The chemokine CCL2 increases Nav1.8 sodium channel activity in primary sensory neurons through a Gβγ-dependent mechanism

Abstract

Changes in function of voltage-gated sodium channels in nociceptive primary sensory neurons participate in the development of peripheral hyperexcitability that occurs in neuropathic and inflammatory chronic pain conditions. Among them, the tetrodotoxin-resistant (TTX-R) sodium channel Na(v)1.8, primarily expressed by small- and medium-sized dorsal root ganglion (DRG) neurons, substantially contributes to the upstroke of action potential in these neurons. Compelling evidence also revealed that the chemokine CCL2 plays a critical role in chronic pain facilitation via its binding to CCR2 receptors. In this study, we therefore investigated the effects of CCL2 on the density and kinetic properties of TTX-R Na(v)1.8 currents in acutely small/medium dissociated lumbar DRG neurons from naive adult rats. Whole-cell patch-clamp recordings demonstrated that CCL2 concentration-dependently increased TTX-resistant Na(v)1.8 current densities in both small- and medium-diameter sensory neurons. Incubation with CCL2 also shifted the activation and steady-state inactivation curves of Na(v)1.8 in a hyperpolarizing direction in small sensory neurons. No change in the activation and inactivation kinetics was, however, observed in medium-sized nociceptive neurons. Our electrophysiological recordings also demonstrated that the selective CCR2 antagonist INCB3344 [N-[2-[[(3S,4S)-1-E4-(1,3-benzodioxol-5-yl)-4-hydroxycyclohexyl]-4-ethoxy-3-pyrrolidinyl]amino]-2-oxoethyl]-3-(trifluoromethyl)benzamide] blocks the potentiation of Na(v)1.8 currents by CCL2 in a concentration-dependent manner. Furthermore, the enhancement in Na(v)1.8 currents was prevented by pretreatment with pertussis toxin (PTX) or gallein (a Gβγ inhibitor), indicating the involvement of Gβγ released from PTX-sensitive G(i/o)-proteins in the cross talk between CCR2 and Na(v)1.8. Together, our data clearly demonstrate that CCL2 may excite primary sensory neurons by acting on the biophysical properties of Na(v)1.8 currents via a CCR2/Gβγ-dependent mechanism.

Figure 1.

Cellular distribution of Na_v1.8 and CCR2 in rat DRG neurons. Immunofluorescence staining of CCR2 (green) (A, D) and Na_v1.8 (red) (B, E) on acutely dissociated primary afferent neurons. The merged images show dually labeled small- and medium-sized sensory neurons (yellow) (C, F). Note that CCR2 and Na_v1.8 are mainly detected at the periphery of the cell. Immunohistochemical labeling performed on DRG sections from naive rats (G–I). Numerous small- to medium-diameter DRG neurons display both Na_v1.8 and CCR2 immunoreactivities. Scale bars, 20 μm.

Figure 2.

Isolation of TTX-resistant Na_v1.8 currents in small-sized rat sensory neurons. Whole-cell voltage-clamp current traces of Na_v1.8 in small-diameter sensory neurons recorded following a 30 min incubation with 100 nm CCL2 in the absence (n = 11) or presence of INCB3344 (100 nm; n = 7), PTX (250 ng/ml; n = 9), or gallein (20 μm; n = 6). Representative I–V curves of currents are determined using the pulse protocol indicated in the inset.

Figure 3.

Na_v1.8 currents are increased in small sensory neurons following exposure to CCL2. A, Current–voltage relationships of Na_v1.8 were determined before (black circles) and after application of 10 nm (gray squares) or 100 nm (gray triangles) of CCL2. B, Peak Na_v1.8 current densities were significantly enhanced in the presence of 10 and 100 nm CCL2 (*p < 0.05 and ***p < 0.001, respectively, vs control; Student's t test; n = 6–13). Error bars indicate SEM. C, D, CCL2 shifted the activation (C) and steady-state inactivation (D) curves in a hyperpolarizing direction. The midpoint values of the activation (V_1/2act) and inactivation (V_1/2inact) curves are illustrated in Table 1.

Figure 4.

INCB3344 treatment blocks the changes in the biophysical properties of Na_v1.8 induced by CCL2. A, I–V curves of Na_v1.8 currents obtained from small rat DRG neurons. B, Histogram showing the effects of different concentrations of INCB3344 (1, 10, 100 nm) on the increased Na_v1.8 peak current induced by 100 nm CCL2 (***p < 0.001, CCL2 alone vs control; ^###p < 0.001, ^#p < 0.05, INCB3344 plus CCL2 vs CCL2 alone; Student's t test; n = 6–11; pound signs correspond to the values in the Table 1). Error bars indicate SEM. C, D, INCB3344 also significantly inhibits the leftward shift of the activation (C) (^##p < 0.01) and inactivation (D) (^###p < 0.001 compared with CCL2 alone) curves of Na_v1.8 current observed in the presence of 100 nm CCL2. The experimental conditions corresponding to control and 100 nm CCL2 have already been presented in Figure 3. Half-activation and half-inactivation potentials and slope factors are summarized in Table 1.

Figure 5.

Involvement of G_i/o-proteins in CCL2-induced potentiation of Na_v1.8 currents. A, Neuronal cAMP measurement following application of CCL2 in the presence or absence of PTX. CCL2-induced inhibition of adenylyl cyclase activity is blunted by PTX pretreatment (*p < 0.05, CCL2 alone vs control; ^#p < 0.05, PTX plus CCL2 vs CCL2 alone; one-way ANOVA followed by Dunnett's posttest, performed in triplicate). Error bars indicate SEM. B, C, I–V curves (B) and histogram (C) showing the action of CCL2 on Na_v1.8 currents (***p < 0.001 compared with Control; Student's t test) and the inhibition of this effect with PTX (250 ng/ml) pretreatment (^###p < 0.001 compared with CCL2 alone; Student's t test; n = 7–11). D, Preincubation with PTX also impedes the action of CCL2 on the normalized conductance (^##p < 0.01; G/G_max). Note that the experimental conditions corresponding to control and 100 nm CCL2 have been shown previously in Figure 3.

Figure 6.

Gβγ participates in CCL2-induced Na_v1.8 currents. A, B, I–V curves (A) and histogram (B) of Na_v1.8 currents obtained from small rat DRG neurons showing that pretreatment with the Gβγ inhibitor gallein (20 μm) prevents the effects of CCL2 on Na_v1.8 currents (***p < 0.001, CCL2 alone vs control; ^###p < 0.001 gallein plus CCL2 compared with CCL2 alone; Student's t test; n = 6–11). Error bars indicate SEM. C, Gallein also completely reverses the leftward shift of activation curve of Na_v1.8 observed in the presence of 100 nm CCL2 (^###p < 0.001). Note that the experimental conditions corresponding to control and 100 nm CCL2 have been shown previously in previous figures. Half-activation and half-inactivation potentials and slope factors are summarized in Table 1.

Figure 7.

Representative families of Na_v1.8 sodium currents in medium-sized sensory neurons. Average I–V curve family of currents recorded from medium neurons under CCL2 stimulation alone (100 nm; n = 11), and in the presence of INCB3344 (100 nm; n = 7), PTX (250 ng/ml; n = 5), or gallein (20 μm; n = 11). INCB3344, PTX, and gallein all efficiently reverse CCL2-induced enhancement of Na_v1.8 currents.

Figure 8.

Density and kinetic properties of Na_v1.8 currents in medium-sized sensory neurons. A, B, CCL2 increases the maximal peak current amplitude of Na_v1.8 (***p < 0.001, CCL2 alone vs control). The activation of Na_v1.8 currents induced by CCL2 is inhibited after treatment with INCB3344 (^###p < 0.001 compared with CCL2 alone; Student's t test). C, No change in the steady-state inactivation curve of Na_v1.8 was seen following CCL2 treatment (n = 6–11). D, E, Both PTX and gallein reverse CCL2-induced Na_v1.8 current activation (^#p < 0.05 and ^###p < 0.001 compared with CCL2 alone, respectively). Half-maximal potentials (V_1/2) and slope (k) values are reported in Table 2. Error bars indicate SEM.