1R) functionally stimulated voltage-gated T-type Ca2+ (CaV3) channels in mouse dorsal root ganglia (DRG) neurons through a mechanism dependent on heterotrimeric G protein (heterotrimeric guanine nucleotide–binding protein) signaling. IGF-1 increased T-type channel currents in small-diameter DRG neurons in a manner dependent on IGF-1 concentration and IGF-1R but independent of phosphatidylinositol 3-kinase (PI3K). The intracellular subunit of IGF-1R coimmunoprecipitated with Gαo. Blocking G protein signaling by the intracellular application of guanosine diphosphate (GDP)-β-S or with pertussis toxin abolished the stimulatory effects of IGF-1. Antagonists of protein kinase Cα (PKCα), but not of PKCβ, abolished the IGF-1–induced T-type channel current increase. Application of IGF-1 increased membrane abundance of PKCα, and PKCα inhibition (either pharmacologically or genetically) abolished the increase in T-type channel currents stimulated by IGF-1. IGF-1 increased action potential firing in DRG neurons and increased the sensitivity of mice to both thermal and mechanical stimuli applied to the hindpaw, both of which were attenuated by intraplantar injection of a T-type channel inhibitor. Furthermore, inhibiting IGF-1R signaling or knocking down CaV3.2 or PKCα in DRG neurons abolished the increased mechanical and thermal sensitivity that mice exhibited under conditions modeling chronic hindpaw inflammation. Together, our results showed that IGF-1 enhances T-type channel currents through the activation of IGF-1R that is coupled to a G protein–dependent PKCα pathway, thereby increasing the excitability of DRG neurons and the sensitivity to pain.

INTRODUCTION

Insulin-like growth factor 1 (IGF-1) is a 70–amino acid polypeptide hormone produced primarily by the liver that acts as an endocrine growth factor in both paracrine and autocrine manners (1). IGF-1 binds with high affinity to the IGF-1 receptor (IGF-1R) and with lower affinity to the insulin receptor (IR) (2), and has been shown to participate in neurotrophy, neurogenesis, and metabolic and other modulatory functions in the nervous system (3). In vitro experiments also suggest that IGF-1 might determine the nociceptive sensitivity of primary afferent neurons (4, 5), which preferentially and abundantly express IGF-1Rs (6). Further, increased peripheral IGF-1 production sensitizes primary afferent neurons to facilitate pain hypersensitivity (7), whereas the application of IGF-1 produces pro-nociceptive effects (8). IGF-1–induced hyperalgesia in animal experiments is consistent with clinical trial findings that describe jaw pain after the administration of recombinant human IGF-1 (9). Even so, and despite the mounting evidence regarding IGF-1 hyperalgesic actions, the contributing underlying mechanisms are not well understood.

T-type Ca2+ channels (T-type channels), also referred to as low voltage–activated (LVA) Ca2+ channels, have the unique ability to become activated after a small depolarization of the cell membrane. This enables T-type channels to function close to the resting membrane potential and to influence neuronal excitability. Abnormal T-type channel expression has been implicated in pathological conditions, such as epilepsy (10) and neuropathic pain (11). Further, pharmacological (12), molecular (13), genetic (14), and functional (15) approaches all suggest that T-type channels can facilitate and amplify pain signals originating from the periphery. Several groups have described T-type channel currents in small dorsal root ganglion (DRG) neurons (~15- to 30-μm soma diameters) (16, 17), most of which are nociceptors (18). It has been suggested that T-type channels directly affect pain sensation by altering membrane excitability, and they are considered potential therapeutic targets for treating both acute and chronic pain (15, 19).

Here, we examined the effect of IGF-1 on T-type channel currents in small-diameter (<30 μm) DRG neurons from mice to investigate whether T-type channels participate in the reported hyperalgesic actions of IGF-1. Our findings suggest that IGF-1 increases T-type channel currents to enhance DRG excitability and contribute to mechanical and thermal hypersensitivity in an IGF-1R–dependent manner. The responses were mediated by the βγ subunit of Go proteins and downstream activation of the classic protein kinase Cα (PKCα) signaling pathway.

RESULTS

IGF-1 enhances T-type channel currents in DRG neurons

In vitro studies of nociceptive and other forms of sensory processing often examine distinct subtypes of DRG neurons (17). Here, we sorted adult mouse lumbar (L4–6) DRG neurons into groups on the basis of soma diameter [small diameter (<30 μm) and medium diameter (30 to 40 μm) (2022)] and restricted our electrophysiological experiments to cells that were less than 30 μm in soma diameter, because these neurons are primarily involved in nociceptive signaling (11, 12, 17). To isolate T-type channel currents, we recorded whole-cell barium currents elicited by a 40-ms depolarizing step pulse from the holding potential (−110 mV) to the command voltage of −40 mV after applying nifedipine (5 μM) to block L-type channels and ω-conotoxin MVIIC (0.2 μM) to block both N- and P/Q-type channels. Addition of NiCl2 inhibited the remaining inward current in a dose-dependent manner (Fig. 1A), confirming the isolation of T-type channel currents.

Fig. 1 IGF-1 increases T-type channel currents in small DRG neurons.

(A) Effect of NiCl2 (Ni2+) on T-type channel currents elicited by whole-cell patch-clamp recordings. A representative trace is shown alongside quantification of current density. Data are means ± SEM from five (10 μM) or seven (100 μM) cells. *P < 0.05, paired t test. (B and C) Time course and summary data show the effect of IGF-1 on T-type channel currents. Inset shows the current traces. Numbers in the plot indicate the points used for sample traces. Data are means ± SEM from nine cells. *P < 0.05, paired t test. (D) Dose-response curve for the stimulatory effects of IGF-1 on T-type channel currents. The line represents the best fit of the data points to the Hill equation. In brackets is the number of cells tested at each concentration of IGF-1. Ig, immunoglobulin. (E) Current-voltage (I-V) curve for the stimulatory effects of IGF-1 on T-type channel currents (n = 9 cells). (F to H) Effect of IGF-1 on T-type channel inactivation (F) and activation (G) properties (Vhalf). Insets show the stimulation waveform. Data (H) are means ± SEM from nine cells each. *P < 0.05, paired t test.

Bath application of IGF-1 (100 nM) increased the peak amplitude of T-type channel currents in small DRG neurons, and this amplitude only partially reversed within 3 min after IGF-1 was washed out (Fig. 1, B and C). Further examination of the IGF-1 effect demonstrated that IGF-1 increased T-type channel currents in a concentration-dependent manner (Fig. 1D). The relationship between the concentration of IGF-1 and the degree of inhibition is described by a sigmoidal Hill equation, whereby the concentration of IGF-1 that produced half-maximal inhibition (IC50) was 29.79 nM and with the apparent Hill coefficient of 0.91 (Fig. 1D). Examination of T-type channel current properties modified by IGF-1 stimulation showed that IGF-1 increased the current density (Fig. 1E). Other IGF-1–mediated properties examined for alterations included the voltage dependences of activation and inactivation. We observed a significant ~9 mV depolarizing shift in the steady-state inactivation profile of T-type channel currents, whereas we found no changes in activation properties (Fig. 1, F to H). These results suggest that a positive shift of the voltage dependence of inactivation may be one of the factors accounting for the IGF-1–mediated increase in T-type channel whole-cell currents in small DRG neurons, although further investigation is required to more fully understand the underlying biophysical mechanisms.

IGF-1R mediates the T-type channel current increase

The IR and IGF-1R subtypes have been identified as endogenous receptors for IGF-1 (3). We examined the extent to which IGF-1R and IR might participate in T-type channel responses to IGF-1 by first examining their respective protein abundance profiles in DRG neurons. Western blot analysis of DRG lysates revealed that both IRβ and IGF-1Rβ are endogenously expressed in DRGs (Fig. 2A). Because both receptors are found in the mouse brain, tissue lysates from the same mice were used as a positive control (Fig. 2A). The native profile was further confirmed with immunohistochemistry staining of mouse DRG sections. The results demonstrate that both IR and IGF-1R were localized to the membrane of neurons of intact DRGs with various diameters (fig. S1). We next determined the relative involvement of these receptor subtypes on the effect of IGF-1 on DRG T-type channel currents. Whereas currents were still increased by IGF-1 in the presence of S961, a specific IR antagonist, they were abolished by the application of JB-1, a selective IGF-1R antagonist (Fig. 2, B to D). Together, these data indicate that IGF-1R, but not IR, is involved in the IGF-1–mediated effects on T-type channel currents in small DRGs. To confirm this notion, we used small interfering RNA (siRNA)–mediated knockdown to attenuate IGF-1R expression in DRG neurons. Western blot analysis revealed that the abundance of IGF-1R was significantly reduced in cells transfected with IGF-1R–specific siRNA at 48 hours after transfection compared with cells transfected with control siRNA (fig. S2). Knockdown of IGF-1R in small DRG neurons resulted in a near-complete elimination of the IGF-1–mediated increase on T-type channel currents (Fig. 2E). Notably, there were no significant differences in T-type channel current density in cells treated with the control siRNA (Fig. 2E) compared with nontransfected control cells (Fig. 1C). These results support the conclusion that the IGF-1–mediated increase in T-type channel currents is mediated by IGF-1R in small DRG neurons. To determine whether IGF-1R signaling is altered under conditions of chronic inflammatory pain, we analyzed IGF-1R protein abundance after complete Freund’s adjuvant (CFA) injection into the paw. DRGs were extracted 2 days after CFA injection. The paw withdrawal threshold (PWT) at this point was at the bottom of the time curve (Fig. 2F), and CFA injection markedly increased IGF-1R abundance in mouse DRGs (Fig. 2G).

Fig. 2 IGF-1 increases T-type channel currents via IGF-1R.

(A) Detection of both IR and IGF-1R protein abundance. The blots shown are representative of three experiments. (B to D) Effect of 100 nM IGF-1 on T-type channel currents in the presence of S961 (B, 1 μM) or JB-1 (C, 1 μM). Insets show the current traces. Numbers in the plot indicate which points were used for sample traces. Data (D) are means ± SEM from nine cells each. *P < 0.05, paired t test. (E) Summary data show the effect of control siRNA (ctrl siRNA, n = 11 cells) or IGF-1R siRNA (n = 9 cells) on IGF-1–induced T-type channel current increase. #P < 0.05 versus ctrl siRNA, one-way analysis of variance (ANOVA). (F) Mechanical hypersensitivity induced by CFA injection. *P < 0.05, **P < 0.01 versus normal saline (NS), one-way ANOVA. (G) Abundance of IGF-1R protein in CFA-injected mice (n = 4 mice for each group). *P < 0.05 versus NS, unpaired t test. (H) Colocalization of IGF-1R (red) and CaV3.2 (green) with three markers (blue; NF-200, CGRP, and IB4) in naïve mouse DRGs. Arrows show the colocalization. Scale bar, 40 μm.

The CaV3.2 T-type channel isoform is the main LVA Ca2+ channel present in small peripheral sensory neurons and is implicated in nociceptive signaling (13, 17). To determine the types of neurons that might be sensitive to IGF-1R signaling, we performed costaining for IGF-1R and CaV3.2 on both small nociceptive neurons and larger myelinated neurons. Double staining in mouse intact DRGs indicated that IGF-1R was heavily colocalized with CaV3.2 channels: CaV3.2 was abundant in about 63% of all IGF-1R–positive neurons; notably, IGF-1R was present in 93% of all CaV3.2-positive neurons (Fig. 2H and fig. S3). Small unmyelinated DRGs can be classified on the basis of their peptide content into non-peptidergic neurons expressing isolectin B4 (IB4) antigen-positive and peptidergic calcitonin gene–related peptide (CGRP)–positive neurons. Both IGF-1R– and CaV3.2-positive DRG neuron populations contained subsets of IB4- and CGRP-positive neurons (Fig. 2H). In mouse intact DRGs, ~47% of IB4-positive neurons exhibited immunoreactivity for both IGF-1R and CaV3.2, and ~32% of CGRP-positive neurons were positive for both IGF-1R and CaV3.2 (Fig. 2H). Few neurons in which both IGF-1R and CaV3.2 were detected showed positivity for neurofilament 200 (NF200), a marker of myelinated A fibers (Fig. 2H).

IGF-1R–mediated T-type channel current increase requires Gαo

Activation of IGF-1Rs is typically associated with receptor autophosphorylation, recruitment, and phosphorylation of insulin receptor substrate 1 (IRS-1) and activation of phosphatidylinositol 3-kinase (PI3K) (23, 24). Hence, we examined whether PI3K-dependent activation of the kinase Akt contributes to the effects of IGF-1R on DRG T-type channel currents. We initially assayed the activity of Akt in cells treated with IGF-1 and found that phosphorylated Akt increased in response to IGF-1, whereas the total abundance of Akt was unchanged (Fig. 3A). This effect was abolished by both the IGF-1R–specific tyrosine kinase inhibitor tyrphostin AG1024 and the PI3K inhibitor LY294002 (Fig. 3A). To further explore the role PI3K plays in IGF-1 modulation of T-type channel currents, DRG neurons were preincubated for 30 min with AG1024 (Fig. 3B) or LY294002 (Fig. 3C) and then exposed to IGF-1. Neither AG1024 nor LY294002 affected the ability of IGF-1 to increase T-type channel currents (Fig. 3, B to D). These results indicate that IGF-1–induced T-type channel current responses do not occur through pathways typically associated with IGF-1R tyrosine kinase and are independent of downstream PI3K activation.

Fig. 3 IGF-1R–mediated T-type channel current increase requires Gαo proteins.

(A) Effect of 100 nM IGF-1 on phosphorylation of Akt (p-Akt) or total Akt (t-Akt) abundance in the presence of AG1024 (1 μM) or LY294002 (20 μM). Data are means ± SEM from three experiments. *P < 0.05, unpaired t test. (B to D) Effect of IGF-1 on T-type channel currents in the presence of AG1024 (B; 1 μM, n = 9 cells) or LY294002 (C; 20 μM, n = 8 cells). Insets show the current traces. Numbers in the plot indicate which points were used for sample traces. (E and F) Effect of IGF-1 on T-type channel currents in the presence of GDP-β-S (1 mM, n = 8 cells), cholera toxin (CTX) (0.5 μg/ml for 24 hours, n = 7 cells), or pertussis toxin (PTX) (0.2 μg/ml for 24 hours, n = 8 cells). *P < 0.05, paired t test. (G to I) Effect of IGF-1 on T-type channel currents in the presence of antibody against Gαo (anti-Gαo) (G; n = 6 cells) or antibody against Gαi (anti-Gαi) (H; n = 8 cells). **P < 0.01, paired t test. (J) Association of Gαo with the IGF-1Rβ. The blots shown are representative of three experiments.

It has been suggested that IGF-1R may also couple to heterotrimeric G proteins (heterotrimeric guanine nucleotide–binding proteins) to modulate cellular processes through an alternate signaling pathway (25). To investigate the potential involvement of heterotrimeric G proteins, we first dialyzed small DRG neurons with the nonhydrolyzable guanosine diphosphate (GDP) analog guanosine-5′-O-(2-thiodiphosphate) (GDP-β-S). GDP-β-S completely abolished the increase in T-type channel currents mediated by IGF-1 (Fig. 3, E and F), indicating that G protein activation is required for the effects of IGF-1 on T-type channels. We next examined different subtypes of G proteins for involvement in the IGF-1–mediated T-type channel current modulation. Inactivation of GαS by pretreating DRG neurons with cholera toxin did not affect the ability of IGF-1 to induce T-type channel currents (Fig. 3F), suggesting that IGF-1 effects are independent of GαS. Contrastingly, pretreating cells with pertussis toxin to inactivate Gαi/o abolished the stimulatory effect of IGF-1 (Fig. 3F). Additionally, dialysis into small DRG neurons of an antibody specific to Gαo [but not Gαi (26)] attenuated the effect of IGF-1 on T-type channel currents, whereas a Gαi-specific antibody had no significant effect (Fig. 3, G to I). Together, these results suggest that a Gαo, but not GαI, protein mediates the response to IGF-1R. To further test this notion, we examined whether IGF-1R interacted directly with Gαo using coimmunoprecipitation between Gαo protein and IGF-1Rβ, the intracellular subunit of IGF-1R. In lysates from mouse DRGs, endogenous Gαo, but not GαI, immunoprecipitated with an antibody against IGF-1Rβ (Fig. 3J). Together, these results imply that IGF-1Rβ and the Gαo subunit, but not the Gαi subunit, form a signaling complex in situ. The possibility of an indirect interaction involving one or more accessory binding proteins cannot, however, be excluded.

Gβγ-dependent PKC is required for the IGF-1R–mediated response

We further examined the potential role of the native βγ subunits (Gβγ) of Go protein in mediating the IGF-1R–induced increase in T-type channel currents. The synthetic peptide QEHA competitively binds Gβγ and blocks Gβγ-mediated signaling (27). Application of QEHA through the recording pipette blocked the IGF-1–mediated effect, whereas similar dialysis of a scrambled peptide (SKEE) did not alter the ability of IGF-1 to enhance DRG T-type channel currents (Fig. 4, A to C). This finding suggests that the βγ subunits of the Gαo complex together mediate the increase in IGF-1R T-type channel current. Previous studies have suggested the involvement of a downstream protein kinase A (PKA)–dependent regulatory contribution (28). However, preincubating DRG neurons with the PKA inhibitor KT-5720 did not alter the ability of IGF-1 to enhance T-type channel currents (Fig. 4, D and E). That KT-5720 was functional in this assay was evidenced by the observation that pretreating cells with KT-5720 abolished the ability of forskolin to increase PKA activity (Fig. 4F).

Fig. 4 The Gβγ-dependent PKC is required for the IGF-1R–mediated response.

(A to C) Effects of IGF-1 on T-type channel currents in the presence of QEHA (A; 50 μM, n = 9 cells) or SKEE (B; 50 μM, n = 7 cells). *P < 0.05, paired t test. (D and E) Effects of IGF-1 on T-type channel currents in the presence of KT-5720 (n = 9 cells). (F) Bar graph shows the effect of 20 μM forskolin on the activity of PKA with or without KT-5720 (1 μM). Data are means ± SEM from four separate experiments. #P < 0.05 versus vehicle, one-way ANOVA. (G) Effect of GF109203X (1 μM) on 100 nM IGF-1–induced response in the inactivation curve (n = 9 cells). (H to L) Effect of IGF-1 on T-type channel currents in the presence of GF109203X (H; 50 μM, n = 7 cells), bisindolylmaleimide V (I; 1 μM, n = 8 cells), Go6976 (J; 200 nM, n = 9 cells), BAPTA (K; 20 mM, n = 9 cells), or dn-BAPTA (L; 20 mM, n = 9 cells). *P < 0.05, paired t test. Insets show exemplary current traces. Numbers in the plot indicate which points were used for sample traces.

PKC activation has been shown to modulate T-type channel currents (29, 30) and can act as a downstream effector of Gβγ activation (31). Pretreating DRGs with an inhibitor of both nonclassical and classical PKC isoforms, GF109203X, prevented the IGF-1–induced depolarizing shift in the inactivation curve (Fig. 4G) and abolished the ability of IGF-1 to enhance T-type channel currents (Fig. 4H), whereas the inactive analog bisindolylmaleimide V had no effect (Fig. 4I). Similar attenuation of the IGF-1 response was noted for application of the classical PKC isoform antagonist, Go6976 (Fig. 4J). Compared to nonclassical PKC isoforms, activation of classic PKC isoforms requires cytoplasmic Ca2+. Intracellular dialysis of the fast Ca2+ chelator BAPTA [1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid], but not its inactive analog dn-BAPTA, completely abolished the IGF-1–mediated T-type channel current increase (Fig. 4, K and L), supporting the involvement of classic PKC isoforms in this process.

The PKCα pathway is involved in IGF-1–mediated T-type channel modulation

To define the PKC isoforms involved, we first investigated the protein abundance profiles of the classic PKC isoforms in mouse DRG neurons. Western blot analysis revealed that PKCα, PKCβ1, and PKCβ2 proteins were abundant in DRG neurons, whereas PKCγ was not detected (Fig. 5A). Lysates from the mouse spinal cord, in which all four PKC isoforms are present, were used as a positive control (Fig. 5A). Pretreating DRG neurons with the PKCβ antagonist LY333531 did not affect the stimulatory effects of IGF-1 on T-type channel currents, whereas pretreating cells with the PKCα and PKCγ inhibitor HBDDE abolished the IGF-1–mediated T-type channel current response (Fig. 5, B to D). To further confirm the specific PKCα-mediated pathway involved, we used siRNA to knock down PKCα in small DRG neurons. Western blot analysis revealed that the abundance of PKCα was significantly reduced in cells transfected with PKCα-specific siRNA compared with neurons transfected with control siRNA (fig. S4). Knockdown of PKCα in small DRG neurons almost completely eliminated the IGF-1R–mediated increase in T-type channel currents (Fig. 5E).

Fig. 5 IGF-1 increases T-type channel currents through the classic PKCα pathway.

(A) Western blot analysis of the classic PKC isoforms. The blots shown are representative of three independent experiments. (B to D) Time course and summary data show the effects of IGF-1 on T-type channel currents in the presence of LY333531 (B; 0.2 μM, n = 9 cells) or HBDDE (C; 1 μM, n = 7 cells). Insets show exemplary current traces. Numbers in the plot indicate which points were used for sample traces. *P < 0.05, paired t test. (E) Exemplary current traces and summary data show the effect of PKCα siRNA on 100 nM IGF-1–induced T-type channel current increase in small DRG neurons. T-type channel currents were recorded from DRG neurons transfected with control siRNA (ctrl siRNA, n = 8 cells) or PKCα siRNA (n = 9 cells) at 48 hours after transfection. #P < 0.05 versus ctrl siRNA, one-way ANOVA. (F) Effects of 15-min application of IGF-1 or vehicle (control) on membrane PKCα abundance. Data are means ± SEM of triplicate samples and are representative of three independent experiments. *P < 0.05, unpaired t test.

The cellular activation of PKCα is intimately related to its translocation and binding to the plasma membrane. Thus, to further support the results so far, we examined the translocation of PKCα from the cytosolic to the membrane fractions in DRG neurons after treatment with IGF-1. Western blot analysis showed an increase of membrane-bound PKCα and a decrease in the cytosolic fraction when IGF-1 was applied to cells (Fig. 5F). Together, the results suggest that IGF-1R–mediated IGF-1–evoked T-type channel current increase occurs through the classical PKCα pathway.

IGF-1 increases membrane excitability of small DRG neurons

T-type channels are well known to play crucial roles in regulating membrane excitability in many neuron types, including primary sensory neurons (10, 11). To define the functional implications of the T-type channel current increase induced by IGF-1, we examined whether IGF-1 affected membrane excitability in small DRG neurons. Application of IGF-1 did not affect whole currents through voltage-gated Na+ (NaV) channels in small DRG neurons (Fig. 6A). In an external solution containing 4-aminopyridine (4-AP) to block transient A-type K+ channels, nifedipine (L-type channel blocker), and ω-conotoxin MVIIC (N- and P/Q-type channel blocker), IGF-1 significantly increased action potential firing in response to a 1-s current injection compared to control (Fig. 6B). After washout, the pre–IGF-1 firing rate was partially restored (Fig. 6B). Other membrane properties of neuronal excitability, including resting membrane potential and input resistance, were not significantly changed by IGF-1 in small DRG neurons (Fig. 6C and table S1). To verify that IGF-1 induced hyperexcitability occurred because of increased T-type channel currents, NiCl2 was applied in the external solution and found to completely abolish hyperexcitability in small DRGs (Fig. 6D). These results suggest that IGF-1 acts to stimulate T-type channels and subsequently induce hyperexcitability in small DRGs.

Fig. 6 IGF-1 induces neuronal hyperexcitability in small DRG neurons.

(A) Time course and summary data show that IGF-1 had no effect on voltage-gated Na+ channel (NaV) currents (n = 7 cells). Inset shows representative current traces. Numbers in the plot indicate which points were used for sample traces. (B) Left panel: Representative traces of action potential firing at holding potential of −75 mV under control conditions, during exposure to 100 nM IGF-1, and after washout. Right panel: Summary data show the effect of IGF-1 (100 nM) on firing rate (n = 17 cells). *P < 0.05, paired t test. (C) IGF-1 (100 nM) did not significantly affect the resting membrane potential (RMP) and input resistance. (D) Representative current traces and summary data show that application of 10 μM NiCl2 (Ni2+) abolished 100 nM IGF-1–induced hyperexcitability (n = 9 cells). (E) Left panel: Traces are from a single IGF-1–sensitive mouse DRG neuron. Right panel: Threshold was +1.38 nA in control and +1.18 nA in the presence of 100 nM IGF-1 (n = 13 cells). *P < 0.05, paired t test. (F) Summary data show that 10 μM Ni2+ abolished 100 nM IGF-1–induced lower threshold (n = 9 cells).

To further test this hypothesis, a shorter current injection (1 ms) was applied to avoid contaminating the action potential waveform with the stimulus. Neurons were manually hyperpolarized to membrane potentials of −75 mV to maximize the number of T-type channels available for activation. With increasingly stronger current injections (+200 pA), the threshold current necessary to evoke an overshooting AP was determined for each neuron in the presence or absence of IGF-1. In 9 of 13 cells tested, IGF-1 lowered the excitability threshold. Among these, the threshold was decreased by an average of 230 pA in the presence of IGF-1 compared with control (Fig. 6E). Blockade of T-type channels with NiCl2 completely abolished the IGF-1–induced lower threshold for AP firing in the small DRGs (Fig. 6F).

T-type channels are involved in IGF-1–mediated pain hypersensitivity

To examine the functional significance of IGF-1R at the whole-animal level, we examined whether IGF-1 directly contributed to pain sensitivity in mice. Sensitivity to mechanical or thermal stimuli was assessed by paw withdrawal from von Frey hairs or heat (through a hot plate), respectively. Intraplantar injection of IGF-1 induced a marked hypersensitivity to acute mechanical stimulus and heat (Fig. 7A), which recovered after 3 hours. The IGF-1–induced hypersensitivity to thermal or mechanical pain was abolished by previous intraplantar injection with the IGF-1R antagonist JB-1 (Fig. 7B). We next examined whether PKCα was involved in IGF-1–induced pain hypersensitivity. Previous observations indicate that lumbar intrathecal injection of antisense oligonucleotides leads to high uptake into DRGs (13, 32). Mice were pretreated with PKCα siRNA by intrathecal administration. PKCα knockdown in lumbar DRGs resulted in a significant decrease in PKCα protein abundance compared with those transfected with control siRNA (Fig. 7C). Intrathecal delivery of PKCα siRNA blocked both the mechanical hypersensitivity and thermal hyperalgesia induced by IGF-1, whereas delivery of the control siRNA had no significant effect in either nociceptive behavioral tests (Fig. 7C).

Fig. 7 Involvement of peripheral IGF-1R activation in pain hypersensitivity.

(A and B) Pretreatment with JB-1, TTA-P2, or Z941 significantly attenuated 1 nmol IGF-1–induced mechanical hypersensitivity and thermal hyperalgesia. *P < 0.05, **P < 0.01, IGF-1 injection versus vehicle; +P < 0.05, ++P < 0.01 versus IGF-1 + TTA-P2 at 1 hour; #P < 0.05 versus IGF-1 + Z941 at 1 hour, one-way ANOVA. (C) PKCα siRNA blocked the mechanical hypersensitivity and thermal hyperalgesia induced by 1 nmol IGF-1. Insets show the PKCα siRNA application significantly decreases the abundance of PKCα protein. The blots shown are representative of three independent experiments. *P < 0.05 versus vehicle; @P < 0.05 versus ctrl siRNA, one-way ANOVA. (D) JB-1 significantly suppresses the mechanical hypersensitivity and thermal hyperalgesia in CFA-treated mice. The injection (JB-1 or vehicle) was indicated with an arrow. *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA. (E) The CaV3.2 siRNA application resulted in a significant decrease in CaV3.2 protein abundance. The blots shown are representative of three independent experiments. #P < 0.05, unpaired t test. (F) Knockdown of CaV3.2 attenuated JB-1–induced alleviation of mechanical hypersensitivity and thermal hyperalgesia. ++P < 0.01 versus CFA 2 days; **P < 0.01, ***P < 0.001 in CaV3.2 siRNA versus ctrl siRNA in CFA mice; #P < 0.05 at 1 hour after injection in control siRNA-treated CFA mice compared to 0-hour point, two-way ANOVA. For all animal behavior data, n = at least eight mice.

The involvement of T-type channels toward the IGF-1–induced hypersensitivity to pain was investigated using TTA-P2, a potent blocker of T-type channels. Intraplantar injection of TTA-P2 had no significant effect on mechanical PWT or thermal paw withdrawal latency (PWL) (Fig. 7B), which was consistent with observations of a previously published study (30). In contrast, intraplantar pretreatment with TTA-P2 significantly attenuated both thermal hyperalgesia and mechanical hypersensitivity (Fig. 7B) induced by IGF-1. Similar results were obtained with another potent small organic T-type channel blocker, Z941 (33). Intraplantar injection of Z941 had no significant effect on mechanical PWT or thermal PWL (Fig. 7B), but significantly attenuated the mechanical hypersensitivity and thermal hyperalgesia (Fig. 7B) induced by IGF-1. These results together suggest that T-type channels contribute to IGF-1R–mediated hypersensitivity to acute pain.

Next, we examined the role of IGF-1 signaling in chronic inflammatory pain. We showed that mice exhibited significant mechanical allodynia to CFA (the sensation of pain to a normally nonpainful stimulus) (Fig. 2F) with a concomitant increase in IGF-1R abundance in DRGs at the peak of allodynia (Fig. 2G). Thus, the effects of IGF-1R blockade on nociceptive responses in CFA-treated mice were investigated. Either JB-1 or the vehicle was administered, and pain behavioral phenotype was assessed on an hourly basis. In the CFA-induced inflamed paws, JB-1 significantly suppressed mechanical allodynia (Fig. 7D). Similarly, when thermal sensitivity was assessed, we observed significant alleviation of thermal hyperalgesia by JB-1 in CFA-treated mice (Fig. 7D), and the effect was sustained for 2 hours. These results together indicate that blockade of IGF-1R under chronic inflammatory pain conditions mediates protection to pain hypersensitivity.

To further validate the CaV3.2 T-type channel as an important cellular target for pain-alleviating effects of IGF-1 signaling in chronic inflammatory pain, we intrathecally administered siRNA specific for the CaV3.2 T-type channel (or a nonspecific control siRNA as a control). We first determined the temporal profile of siRNA uptake into the lumbar DRG using fluorescein isothiocyanate (FITC)–labeled CaV3.2 siRNA. Bilateral L5 and L6 DRGs were dissected after 4 days of twice-daily intrathecal injections of siRNA. Examining these sections by confocal microscopy, we found that CaV3.2 siRNA had penetrated L5 and L6 DRGs, indicating effective uptake of CaV3.2 siRNA (fig. S5), and resulted in a significant decrease in CaV3.2 T-type channel protein abundance in lumbar DRGs compared with control siRNA (Fig. 7E). In addition, CaV3.2 siRNA–injected, CFA-treated mice exhibited a significant decrease in mechanical and heat hypersensitivity as compared with controls (Fig. 7F). Sensitivity assessed hourly after administration of JB-1 showed that JB-1 had no significantly additive effect to that of CaV3.2 siRNA on mechanical or thermal sensitivity in CFA-treated mice (Fig. 7F). This suggests that JB-1 and CaV3.2 siRNA likely target molecules in the same cellular signaling pathway in vivo. In contrast, in mice that either received no siRNA or were injected with control siRNA, JB-1 similarly induced significant sensitivity to mechanical and heat stimuli in addition to that induced by CFA (Fig. 7F), further suggesting that the CaV3.2 T-type channel is coupled to the JB-1 molecular target (IGF-1R). These results together suggest that CaV3.2 is involved in IGF-1R–mediated pain hypersensitivity in CFA-induced inflammatory pain behaviors.

DISCUSSION

The present study demonstrates that IGF-1 plays a role in modulating T-type channel currents in small DRG neurons. Our findings suggest that the response is mediated by IGF-1R coupled to βγ subunits of Gαo proteins, leading to activation of PKCα. Physiologically, IGF-1 mediates hyperexcitability in DRG neurons and pain hypersensitivity in mice (Fig. 8).

Fig. 8 Illustration of the hypothesized mechanism of IGF-1R.

IGF-1 acting through the IGF-1R activates Gαo proteins and releases the Gβγ subunits. Activation of PKCα by the Gβγ of Gαo protein subsequently stimulates the T-type channel currents and induces neuronal hyperexcitability and pain hypersensitivity. Neither PKA nor PI3K, nor the direct interaction between Gβγ and T-type channels, is necessary for IGF-1R–mediated increase of T-type channel currents. Whether PKCα directly phosphorylates T-type channels or acts on an intermediate protein needs to be further determined.

IGF-1Rs are generally considered classical tyrosine kinase receptors that undergo autophosphorylation and initiate a number of signaling events including the PI3K-dependent Akt signaling cascade (24). Here, the PI3K inhibitor LY294002 inhibited IGF-1–induced Akt activation, but this had no effect on T-type channel currents, indicating that the PI3K-dependent Akt signaling pathway is not involved in the IGF-1–mediated actions. IGF-1Rs also participate in kinase-independent events (34) and can couple with heterotrimeric G proteins to modulate cellular processes (25, 35). We found that G proteins contribute to IGF-1R–dependent increases in T-type channel currents, evidenced by the observation that the nonselective G protein inhibitor GDP-β-S blocked the effect. Further, the βγ subunit of Gαo is necessary for the IGF-1R–mediated response because intracellular application of either a competing peptide (QEHA) or an antibody against Gαo abolished the IGF-1–induced increase in the T-type channel current. These results are consistent with previous findings. For example, pertussis toxin, which inhibits Gαi/o-sensitive signaling, represses the cellular effects of IGF-1R activation (24). An association between heterotrimeric Gαi and IGF-1R was demonstrated in another study, in which a discrete pool of Gβγ subunits were necessary for downstream signaling after receptor stimulation with IGF-1 (36). It should be noted, however, that in the present study, application of an antibody specific for Gαi had no effect on IGF-1 stimulation of T-type channel currents, suggesting that the response is Gαi-independent. Consistent with this notion, coimmunoprecipitation studies indicated that IGF-1R interacts directly with Gαo but not Gαi. Discrepancies between our findings and those of other studies might be explained by cell types that have different IGF-1R isoforms that couple to different G proteins. Gβγ subunits can activate PKA to modulate various targets, including T-type channels (29, 30). For example, application of PKA inducers [8-Br-cAMP (8-bromo-adenosine 3′,5′-cyclic monophosphate) or forskolin] mimics the effect of acetylcholine, which activates T-type channels, confirming that PKA activation can contribute to increasing T-type channel currents (37). Similarly, activation of the serotonin 5-HT7 (5-hydroxytryptamine 7) receptors increases T-type channel currents in rat adrenal glomerulosa cells, an effect that is prevented by the PKA inhibitor H89 (38). A similar transduction cascade was reconstituted exogenously in Xenopus oocytes (39). In contrast, T-type channel current inhibition by dopamine in retinal horizontal cell is prevented by PKA inhibitors (29). Similarly, T-type channel current inhibition by adrenaline in newt olfactory receptor cell is mimicked by 8-Br-cAMP and by the intracellular application of the catalytic subunit of PKA (29). Here, we found that the stimulatory effects of IGF-1 on T-type channel currents were independent of PKA, suggesting that mechanisms other than the PKA pathway are involved.

From our results, we propose that IGF-1 increases T-type channel currents in small DRG neurons through activation of the classic PKCα isoform. The activation of PKC is intimately related to its translocation and binding to the plasma membrane; at room temperature, translocation can rapidly occur. For example, C-type natriuretic peptide treatment leads to translocation of PKC to cell membrane in mouse DRG neurons at room temperature within 5 min (40). In central neurons, low-frequency stimulation that induces long-term depression in the CA1 region of hippocampal slices also causes conventional PKC isoforms to translocate from the cytosol to the membrane. Translocation is transient, lasting less than 15 min (41), and supports a similar potential type of involvement of PKCα in the IGF-1–mediated T-type channel response observed in DRGs. Studies examining the PKC-dependent modulation of T-type channel activity show conflicting results, in that T-type channel currents have been reported to be either increased or decreased. In small-diameter DRGs, we found that pretreatment with either prototypical or nonclassical PKC isoform inhibitors completely abolished the T-type channel current increase evoked by IGF-1. This is consistent with findings in ventricular myocytes, wherein PKC activation by phorbol myristate acetate (PMA) increases T-type channel currents, whereas an inactive analog had no effect (42). Similar results have been reproduced in Xenopus oocytes expressing recombinant T-type channels (43). In contrast, in rat pituitary GH3 cells, PMA and OAG (an analog of diacylglycerol, which activates PKC) significantly inhibited an Ni2+-resistant T-type channel current (29, 30). Similar results were reported for Ni2+-sensitive T-type channel currents in chicken DRG neurons (44) and rat hippocampal neurons (29). The differential modulation of T-type channel activity by PKC may depend on different parameters. First, the expression or activation of endogenous PKC isoforms is tissue-specific, and there exists remarkable heterogeneity across PKC-dependent signal transduction pathways including that for ion channel modulation (45, 46). Second, PKC modulation of T-type channels may involve PKC-interacting proteins as found for the CaV2.2 N-type channel (47). PKC-interacting proteins confer specificity on individual PKC isoforms by regulating their activity and cellular location, endowing isoforms with the ability to mediate specific cellular functions (48). Finally, cell-specific splice variants of T-type channels might lead to the activation of distinct signaling pathways, thereby resulting in different downstream responses (49, 50). Parenthetically, we cannot exclude the possibility that an intermediate protein phosphorylated by a different PKC isoform may be involved in the observed IGF-1–mediated response.

In most cell types, T-type channels act over a range of membrane potentials near the resting membrane potential (51). Proposed roles for T-type channels include promotion of Ca2+-dependent burst firing, elevation of Ca2+ entry boosting dendritic signals that contribute to pacemaker activity, seizure susceptibility, and pain transmission (52). Knockout and knockdown studies have firmly established a prominent role for the CaV3.2 T-type channel (13, 14) in amplifying nociceptive signals in the periphery and in contributing to central sensitization in the spinal dorsal horn (53). Recent evidence also suggests that modulation of peripheral CaV3.2 T-type channels influences somatic and visceral nociceptive inputs and that inhibition of T-type channel currents results in significant antinociception in a variety of animal neuropathic pain models (15, 39). Here, we found that—consistent with the IGF-1–induced increase in T-type channel current—activation of IGF-1R increased the excitability of DRG neurons and induced acute mechanical hypersensitivity and thermal hyperalgesia, which were abolished by T-type channel blockade. Furthermore, IGF-1R signaling blockade abolished the mechanical hypersensitivity and thermal hyperalgesia under conditions of chronic inflammatory pain. These effects were abolished by the selective knockdown of CaV3.2 T-type channels. Hence, it is reasonable to infer that the nociceptive effects of IGF-1R activation are mediated, at least in part, through PKCα-mediated stimulation of T-type channels. These findings are consistent with previous reports showing that application of IGF-1 can induce pronociceptive effects (7, 8). Antinociceptive effects of IGF-1 also have been reported (54). Although this discrepancy has yet to be explained, the analgesic effect of IGF-1 may involve central rather than peripheral actions of IGF-1, because increased IGF-1 production in peripheral tissues sensitizes primary afferent neurons and facilitates hypersensitivity to pain (7). Although the exact subtype of hyperalgesia induced by PKCα downstream of IGF-1R requires further investigation, previous reports indicate that IGF-1R can produce nociceptive effects (8). Examining different PKC inhibitors, Yajima et al. (55) reported that thermal hyperalgesia induced by CFA was not dependent on PKC activation. In contrast, using targeted gene knockout techniques in mice, Zhao et al. (56) have reported that deletion of PKCα enhanced neuropathic mechanical allodynia induced by spared nerve injury, which suggests that distinct modes of pain sensitivity and transmission are not necessarily stimulated by similar pathways involving PKC.

In summary, we present new insights underlying the effect of IGF-1 on T-type channel currents in small DRG neurons. We provide evidence that IGF-1 increases T-type channel currents through stimulation of IGF-1R and Gβγ-mediated activation of PKCα. This mechanism is proposed to underlie neuronal excitability induced by IGF-1 and suggests that the regulation of IGF-1R might be a potential therapeutic target for the clinical management of pain.

MATERIALS AND METHODS

Reagents

All drugs were obtained from Sigma unless otherwise indicated. LY333531 and ω-conotoxin MVIIC were obtained from Tocris Bioscience. The QEHA and SKEE peptides (27) were synthesized by GenScript Corp. Stock solutions of IGF-1, 4-AP, GDP-β-S, pertussis toxin, cholera toxin, BAPTA, dn-BAPTA, and ω-conotoxin MVIIC were prepared in distilled deionized water. Stock solutions of S961, JB-1, AG1024, LY294002, nifedipine, forskolin, GF109203X, bisindolylmaleimide V, Go6976, LY333531, Z941 (from T. P. Snutch), HBDDE, and KT-5720 were prepared in dimethyl sulfoxide (DMSO). Nifedipine stock solution was stored in the dark. The final concentration of DMSO in the bath solution was less than 0.01% and had no functional effects on T-type channel currents.

Preparation of DRG neurons

All animal care and handling conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and the study was approved by the Animal Care and Use Committee of Soochow University. Neurons were isolated from lumbar (L4–6) DRG of adult mice (ICR, 6 to 8 weeks of age) as described in our previous studies (57). Briefly, the freshly removed ganglia were minced and enzymatically digested first with collagenase I (Invitrogen) for 30 min (37°C) and then with trypsin (2.5 mg/ml) (Sigma) for 15 min (37°C). They were then gently triturated with a fire-polished pipette to obtain a suspension with single cells. In most experiments, acutely dissociated DRGs were used within 6 to 8 hours for whole-cell recordings. Most of the data were obtained from small-diameter neurons (<30 μm) without visible processes (17, 22). In siRNA transfection experiments, isolated cells were collected by centrifugation and suspended in minimum essential medium supplemented with fetal bovine serum (10%), B27 (2%), Glutamax (1%), penicillin (20 U/ml), and streptomycin (0.2 mg/ml). Dissociated neurons were plated on polylysine (0.1 mg/ml)–coated glass coverslips and kept in 95% air and 5% CO2 incubators at 37°C. DRGs were replated 48 hours after siRNA transfection for patch clamping.

Electrophysiological recordings

Whole-cell patch-clamp recordings were performed at room temperature (22° to 24°C). Recording pipettes (World Precision Instruments) had 3- to 5-megaohm resistance when filled with internal solution. pClamp 10.2 was used to acquire and analyze data. Neurons were recorded with a MultiClamp 700B amplifier (Molecular Devices). Signals were filtered at 1 kHz and digitized at 10 kHz. Series resistance (Rs) and capacitance (Cm) values were taken directly from readings of the amplifier after electronic subtraction of the capacitive transients. Series resistance was compensated to the maximum extent (at least 75%). Current traces were corrected using on-line P/6 trace subtraction. Multiple independently controlled glass syringes served as reservoirs for a gravity-driven local perfusion system. Solution exchange was accomplished by constant suction through a glass capillary tube placed at the opposite end of the recording chamber. The recording chamber was perfused with extracellular solution (0.5 ml/min) containing: 140 mM tetraethylammonium chloride (TEA-Cl), 5 mM BaCl2, 0.5 mM MgCl2, 5.5 mM glucose, 5 mM CsCl, and 10 mM Hepes, pH adjusted to 7.35 with TEA-OH. The pipette solution contained the following: 110 mM CsCl, 25 mM Hepes, 4 mM Mg-ATP (adenosine triphosphate), 0.3 mM Na2-GTP (guanosine triphosphate), and 10 mM EGTA, pH adjusted to 7.4 with CsOH. To isolate T-type channel currents, we recorded the barium currents elicited by a 40-ms-long depolarizing step pulse from the holding potential of −110 to −40 mV after bath application of 5 μM nifedipine (L-type Ca2+ channel blocker) and 0.2 μM ω-conotoxin MVIIC (N- and P/Q-type channel blocker) in the external solution. This protocol was used for isolation of T-type channel currents in all figures. For inactivation curve, T-type channel currents were evoked by 40-ms test pulse to −40 mV after the 3-s conditioning pulses ranging from −120 to −30 mV with +10 mV increments. In experiments in which neurons were dialyzed with compounds or peptides, recording pipettes had 2- to 3-megaohm resistance for intracellular application of compounds, and current measurements were started at least 5 min after breaking the patch. T-type channel currents were measured at their peak amplitude within the 40-ms test pulse and were used to determine the percentage of T-type channel current increase. Action potentials were recorded in current-clamp mode. For both current-clamp and Na+ current recordings, the patch pipette solution contained the following: 110 mM KCl, 10 mM NaCl, 2 mM EGTA, 25 mM Hepes, 4 mM Mg-ATP, and 0.3 mM Na2-GTP, pH adjusted to 7.3 with KOH (295 mosmol). The external solution contained 128 mM NaCl, 2 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 30 mM glucose, 25 mM Hepes, pH adjusted to 7.4 with NaOH (305 mosmol). Na+ currents were elicited by a 100-ms depolarizing step pulse from the holding potential of −60 to 0 mV. We added 5 mM 4-AP (A-type K+ channel blocker), 5 μM nifedipine (L-type Ca2+ channel blocker), and 0.2 μM ω-conotoxin MVIIC (N- and P/Q-type channel blocker) in the external solution during current-clamp recordings.

Western blot analysis

DRGs were homogenized in ice-cold lysis buffer [50 mM tris (pH 7.4), 150 mM NaCl, 1.5 mM MgCl2, 10% glycerol, 1% Triton X-100, 5 mM EGTA, leupeptin (0.5 μg/ml), 1 mm phenylmethylsulfonyl fluoride (PMSF), 1 mm Na3VO4, 10 mm NaF, and proteinase inhibitor mixture] and rotated at 4°C for 1 hour before the supernatant was extracted. Equivalent amounts of extracted proteins (20 μg) were separated by 7.5% SDS–polyacrylamide gel electrophoresis and electroblotted onto polyvinylidene difluoride membranes (Amersham Biosciences). The membranes were blocked with 5% skim milk in phosphate-buffered saline (PBS) for 1 hour at room temperature. Blotted proteins were probed with the following primary antibodies: antibody against IGF-1Rβ (rabbit, 1:500, Cell Signaling Technology), antibody against IRβ (rabbit, 1:1000, Cell Signaling Technology), antibody against PKCα (rabbit, 1:500, Santa Cruz Biotechnology), antibody against PKCβI (rabbit, 1:500, Santa Cruz Biotechnology), antibody against PKCβII (rabbit, 1:500, Santa Cruz Biotechnology), antibody against PKCγ (rabbit, 1:500, Santa Cruz Biotechnology), antibody against phospho-Akt (rabbit, 1:1000, Cell Signaling Technology), antibody against Akt (rabbit, 1:1000, Cell Signaling Technology), antibody against Gαo (mouse, 1:1000, Santa Cruz Biotechnology), antibody against Gαi (mouse, 1:1000, Santa Cruz Biotechnology), and antibody against CaV3.2 (mouse, 1:500, S55-10, Novus Biologicals). Secondary antibodies were goat anti-rabbit (1:5000, Santa Cruz Biotechnology) or goat anti-mouse (1:10,000; Santa Cruz Biotechnology) IgG (immunoglobulin G) horseradish peroxidase–conjugated antibodies. An antibody against GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (1:2000, Cell Signaling Technology) was used as an internal control for the concentration of proteins loaded. Chemiluminescent signals were generated using a SuperSignal West Pico trial kit (Pierce) and detected by exposure to x-ray film. Quantity software (Bio-Rad) was used for background subtraction and for quantification of immunoblotting data.

Coimmunoprecipitation

For coimmunoprecipitation, protein extracts from DRG were prepared as described above. Extract containing 400 to 500 μg of protein was incubated at 4°C for 3 hours with 3 μg of rabbit polyclonal antibody against IGF-1Rβ, followed by incubation with protein A–Sepharose beads (Amersham Biosciences) for 4 hours at 4°C. Immunoprecipitates were washed five times with tris-buffered saline containing 0.1% Triton X-100 and then analyzed by Western blotting. Immunoreactive proteins on membranes were developed as described above.

Subcellular fractionation

Cells were treated with 100 nM IGF-1 for 15 min. The cells were extracted with lysis buffer: 50 mM tris-HCl (pH 7.5), 5 mM EDTA, 2 mM EGTA, 1 mM PMSF, 1 mM DTT (dithiothreitol), 0.25 M sucrose, aprotinin (10 μg/ml), and leupeptin (10 μg/ml), and sonicated on ice (3 × 10-s cycles). The mixture was centrifuged for 10 min at 800g, and the supernatant was saved and centrifuged at 100,000g for 1 hour. The supernatant was taken as the cytosol fraction. The pellet was resuspended in lysis buffer plus 1% Triton X-100 and centrifuged as before. The supernatant was collected as the membrane fraction. Protein concentrations of the fractions were determined using Bio-Rad assay. Cells incubated with medium only were used as controls.

Immunohistochemistry

Mice were anesthetized with isoflurane and perfused through the ascending aorta with 0.9% saline, followed by 4% paraformaldehyde (PFA; Sigma-Aldrich) in PBS. The lumber DRGs (L4–6) were removed and postfixed in 4% PFA overnight at 4°C and then were transferred into 30% sucrose (in PBS) at 4°C for at least 24 hours. Samples were sectioned (7 μm) in a cryostat (CM 1950; Leica). To improve CaV3.2 and IGF-1Rβ immunostaining, slides were first processed for antigen retrieval in citrate sodium buffer (0.01 M citrate sodium, 0.05% Tween 20, pH 6.0) at 100°C in microwave for 10 min, and then washed three times with PBS at room temperature. The sections were blocked with 10% donkey serum in PBS for 1 hour at room temperature and then incubated overnight at 4°C with antibody against IGF-1Rβ (rabbit, 1:150, Cell Signaling Technology), antibody against CaV3.2 T-type channel α1H (goat, 1:150, N-18, Santa Cruz Biotechnology), antibody against NF200 (mouse, 1:4000, Sigma), or antibody against CGRP (guinea pig, 1:1000, Bachem). Sections were washed three times with PBS at room temperature, followed by Cy3, Cy5, or Alexa Fluor 488–conjugated secondary antibodies (donkey, 1:200, Jackson ImmunoResearch) or FITC-IB4 (1:200, Sigma) in PBS at room temperature for 2 hours. After sections were washed three times with PBS at room temperature, images were captured with an E80i Nikon confocal microscope.

siRNA transfection

Chemically synthesized IGF-1R siRNA (sequence: GAACCUUCGUCUCAUCUUAdTdT) and PKCα siRNA (sequence: 5′-ACAACCUGGACAGAGUGAAdTdT-3′) were purchased from Gene Pharm. The siRNA sequences were subjected to Basic Local Alignment Search Tool (BLAST) analysis to minimize any potential off-target effects. We also used negative-control siRNAs (for IGF-1R, sequence: CAGCUAUCUCACUUGUCAUdTdT; for PKCα, sequence: GACAGCAGAUGAACCUAGAdTdT), which were screened against the Expressed Sequence Tag GenBank database and had no homology to the vertebrate genome. DRG neurons were transfected with either 0.6 μg of 6-FAM–modified siRNA or negative control siRNA at a final concentration of 100 nM using the oligofectamine reagent per the manufacturer’s instructions (Invitrogen). Small neurons showing green fluorescence under an inverted fluorescence microscope (Ti-2000, Nikon) 48 hours after transfection were subjected to whole-cell patch-clamp analysis. siRNA efficacy on IGF-1R or PKCα expression was analyzed by Western blotting.

PKA kinase activity assay

DRG neurons were cultured in 12-well plates and were pretreated with either vehicle (0.1% DMSO) or KT-5720 for 15 min at 37°C followed by treatment with either vehicle (0.1% DMSO) or forskolin for 15 min. The cells were washed with ice-cold PBS and were placed on ice. Lysis buffer (200 μl) [20 mM Mops, 50 mM β-glycerolphosphate, 50 mM sodium fluoride, 1 mM sodium vanadate, 5 mM EGTA, 2 mM EDTA, 1% NP-40, 1 mM DTT, 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, leupeptin (10 μg/ml), and aprotinin (10 μg/ml)] was added. After 10 min of incubation on ice, cells were scraped off and transferred to microcentrifuge tubes. The cell lysates were centrifuged for 15 min, and aliquots of the supernatants containing 0.2 μg of protein were assayed for PKA activity using an enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer’s instructions (JC Biotech).

CFA-induced chronic inflammation

Chronic inflammation was induced with CFA, as previously described (58). Briefly, mice were gently anesthetized with oxygen (3%) and isoflurane (2%) inhalation, and then administered a unilateral 30 μl of intraplantar injection of CFA (0.5 mg/ml; Sigma) into the left hindpaws. Mice serving as the control group were injected with 30 μl of saline to the left hindpaws. Thermal and mechanical paw withdrawal latencies and thresholds were determined before and up to 2 weeks after CFA administration.

Behavioral studies

ICR mice were housed in cages with access to food and water ad libitum. Six- to 8-week-old mice weighing 20 to 25 g were used for this study. The experimental procedures in this study were approved by the Soochow University Animal Care and Use Committee. Mice were allowed to acclimate for at least 3 days before any behavioral tests. Behavioral tests began with a habituation period, in which mice were placed in Plexiglass cubicles for at least 1 hour. For testing mechanical sensitivity, the plantar surface of the hindpaw was stimulated with a series of von Frey hairs (0.02 to 2.56 g; Stoelting), and the PWT was determined using Dixon’s up-down method.

The nociceptive response to thermal stimulation was tested in a commercially available paw thermal stimulation system (IITC Life Science). Mice were placed in the chamber and given 30 min to accommodate. The latency for PWL was then measured. To prevent injury, the thermal source is automatically discontinued after 20 s if the mouse fails to withdraw its paw. All drugs were administered subcutaneously in a volume of 10 μl into the hindpaw plantar surface using a 25-μl Hamilton syringe with a 28-gauge needle. The bleb disappeared within 5 min after injection. In separate experiments, multiple injections were made in the same hindpaw, at similar sites with a bleb encompassing the same area. In some mice, the inhibitors (10 μl) were injected 5 min before IGF-1. In the control group, the vehicle solution did not affect the basal PWL. All solutions were pH-balanced to 7.4 to avoid skin irritation. No signs of skin inflammation, discoloration, or irritation were noted at the sites of injection with test compounds. siRNAs targeting mouse CaV3.2 or PKCα mRNA and respective nonselective control siRNAs were obtained from Thermo Scientific Dharmacon. siRNA (5 μg in 5 μl) was mixed with 9 μl of polyethyleneimine (PEI, 10 mM) (Fermentas) for 10 min at room temperature before being delivered. PEI was used as a delivery vehicle to prevent degradation and enhance cell membrane penetration of siRNAs (5961). The siRNA-PEI complexes were delivered by intrathecal administrations via direct transcutaneous injection between the L5 and L6 dorsal spinous processes. Mice were maintained in a surgical plane of anesthesia with isoflurane (2 to 3% in oxygen delivered via nose cone) throughout the injection procedure. This treatment was repeated every 12 hours for 4 days, for a total of eight injections. The protein abundance measurement was performed 10 hours after the last siRNA injection. This protocol was based on previous studies demonstrating an efficient knockdown of potassium channels (61) and T-type channels in sensory neurons in vivo (13).

Statistical analysis

All data are presented as means ± SEM. GraphPad Prism 5.0 was used for data plotting. A paired or two-sample t test was used when comparisons were restricted to two means. Treatment effects were statistically analyzed using one-way ANOVA followed by a post hoc Bonferroni multiple comparison test. Differences in values over time between the treatment groups were tested using two-way ANOVA. Error probabilities of P < 0.05 were considered statistically significant. Concentration-response curves were fitted by sigmoidal Hill equation I/Icontrol = 1/(1 + 10(log IC50−X)nH), where X is the decadic logarithm of the concentration used, IC50 is the concentration at which the half-maximum effect occurs, and nH is the Hill coefficient. Activation data were fitted by the following modified Boltzmann equation: G/Gmax = 1/{1 + exp[−(V1/2Vm)/k]}, where Gmax is the fitted maximal conductance, V1/2 is the membrane potential for half-activation, and k is the slope factor. Steady-state inactivation of T-type channel currents was fitted with the following negative Boltzmann equation: I/Imax = 1/{1 + exp[(V1/2Vm)/k]}, where Imax is the maximal current, V1/2 is the membrane potential for half-inactivation, and k is the slope factor.

SUPPLEMENTARY MATERIALS

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Fig. S1. Cell surface abundance of IR and IGF-1R in DRGs.

Fig. S2. Knockdown of IGF-R1 in DRGs.

Fig. S3. Colocalization of IGF-1R with CaV3.2 in mouse DRGs.

Fig. S4. Knockdown of PKCα in DRGs.

Fig. S5. Uptake of siRNA in DRGs.

Table S1. Membrane properties of small DRG neurons induced by IGF-1 in mice.

REFERENCES AND NOTES

Acknowledgments: We express our deep and sincere gratitude to D. Jiang, University of Ulm, for his helpful comments. Funding: This work was supported by the National Natural Science Foundation of China (nos. 31371122, 81171056, 31271258, 81322015, and 81200852), National Natural Science Foundation of China (NSFC)–National Center for Scientific Research (CNRS) Joint Program (no. 81311130114), Natural Science Funding of Jiangsu Province (BK2011293), Natural Science Funding for Colleges and Universities in Jiangsu Province (nos. 10KJB310013 and 12KJB320010), Scientific Research Foundation for the Returned Overseas Chinese Scholars of State Education Ministry (to J.T.), and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. Work in the laboratory of T.P.S. was support by a grant from the Canadian Institutes of Health Research (#10677) and by a Canada Research Chair in Neurobiology and Genomics–Biotechnology. Author contributions: Y.Z., W.Q., and J.T. conceived the project and designed and performed the experiments; Y.Z., H.W., and W.Q. performed the electrophysiological studies; Y.Z., X.L., and Y.-G.S. performed the immunohistochemistry; W.Q., S.G., and Z.Q. performed the Western blot analysis and behavior test; X.J. and J.T. analyzed and interpreted the data; T.P.S. provided Z941; and Y.-G.S., T.P.S., and J.T. wrote (and all authors edited) the manuscript. Competing interests: The authors declare that they have no competing interests.
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