CaV3.2 T-type calcium channels contribute to CGRP- induced allodynia in a rodent model of experimental migraine

Abstract

Background: Migraine is a painful neurological syndrome characterized by attacks of throbbing headache, of moderate to severe intensity, which is associated with photo- and phono- sensitivity as well as nausea and vomiting. It affects about 15% of the world's population being 2-3 times more prevalent in females. The calcitonin gene-related peptide (CGRP) is a key mediator in the pathophysiology of migraine, and a significant advance in the field has been the development of anti-CGRP therapies. The trigeminal ganglion (TG) is thought to be an important site of action for these drugs. Moreover, experimental migraine can be induced by CGRP injection in the TG. The signaling pathway induced by CGRP in the TG is not fully understood, but studies suggest that voltage-gated calcium channels contribute to CGRP effects relevant to migraine.

Objective: We hypothesised that CGRP injection in the TG enhances Ca_V3.2 T-type calcium channel currents to contribute to the development of periorbital mechanical allodynia.

Results: A Co-Immunoprecipitation assay in tsA-201 cells revealed that Ca_V3.2 channels form a complex with RAMP-1, a component of the CGRP receptor. Constitutive CGRPR activity was able to inhibit Ca_V3.2 channels and induce a depolarizing shift in both activation and inactivation curves. Incubation of TG neurons with CGRP increased T-type current density by ~ 3.6 fold, an effect that was not observed in TG neurons from Ca_V3.2 knockout mice. Incubation of TG neurons with Z944, a pan T-type channel blocker, resulted in an approximately 80% inhibition of T-type currents. In vivo, this treatment abolished the development of periorbital mechanical allodynia induced by CGRP in male and female mice. Likewise, Ca_V3.2 knockout mice did not develop periorbital mechanical allodynia after intraganglionic CGRP injection. Finally, we demonstrated that the CGRP effect depends on the activation of its canonical GPCR, followed by protein kinase A activation.

Conclusion: The present study suggests that CGRP modulates Ca_V3.2 in the TG, an effect possibly mediated by the canonical CGRP receptor and PKA activation. The increase in T-type currents in the TG may represent a contributing factor for the initiation and maintenance of the headache pain during migraine.

Keywords: Calcitonin gene-related peptide; Electrophysiology; Mice; Periorbital mechanical allodynia; RAMP-1; Trigeminal ganglion; Voltage-gated calcium channels.

Fig. 1

Ca_V3.2 co-immunoprecipitates with a component of the CGRP receptor. (A) Protein from tsA-201 cells expressing Ca_V3.2 and CLR + RAMP-1-FLAG was immunoprecipitated using a FLAG antibody followed by western blot analysis with a Ca_V3.2 antibody. (B) Input: Expression of Ca_V3.2 at lysates. (C) Protein from tsA-201 cells expressing Ca_V3.2 and RAMP-1-FLAG was immunoprecipitated using a Ca_V3.2 antibody followed by western blot analysis with a FLAG antibody. (D) Input: Expression of RAMP-1-FLAG at lysates. These experiments were repeated 3 times with identical results

Fig. 2

Effect of CGRPR in the Ca_V3.2 channel biophysical properties. (A) Current density-voltage (IV) relation curves of cells expressing hCa_V3.2 or co-expressed with hCGRPR. (B) Average peak current density from cells cells expressing hCa_V3.2 or co-expressed with hCGRPR. (C) Voltage dependence of activation curves from cells expressing hCa_V3.2 or co-expressed with hCGRPR. Activation curves were constructed based on the parameters obtained from the IV curves. (D) Average voltage for half activation from cells expressing hCa_V3.2 or co-expressed with hCGRPR. (E) Steady-state inactivation curves from cells expressing hCa_V3.2 or co-expressed with hCGRPR. (F) Average voltage for half inactivation from cells expressing hCa_V3.2 or co-expressed with hCGRPR. Data is expressed as mean ± SEM, two-tailed Student’s t test *p < 0.05 and ****p < 0.0001 vs. Ca_V3.2

Fig. 3

CGRP receptor modulates T-type currents in trigeminal ganglion neurons. (A) T-type calcium channels current density in trigeminal ganglion neurons from wild-type (WT) and Ca_V3.2^−/− mice treated with vehicle (water) or 1µM rat CGRP. (B) Acute treatment with 10 µM Z944 on T-type calcium channels current in TG neurons treated with vehicle (water) or 1 µM rat CGRP. Data is expressed as mean ± SEM, two-tailed Student’s t test. **p < 0.01 vs. Vehicle, ##p < 0.01 vs. WT

Fig. 4

Z944 blocks periorbital mechanical allodynia induced by CGRP in male and female mice. Mice were treated with Z944 (10 mg/kg; i.p.) and fifteen minutes later received an intraganglionar injection of CGRP (0.1 nmol/5 μL; i.g.). Control groups received the corresponding vehicles. Periorbital mechanical allodynia was assessed in male mice (A) and in female mice (B), before the treatments (BL) and after CGRP injection. Data is expressed as mean ± SEM (n = 10). Two-way ANOVA with repeated measures followed by the Bonferroni post hoc test. *p < 0.05, compared to Vehicle (VEH + VEH). The arrow indicates CGRP injection

Fig. 5

Ca_V3.2 knockout mice does not develop periorbital mechanical allodynia after CGRP injection. CGRP (0.1 nmol/5 µL) was injected in the TG of wild-type (WT) and Ca_V3.2^−/− mice. Periorbital mechanical allodynia was assessed in male (A) and in female (B) mice before the treatments (BL) and after CGRP injection. Data is expressed as mean ± SEM (n = 10). Two-way ANOVA with repeated measures followed by the Bonferroni post hoc test. *p < 0.05, compared to the respective Vehicle. The arrow indicates CGRP injection

Fig. 6

Periorbital mechanical allodynia induced by CGRP involves the activation of the canonical CGRP receptor and PKA. Female mice were treated with (A) CGRP 8–37 (10 nmol/5 µl; i.g.) or (B) H89 (2.5 µg/5 µl; i.g.). and 15 min later received an intraganglionar injection of CGRP (0.1 nmol/ 5 μL; i.g.). Control groups received the corresponding vehicles. Periorbital mechanical allodynia was assessed in female mice before the treatments (BL) and after CGRP injection. Data is expressed as mean ± SEM (n = 10). Two-way ANOVA with repeated measures followed by the Bonferroni post hoc test *p < 0.05, compared to CGRP (VEH + CGRP). The arrow indicates CGRP injection