Ca_V3.2 calcium channels: new players in facial pain

Trigeminal neuralgia is the most common type of neuropathic pain involving the craniofacial region. Trigeminal neuralgia is defined as sudden and brief recurrent episodes of stabbing pain in one or more branches of the trigeminal nerve [13]. Recent studies have linked trigeminal neuralgia to dysfunctions in voltage-gated calcium channels [3; 4]. For instance, a CACNA1A (Ca_V2.1 or P/Q-type voltage-gated calcium channel) variant (P2455H) was reported in a patient with trigeminal neuralgia [3] and caused both gain-of-function (GoF) and loss-of-function (LoF) of the channels’ activity [6], suggesting that associated changes in Ca_V2.1 function in the trigeminal system may contribute to the development of trigeminal neuralgia. In CACNA1H (i.e., the Ca_V3.2 T-type voltage-gated calcium channel), nineteen damaging variants have been predicted; however, no study to date has specifically investigated the functional role of any of these Ca_V3.2 channel variants in trigeminal neuralgia. In the current issue of Pain, Gambeta et al. [7] address this gap in knowledge. Using electrophysiological recordings, biochemistry, and animal behavior assays, the authors uncovered a novel role of Ca_V3.2 in trigeminal orofacial pain syndromes.

While Ca_V3.2 channels have been implicated in pain hypersensitivity [1], most of this research has centered around the roles of these channels in regulating dorsal root ganglia (DRG) and spinal dorsal horn neuron excitability in numerous inflammatory and neuropathic pain models [1]. Here, the authors show that Ca_V3.2 missense mutations linked to trigeminal neuralgia in humans impact the functional activity of recombinant Ca_V3.2 channels expressed in Human embryonic kidney (HEK) cells. Of the nineteen CACNA1H mutations reported by Dong et al. [4], four variants were selected for this study and their effect on the biophysical properties of Ca_V3.2 channels were interrogated using the exon 26 (human) /25 (mouse) variant. One mutation (E286K) is located within the pore loop of domain I, and the other three mutations (H526Y, G563R, and P566T) are in the intracellular loop I-II of the channel (Fig. 1A, B). Among the four mutations analyzed, two of them (G563R and P566T) produced a significant increase in macroscopic Ca_V3.2 Ca²⁺ currents, consistent with a GoF of the channel’s variants. In contrast, the E286K mutation resulted in a slight LoF, while no changes were seen with the H526Y mutation. The observed effects were independent of the expression level of the channels at the membrane. These results raise the question of how two opposite mechanisms — LoF and GoF — of Cav3.2 mutations contribute to trigeminal neuralgia. Heterologous expression systems provide only one snapshot of how a mutation affects channel behavior. Because promotion of a pain state rests on perturbation of excitability of neurons, which in turn is determined by a balance between excitation and inhibition, the investigators explored the consequences of expressing this mutant in trigeminal ganglion (TG) neurons. Trigeminal ganglion neurons expressing the E286K Ca_V3.2 variant showed an increase in the overall TG neuronal excitability (Fig. 1B).

Figure 1. Ca_V3.2 channel variants and their role in trigeminal neuralgia.

(A) AlphaFold model of human Ca_V3.2 channel [11] shown as cartoon with E286 (blue), H526 (green), P566 (red) and G563 (purple) residues modeled at confidence ≥70 in gray and those at confidence <70 in wheat. Inset box: AlphaFold model of Ca_V3.2 (gray cartoon) with docked Z944 (red sticks, docking score = −5.9 kcal/mol) aligned to structure of Ca_V3.1 colored by domain with Z944 bound shown as black sticks (PDB: 6KZP [16]). Docking performed with Schrödinger Glide in SP mode. (B) Schematic representation of the location of Ca_V3.2 channel’s mutations and their functional effects in HEK293 cells and in trigeminal ganglion (TG) neurons. Mutant G563R was expressed in TG neurons obtained from mice and action potentials were recorded. (C) Biochemical effects of constriction of the infraorbital nerve (CION) injury on Ca_V3.2 expression and effects of intraperitoneal injection (i.p.) of Z944 in facial hyperalgesia. Images in B and C generated with BioRender.com.

To translate these in vitro findings to in vivo, Z944 – a specific T-type Ca²⁺ channel blocker (Fig. 1A, inset box) – was used to assess the role of Ca_V3.2 channels in trigeminal pain. Intraperitoneal injection of Z944 resulted in an antihyperalgesic effect on male and female mice with constriction of the infraorbital nerve (Fig. 1C). Although Z944 can modulate pain signaling in humans [12], a limitation of using this small molecule for the study by Gambeta et al. [7] is that Z944 is a pan-T-type Ca²⁺ channel inhibitor and thus, the antihyperalgesic effects observed in mice with trigeminal neuralgia cannot be attributed solely to an effect on Ca_V3.2 channels per se. Both Ca_V3.1 and Ca_V3.3 channels are important players in trigeminal neuropathic pain as well [2; 14]: following infraorbital nerve ligation, Ca_V3.1 channel knock-out mice have attenuated mechanical hypersensitivity [2]; trigeminal neuropathic pain was alleviated by inhibition of Cav3.3 channels in mice with a Cav3.3-selective blocking peptide [14]. To resolve if the antihyperalgesic affect could be due to broad block of Ca_V3.x channels by Z944, the authors tested Z944 treatment on Ca_V3.2 knock-out (Ca_V3.2^−/−) mice. Surprisingly, Ca_V3.2^−/− mice with constriction of the infraorbital nerve still developed facial thermal hyperalgesia. The antihyperalgesic effect of Z944 was lost in Ca_V3.2^−/− mice, suggesting that in this model, Z944 acts on Ca_V3.2 channels. A general gene knockout approach is often encumbered by compensatory mechanisms. This compensation has been reported previously by the authors, where Ca_V3.2^−/− mice showed normal withdrawal response to mechanical stimuli in the complete Freund’s adjuvant model of inflammatory pain when compared to wildtype animals [8]. The investigators propose that it is unlikely that Ca_V3.1 and Ca_V3.3 channels are involved given that Z944 blocks all three isoforms and should have mediated analgesia even in the absence of Ca_V3.2. Even though this compensatory mechanism occurred, it appears that Ca_V3.2 channels are essential players in trigeminal neuralgia.

Numerous studies have shown that Ca_V3.2 expression is significantly increased in spinal dorsal horn and dorsal root ganglia neurons in different inflammatory and neuropathic pain models [1]. For example, in nerve ligation models, Ca_V3.2 expression is increased in chronic constriction injury [10], L5/L6 spinal nerve ligation (SNL) [9; 15], partial sciatic nerve ligation [5], among others. To determine if this also occurs in trigeminal neuralgia, the investigators assessed the expression of Ca_V3.2 in the infraorbital nerve (IoN), TG, and the spinal trigeminal subnucleus caudalis (Sp5C) in mice with constriction of the infraorbital nerve. Seven and fourteen days after injury, Ca_V3.2 expression did not change in the IoN or the TG but was increased in the Sp5C in mice with constriction of the infraorbital nerve compared to mice with sham surgery (Fig. 1C). These data correlate with RNAseq analysis showing that CACNA1H expression is not significantly changed in TG isolated from mice with Foramen Rotundum Inflammatory Constriction Trigeminal Infra Orbital Nerve (FRICT-ION) injury [14]. The spinal trigeminal nucleus is a sensory tract located in the lateral medulla of the brain stem that further subdivides into three subnuclei including the Sp5C, and the fact that Ca_V3.2 expression is increased in this nucleus argues for a central role of Ca_V3.2 channels in pain, rather than peripheral sensitization of the trigeminal pathway. The results by Gambeta et al. [7] are consistent with the data obtained by the CNS-permeant, centrally acting, Ca_V3.2 channel blocker Z944. Whether this upregulation is due to increased membrane trafficking of Ca_V3.2 channels remains an open question.

In summary, this is the first study reporting the distribution of Ca_V3.2 channels in the trigeminal pathway; however, to better understand the complexity underlying the molecular mechanisms for increased Ca_V3.2 functional expression in trigeminal neuralgia, further studies are needed. Altogether, the combined approaches (functional, genetic, and behavioral) used in this study reveal how dysfunction of Ca_V3.2 channels could alter nociceptive signaling in a model of orofacial pain in a sex-independent manner and identifies the Ca_V3.2 channel as a potential target in trigeminal neuralgia (Fig. 1B, C).