Keywords

Introduction

Nociception is a key physiological process that has a critical protective function across many different species (Dubin & Patapoutian, 2010). The same neuroanatomical pathways that support nociceptive processing can become dysregulated from an adaptive to a maladaptive state and result in chronic hypersensitivity that serves no useful biological purpose (Kuner & Flor, 2017). Chronic pain is a debilitating condition that affects more than 20% of adult North Americans, and treatment of chronic pain represents one of the most serious health challenges facing society (Schopflocher et al., 2011; Reitsma et al., 2012). It is a major cause of lost productivity, and leads to a substantially reduced quality of life in both adults and children, along with staggering health care and economic costs (Gaskin & Richard, 2012; Mayer et al., 2019).

Painful stimuli are first detected by a set of diverse and specialized nociceptors that have their nerve endings in the skin or internal organs, and their cell bodies in either the dorsal root ganglia (DRG) or trigeminal ganglia (Fig. 1). In response to noxious stimuli such as heat, mechanical pressure, or chemicals, action potentials are initiated in the distal nerve endings, which then propagate along the afferent axons to nerve terminals located in the superficial layers of the spinal dorsal horn and brain stem (for cephalic neurons of the trigeminal ganglia) (Dubin & Patapoutian, 2010; Basbaum et al., 2009). Synaptic transmission then leads to the initiation of action potentials in second-order neurons that project to various regions in the brain, where they are processed to ultimately give rise to the perception of an unpleasant sensation. The brain extends descending projections to the spinal cord, which modulate ascending nociceptive inputs through activation of excitatory and inhibitory spinal interneurons. Afferent sensory fibers are heterogeneous and do not just convey nociceptive inputs, but also other input modalities such as touch, proprioception, and itch, and ascending spinal dorsal columns are similarly organized along modalities (Niu et al., 2013). Nociceptive fibers are classified into (1) non-myelinated, slowly conducting peptidergic and non-peptidergic C-fibers, and (2) myelinated, more rapidly conducting A-fibers (Basbaum et al., 2009). These can be further subdivided based on their diameter and more precisely, based on a number of different markers that in many cases show overlapping expression patterns (e.g., tropomyosin receptor kinase A [TRKA], Ret, calcitonin gene-related peptide [CGRP], substance P, islocetin B4, transient receptor potential cation channel subfamily V member 1 [TRPV1], Mas-related G-protein-coupled receptor [MrgpR] among others). Furthermore, different subtypes of afferent fibers terminate in different laminae in the spinal cord (Molliver et al., 1995; Li et al., 2011). A more detailed description of afferent fiber types is beyond the scope of this chapter but can be found in recent transcriptomic analyses and comprehensive reviews (Finnerup et al., 2021; Sharma et al., 2020; Kupari et al., 2021). Equally complex is the organization of the spinal dorsal horn and its role in processing nociceptive information (Peirs et al., 2015; Gangadharan & Kuner, 2015; Harding et al., 2020). Suffice it to say that dorsal horn neurons are responsible for the integration of nociceptive peripheral inputs, and the activity of a heterogeneous set of spinal interneurons, and their inputs from descending pathways.

Fig. 1
A schematic diagram illustrates afferent fiber and interneuron activities in a calcium channel labeled C a v 3.2. On its right and left are two diagrams labeled nociceptive inputs, skin to ascending and descending pathways of cortex. Below it is a diagram that illustrates dorsal horn synaptic transmission in the calcium channels C a v 2.2 and 3.2.

Schematic representation of the afferent pain pathway, illustrating the roles of Cav3.2 and Cav2.2 calcium channels

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Voltage-gated calcium channels play important roles in these processes (Zamponi, 2016). Cav3.2 (T-type) calcium channels regulate the firing of certain types of afferent sensory neurons and contribute to synaptic transmission in dorsal horn synapses. They also appear to be involved in shaping the firing properties of spinal interneurons. Cav2.2 (N-type) channels are the major calcium channel subtype that is involved in synaptic transmission between nociceptive fibers and second-order neurons in the spinal cord (Fig. 1). In this chapter, we focus primarily on the roles of these two calcium channel isoforms in afferent nociceptive signaling.

N-Type Calcium Channels in the Afferent Pain Pathway

N-type (Cav2.2) channels exhibit a mainly neuronal distribution and they are enriched at presynaptic terminals where they coordinate the synchronous release of neurotransmitters (Nowycky et al., 1985; Westenbroek et al., 1992; Wheeler et al., 1994). In terminals of primary afferent neurons, calcium entry via Cav2.2 channels triggers the release of neurotransmitters such as glutamate, substance P, and CGRP (Gruner & Silva, 1994; Holz et al., 1988; Maggi et al., 1990; Santicioli et al., 1992). Specific Cav2.2 channel blockers such as ω-conotoxin GVIA and MVIIA (Olivera et al., 1994) have been used to demonstrate the involvement of this channel type in the afferent pain pathway (basal thermal and mechanical nociception), and they have also highlighted the potential of N-type blockers as potent analgesics (Chaplan et al., 1994; Malmberg & Yaksh, 1994; Bowersox et al., 1996; Diaz & Dickenson, 1997; Sluka, 1998). The participation of Cav2.2 channels in the pain pathway was later confirmed by the phenotype of Cav2.2 channel knockout mice. Indeed, these mice are hyposensitive to pain (Saegusa et al., 2001; Kim et al., 2001; Hatakeyama et al., 2001), and they only present a relatively mild central nervous system (CNS) phenotype that includes reduced anxiety levels and reduced alcohol withdrawal symptoms (Saegusa et al., 2001; Newton et al., 2004).

The CACNA1B gene, which encodes the Cav2.2α1 subunit, is subject to extensive alternative splicing (Gray et al., 2007). The chapter by T.W. Soong (chapter “Splicing and Editing to Fine-Tune Activity of High Voltage-Activated Calcium Channels”) deals in detail with this regulatory mechanism for voltage-gated calcium channels. In this section, we focus on splice variants that are relevant to the afferent pain pathway. Exon 37 encodes a short section (14 amino acids) of the cytoplasmic carboxy-terminal tail of the channel and can generate two mutually exclusive variants: E37a and E37b (Lipscombe et al., 2002; Bell et al., 2004). In heterologous expression systems, Cav2.2 E37a produces larger calcium currents than its counterpart Cav2.2 E37b, which is probably due to the fact that Cav2.2 E37a is both more resistant to degradation by the proteasome and more efficiently trafficked to the plasma membrane (Marangoudakis et al., 2012; Macabuag & Dolphin, 2015). It is noteworthy that Cav2.2 E37a is selectively expressed in peripheral nociceptive neurons that are positive for both TRPV1 and Nav1.8 channels, and its expression is critical for pain signaling (Bell et al., 2004; Altier et al., 2007). Indeed, using an small interfering RNA (siRNA) knockdown strategy, it was shown that Cav2.2 channels containing E37a mediate basal nociception and inflammatory pain (Altier et al., 2007).

In a chronic pain context, an upregulation of Cav2.2 channel expression has been shown in dorsal root ganglia and in the dorsal horn of the spinal cord (Cizkova et al., 2002; Yokoyama et al., 2003). However, it is still unclear whether the increase of Cav2.2 channels in the dorsal horn results from an increase of channels in central neurons or from an accumulation of channels in primary afferent terminals or a combination of both. Interestingly, while several studies have investigated the transcription level of Cav2.2 channels in chronic pain models, none has shown a correlation between level of Cav2.2 channel proteins and level of Cav2.2 channel mRNAs (messenger RNA), suggesting that chronic pain-induced changes in Cav2.2 channel expression occur at a translational and/or post-translational level (Altier et al., 2007; Umeda et al., 2006). Cav2.2-channel-deficient mice subjected to inflammatory and neuropathic insults exhibit reduced pain hypersensitivity (Hatakeyama et al., 2001; Saegusa et al., 2001; Kim et al., 2001). Moreover, whereas thermal hyperalgesia induced by sciatic nerve ligation relies mainly on the expression of E37a, tactile allodynia depends on both E37a and E37b splice variants (Altier et al., 2007). E37a was also shown to play a critical role in morphine-dependent analgesia (Andrade et al., 2010). Finally, in a model of chronic inflammatory pain, the expression of a Cav2.2 channel splice variant that lacks exon 18a was shown to be increased in DRG neurons (Asadi et al., 2009). Exon 18 encodes a region of the channel that corresponds to the II–III linker, which plays a critical role in presynaptic targeting (Zamponi et al., 2015). Although the physiological relevance of this variant switch is still unclear, it has been suggested that it could constitute a regulatory mechanism to attenuate excitability and reduce synaptic transmission (Asadi et al., 2009).

In the central nervous system, excitatory synaptic transmission usually depends on the dual activity of Cav2.2 and Cav2.1 channels. However, using a multi-electrode array recording system, a study has shown that in the anterior cingulate cortex (ACC), Cav2.2 channels are the only voltage-gated calcium channels involved in glutamatergic excitatory synaptic transmission (Kang et al., 2013). Interestingly, the ACC plays important roles in pain perception and chronic pain (Xu et al., 2008; Li et al., 2010).

Interaction Between CRMP2 and Cav2.2: A Therapeutic Target for Chronic Pain

For more than a decade, the interaction between Cav2.2 channels and collapsin response mediator protein 2 (CRMP2) has attracted increasing interest (Brittain et al., 2009). Initially identified as a protein involved in axonal growth, CRMP2 was then shown to directly interact with Cav2.2 channels and to promote their forward trafficking to the plasma membrane (Brittain et al., 2009). The CRMP2/Cav2.2 interaction was shown to modulate vesicular release in hippocampal neurons (Brittain et al., 2009; Chew & Khanna, 2018). In DRG neurons, Cav2.2-dependent secretion of CGRP was enhanced by the over-expression of CRMP2, which pointed to its involvement in the pain pathway (Chi et al., 2009). These initial studies led to the identification of interaction domains between the two proteins and the development of a short 14 amino-acid peptide (CBD3) derived from CRMP2 that can interfere with the binding of CRMP2 to Cav2.2 (Brittain et al., 2011). Conjugated to the HIV1 TAT protein and injected in vivo, CBD3 peptide is able to suppress inflammatory, neuropathic, postoperative, diabetic, migraine, and HIV-related pain (Brittain et al., 2011; Wilson et al., 2011; François-Moutal et al., 2015; Xie et al., 2016; Piekarz et al., 2012; Ripsch et al., 2012).

CRMP2/Cav2.2 interactions can be modulated by several mechanisms (Moutal et al., 2019). Phosphorylation of CRMP2 by cyclin-dependent kinase 5 (Cdk5) has been shown to reinforce its interaction with Cav2.2 channels (Brittain et al., 2012) and thus potentially enhance calcium currents. Interestingly, activation of Cdk5 was increased in animal models of neuropathic pain and inhibiting Cdk5 activity attenuated mechanical allodynia (Li et al., 2014; Yang et al., 2014; Brittain et al., 2012; Moutal et al., 2016a, b). Cdk5 has multiple cellular targets, and thus inhibiting its phosphorylation activity likely disrupts other pathways that (in addition to reducing CRMP2/Cav2.2 interaction) can account for the improvement of the pain phenotype (Yang et al., 2014).

SUMOylation, the post-translational addition of small ubiquitin-related modifier (SUMO) peptide, can modulate CRMP2 function (Dustrude et al., 2013). A putative SUMO-interaction motif in CRMP2 has been linked to its role in regulating calcium influx via voltage-gated calcium channels (Ju et al., 2013). However, there is still no evidence showing involvement of CRMP2 SUMOylation and Cav2.2 channels in pain pathways.

Finally, the tumor suppressor protein neurofibromin has been shown to interact with CRMP2 via its C-terminal domain, inhibiting its function on Cav2.2 channels (Moutal et al., 2017a, b, 2019). Neurofibromatosis type 1 patients present mutations in the NF1 gene, which encodes neurofibromin. These mutations often result in truncated proteins and afflicted patients suffer from idiopathic chronic pain (Gutmann et al., 2017; Esposito et al., 2015). In mutant mice heterozygous for Nf1 (Nf1+/−), N-type current densities are increased in DRG neurons and vesicular release is enhanced compared with wild-type neurons (Duan et al., 2014). Of note, the use of the CBD3 peptide to treat these Nf1+/− mice restores calcium currents and vesicular release to control levels indicating that neurofibromin contributes to the same regulatory pathway as CRMP2–Cav2.2 (Wilson et al., 2012). Moreover, a clustered regularly (CRISPR)/Cas9 strategy was used to truncate the C-terminal domain of neurofibromin in rats and the effect on calcium channel function was examined (Moutal et al., 2017b). This latter study showed an increase in voltage-gated calcium currents in DRG neurons and the animals developed thermal hyperalgesia. Further investigation of the interaction between neurofibromin and CRMP2 led to the design of a 15 amino-acid peptide (CNRP1) derived from CRMP2 C-terminal sequence, which, when coupled to a TAT peptide, was able to mimic the negative regulation of neurofibromin on CRMP2–Cav2.2 interaction in vivo (Moutal et al., 2017a). Indeed, intrathecal injection of TAT-CNRP1 alleviated nociceptive responses in animal models of inflammatory, post-surgical, and neuropathic pain (Moutal et al., 2017a). Interestingly, CNRP1 was also used in a proteomic analysis of a synaptic membrane library to reveal a novel interaction between CRMP2 and the presynaptic protein syntaxin 1A (Moutal et al., 2017a). Syntaxin 1A, which interacts with the synprint (Synaptic Protein Interaction) domain of Cav2.2 and Cav2.1, plays an important role in trafficking the channels to presynaptic terminals and promotes synaptic vesicle docking (Sheng et al., 1994; Gandini & Zamponi, 2021; Bennett et al., 1992). Altogether, these data reveal the importance of CRMP2 as a presynaptic interaction hub regulating Cav2.2 trafficking and highlight its potential as therapeutic target for pain treatment (Khanna et al., 2020).

Cavα2δ Subunits and Neuropathic Pain

Cavα2δ are critical auxiliary subunits that are required for Cav1.X and Cav2.X channel complexes to be trafficked to the plasma membrane, and for Cav2.X to be targeted to presynaptic terminals (Dolphin, 2016; Ferron et al., 2021). A detailed account on the structure and functions of Cavα2δs is provided in the chapter by A.C. Dolphin (chapter “Regulation of Calcium Channels and Synaptic Function by Auxiliary α2δ Subunits”). In this section, we will focus on Cavα2δ subunits, and particularly Cavα2δ-1, in a context of chronic pain. Cavα2δ-1 mRNA expression is enhanced in DRG neurons in animal models of chronic pain (Newton et al., 2001; Wang et al., 2002; Bauer et al., 2009; Lana et al., 2014). This transcriptional effect is associated with an increase in protein expression in the soma of DRG neurons and also along the axons and in the terminals within the spinal cord (Bauer et al., 2009; Luo et al., 2001). Interestingly, in vivo over-expression of Cavα2δ-1 is sufficient to induce tactile allodynia and hyperalgesia (Li et al., 2006). In addition, mice lacking the Cacna2d1 gene that encodes Cavα2δ-1 have a reduced sensitivity to mechanical and cold stimuli, and delayed mechanical hypersensitivity following sciatic nerve ligation (Patel et al., 2013). Altogether, these data support a key role of Cavα2δ-1 in controlling DRG neuron excitability and its involvement in the development of chronic pain.

Cavα2δ-1 is the target of gabapentinoids (gabapentin and pregabalin), a class of drugs prescribed to treat chronic pain in humans (Rosenberg et al., 1997; Field et al., 2006, 2007; Taylor et al., 2007). Chronic application of gabapentinoids to DRG neurons in culture inhibits synaptic transmission by reducing the trafficking of Cavα2δ-1, and consequently Cav2.2, to the plasma membrane (Hendrich et al., 2008, 2012; Cassidy et al., 2014). In animal models of neuropathic pain, chronic treatment with pregabalin partially prevents the increase of presynaptic Cavα2δ-1 in the dorsal horn of the spinal cord and alleviates allodynia (Bauer et al., 2009).

Besides their role as auxiliary subunits for Cav channels, Cavα2δs have been involved in direct interactions with other ion channels and have been linked to other biological functions. Indeed, the N-terminal domain of KCa1.1 (BK) potassium channels competes with Cav2.2 channels for the binding of Cavα2δ-1 and reduces Cav2.2 surface expression and calcium current density (Zhang et al., 2018). Importantly, over-expressing a membrane-bound BK channel N-terminus peptide has an analgesic effect in mouse models of inflammatory and neuropathic pain. In addition, Cavα2δ-1 has been shown to interact with N-methyl-D-aspartate (NMDA) receptors and to increase their delivery at dorsal horn synapses during neuropathic pain development (Chen et al., 2018). Finally, Cavα2δs have been shown to play a role in synaptogenesis via extracellular matrix protein thrombospondins and thrombospondin-4 has been associated with the development of neuropathic pain (Eroglu et al., 2009; Yu et al., 2018; Kim et al., 2012). Altogether, these studies reinforce the interest of Cavα2δ-1 as a therapeutic target for the treatment of chronic pain, and they also highlight the fact that the benefits of gabapentinoid treatments in vivo are not solely attributable to the normalization of Cav2.2-dependent synaptic transmission in the spinal cord.

N-Type Channel Blockers as Pain Therapeutics

In addition to the gabapentinoids discussed in the preceding section, a number of blockers of Cav2.2 channels have been shown to mediate analgesia in preclinical models, and some in a clinical setting. The chapter by Lewis and Adams (chapter “Pharmacology and Structure-Function of Venom Peptide Inhibitors of N-Type (Cav2.2) Calcium Channels”) discusses in detail the inhibition of voltage-gated calcium channels by peptide toxins. Briefly, let us recall that ω-conotoxins are among the most selective inhibitors of N-type calcium channels. Both ω-conotoxins GVIA (Conus geographus) and MVIIA (Conus magus) potently inhibit native and transiently expressed Cav2.2 channels in an almost irreversible manner by occluding the pore of the channel (Olivera et al., 1984; Reynolds et al., 1986; Feng et al., 2001). The selective action of MVIIA, the analgesic effects in preclinical models (Chaplan et al., 1994; Bowersox & Luther, 1998), and the fact that it can be synthesized in vitro ultimately led this molecule to being developed as a potential therapeutic for pain. Under the commercial name Prialt, MVIIA passed phase III clinical trials and was ultimately approved for use in patients with severe cancer pain (Atanassoff et al., 2000; Miljanich, 2004). A major limitation of Prialt is that it does not cross the blood-brain barrier and therefore has to be delivered intrathecally via an implanted pump. Another is the fact that the therapeutic window is relatively narrow, and surprising side effects such as memory problems, hypotension, and vision problems have been described (Penn & Paice, 2000; Staats et al., 2004). Several additional conotoxins derived from other species such as Conus fulmen and Conus catus have been explored as possible analgesics (Adams et al., 2003; Lee et al., 2010; Sadeghi et al., 2013), with ω-conotoxin CVID having been tested in clinical trials, but ultimately not pursued further (Schroeder et al., 2006). Despite the limitations of MVIIA as a human pain therapeutic, it did serve to validate N-type calcium channels as a possible therapeutic target from pain.

Small organic blockers of N-type calcium channels that can penetrate the blood-brain barrier would be able to circumvent the need for intrathecal delivery. These include several distinct classes of compounds, such as amino-acid derivatives, N-triazoles, piperazines, piperidines, and even some dihydropyridines, that are normally thought to act on L-type channels (for review, see Schroeder et al., 2006). Many of these derivatives are not strictly specific for Cav2.2 channels, such as TROX-1, and N-triazole derivative that acts on all Cav2 channel isoforms but mediates analgesia in rodent models of osteoarthritis pain (Rahman et al., 2015; Abbadie et al., 2010; Swensen et al., 2012). L-cysteine (Seko et al., 2002) and amino-piperidine derivatives (Teodori et al., 2004) are N-type channel-blocking molecules with analgesic properties. The mixed L-/N-type channel-blocking dihydropyridine cilnidipine (Uneyama et al., 1997; Koganei et al., 2009; Yamamoto et al., 2016) has shown promise as an analgesic in preclinical models. A new N-type channel-blocking chemical scaffold was developed based on the structure of piperazine-based antipsychotics (Tytgat et al., 1991) with N-type channel-blocking activity (Zamponi et al., 2009; Pajouhesh et al., 2010). This includes a lead compound termed Z160 that was remarkably efficacious in a number of rodent pain models, but unfortunately failed phase II human trials due to lack of efficacy. The discrepancy between the preclinical and clinical data remains unresolved. To our knowledge, there are no small organic direct blockers of N-type calcium channels approved for use in humans.

Opioid and GABA-B Receptor Regulation of N-Type Calcium Channels

N-type calcium channels are subject to powerful modulation by a wide array of G-protein-coupled receptors (GPCRs) (for review, see Tedford & Zamponi, 2006). A detailed description of GPCR modulation of voltage-gated calcium channels is provided in the chapter by Herlitze et al. (chapter “Modulation of VGCCs by G-protein Coupled Receptors and Their Second Messengers”). In a nutshell, N-type calcium channels are modulated in a voltage-dependent manner through the direct interaction of G-protein βγ subunits with binding sites in the N-terminus and domain I–II linker regions of the Cav2.2 α1 subunit (Herlitze et al., 1996; Zamponi et al., 1997; Agler et al., 2005), leading to an inhibition of voltage-dependent activation of the channels that can be reversed by strong membrane depolarizations. In addition, there are also voltage-independent pathways that involve the activation of classical messenger cascades such as protein kinases (Luebke & Dunlap, 1994). In the context of nociceptive signaling, N-type calcium channel modulation by the family of opioid receptors (ORs) is particularly important. ORs comprise a family of four different receptor subtypes (μ-, δ-, κ-opioid receptors [MOR, DOR, KOR], and opioid receptor like receptors [NOP]; Waldhoer et al., 2004; McDonald & Lambert, 2005), that can undergo alternative splicing to create additional diversity (Gavériaux-Ruff et al., 1997; Pasternak, 2018; Piltonen et al., 2019). The different opioid receptor family members share a common ability to inhibit N-type calcium channels, and can heteromerize to form complexes with altered signaling properties (Evans et al., 2010). Different types of ORs are expressed in different types of afferent fibers, with MORs and DORs modulating different pain modalities (Scherrer et al., 2009). Activation of these receptors by their ligands leads to an activation of G-protein-coupled inwardly rectifying potassium channels (Blanchet & Lüscher, 2002; Marker et al., 2004), and direct voltage-dependent inhibition of presynaptic N-type calcium channels. In the afferent pain pathway, this leads to reduced neuronal excitability and reduced synaptic transmission in the spinal dorsal horn, leading to analgesia (Kondo et al., 2005; Beaudry et al., 2011). There appears to be tonic opioid control over nociceptive inputs such that DOR null mice show tactile allodynia (Nozaki et al., 2012). MORs are the pharmacological target of clinically active opioids such as morphine and fentanyl, whereas the pentazocine is a clinically approved KOR agonist (Goldstein, 1985). Action of opioids on MOR and DOR activation can lead to respiratory depression (Field et al., 1999), and repeated opioid use can lead to addiction (Darcq & Kieffer, 2018). Interestingly, KORs do not affect respiration, which would be an advantage in the development of OR agonists for pain. Several DOR agonists have been explored in clinical trials (Spahn & Stein, 2017), and bivalent ligands of OR subtypes are being explored as possibly less addictive pain medications (Vardanyan et al., 2017).

MOR modulation of Cav2.2 channels expressed in tA-201 cells has been shown to occur in an OR splice isoform-dependent manner (Gandini et al., 2019). Furthermore, MORs couple differentially to Cav2.2 channels with alternatively spliced exon 37 sequences (Gandini et al., 2019; Raingo et al., 2007). This highlights an immense potential for functional diversity in OR coupling to N-type calcium channels, and thus fine tuning of synaptic activity. Further complexity is generated by the formation of signaling complexes between NOP receptors and Cav2.2 channels that leads to (1) agonist-independent modulation of Cav2.2 channel activity, and (2) receptor-mediated regulation of forward trafficking and internalization of the channels (Beedle et al., 2004; Altier et al., 2006). Furthermore, co-expression with NOP can recruit other OR subtypes into Cav2.2 complexes (Evans et al., 2010). How these signaling complexes affect pain modulation remains to be determined.

GABA-B receptors have also been shown to modulate pain transmission via actions on N-type channels in rodents (Terrence et al., 1985), but the clinical utility of agonists such as baclofen has not been borne out due to CNS side effects (Bortolato et al., 2010). The α-conotoxin Vc1.1, which has GABA-B receptor agonist activity (Callaghan et al., 2008), has been explored as a possible pain therapeutic, but despite promising preclinical results (Castro et al., 2017, 2018), has failed clinical development (Carstens et al., 2011). This molecule mediates voltage-independent modulation rather than the classical Gβγ-mediated voltage-dependent inhibition of Cav2.2 activity (Callaghan et al., 2008). In addition to OR and GABA-B receptors, many other types of GPCRs are known to modulate pain transmission (for review, see Pan et al., 2008), including cannabinoid, and adrenergic and muscarinic receptors. Although these receptors all modulate N-type channels, it is unclear if and how their analgesic actions can be directly attributed to the modulation of these channels in the afferent pathway and/or brain.

Other Types of Cav2 Calcium Channels

P/Q-type (Cav2.1) calcium channels are critical mediators of certain types of congenital migraines. Their role in headache is described in chapter “Voltage-Gated Calcium Channels and Migraine” by Dr. Pietrobon and will not be discussed here further. R-type (Cav2.3) calcium channels have emerged as potential players in afferent pain signaling. Due to their hyperpolarized activation range, Cav2.3 channels are well suited toward supporting neuronal firing (e.g., Park et al., 2010; Zaman et al., 2011; Gutzmann et al., 2019), and furthermore, they have been shown to contribute to neurotransmitter release at a subset of CNS synapses (Myoga & Regehr, 2011; Dietrich et al., 2003). They are expressed in peripheral and colonic sensory neurons (Yusaf et al., 2001; Fang et al., 2007; Qian et al., 2013). Mice lacking Cav2.3 channels show resistance to chemically induced seizures (in agreement with the role of these channels in neuronal excitability; Weiergräber et al., 2007; Dibué-Adjei et al., 2017), and partial resistance to inflammatory pain (Saegusa et al., 2000, 2002). Cav2.3 channels can be inhibited by ORs (Berecki et al., 2016), and direct inhibitors of Cav2.3 channels have been shown to mediate antinociception (Murakami et al., 2004, 2007), possibly by acting on Cav2.3 channels in the spinal dorsal horn. A potential role of Cav2.3 channels in afferent pain signaling is also supported by the notion that natural mixed blockers of Cav2.3 and Cav2.2 channels are able to reverse neuropathic pain in rodents (Shan et al., 2019). Given the paucity of selective small organic Cav2.3 channel blockers, the precise role of Cav2.3 in pain signaling is not yet fully understood. In this context we note that gain-of-function mutations in Cav2.3 channels have been linked to seizure disorders in children, without any associated pain hypersensitivity (Helbig et al., 2018). On the other hand, the R-type channel blocker topiramate has been shown to mediate analgesia in patients with polyneuropathy (Nazarbaghi et al., 2017; but see Wiffen et al., 2013). Hence, the jury is out on whether Cav2.3 is a suitable target for treating pain.

Role of T-Type Calcium Channels in Afferent Pain Signaling

T-type calcium channels are uniquely suited toward regulating cellular excitability (Fig. 2). They activate at hyperpolarized membrane potentials and display a hyperpolarized half inactivation potential (Nowycky et al., 1985; Perez-Reyes, 2003). The overlap between steady-state activation and inactivation curves supports a large window current that allows these channels to be active near typical neuronal resting potentials (Iftinca & Zamponi, 2009; Fig. 2a). At rest, a large fraction of T-type calcium channels is tonically inactivated. Upon membrane hyperpolarization, as occurs, for example, during a GABAergic synaptic input, T-type channels recover from inactivation, giving rise to a greatly increased T-type current upon a subsequent membrane depolarization. This in turn triggers the opening of voltage-gated sodium channels and supports a rapid activation discharge, termed a rebound burst (Coulter et al., 1989; Huguenard & Prince, 1992; Fig. 2b). These collective biophysical properties allow T-type calcium channels to regulate the excitability of primary afferent nociceptive fibers, as well as the firing of spinal interneurons (Candelas et al., 2019). T-type calcium channels also associate with the synaptic vesicle release protein syntaxin 1A to support low-threshold exocytosis (Weiss et al., 2012). This is relevant for synaptic transmission in the dorsal horn of the spinal cord (Jacus et al., 2012; García-Caballero et al., 2014). A role of T-type calcium channels in afferent pain signaling in rodents was originally supported by two lines of evidence. First, delivery of known blockers of T-type calcium channels such as ethosuximide mediated analgesic effects in rodent pain models (Matthews & Dickenson, 2001; Dogrul et al., 2003; Flatters & Bennett, 2004). Second, in an elegant study, Bourinet and coworkers delivered siRNA against the three known T-type calcium channel isoforms, and found that either pan knockdown of Cav3 or specific knockdown of the Cav3.2 isoform resulted in potent analgesia in rat neuropathic pain models, thus identifying a single specific Cav3 channel isoform as a key mediator of afferent pain signaling (Bourinet et al., 2005). Along these lines, similar protection was observed upon knockdown of Cav3.2 in a model of visceral pain induced by intracolonic delivery of butyric acid and in streptozotocin-induced diabetic neuropathy in rats (Messinger et al., 2009). Subsequent experiments in Cav3.2 null mice revealed a more complex picture, with mice lacking Cav3.2 showing reduced sensitivity to intraplantar formalin, but relatively normal response to intraplantar injection of Complete Freund’s Adjuvant (CFA) and in neuropathic pain models (Choi et al., 2007; García-Caballero et al., 2014). This is most likely due to developmental compensatory mechanisms.

Fig. 2
A, a graph of g by g max versus voltage in which an S shaped activation curve and a concavely descending inactivation curve are plotted. B is a set of three neuronal output waveforms and three current trajectories of voltage gated calcium channels 3.1, 3.2, and 3.3.

(a) Schematic representation of the steady-state activation and inactivation curves for T-type calcium channels, highlighting the large window current. (Adapted with permission from Iftinca & Zamponi, 2009). (b) Effects of ICaT in a model neuron on rebound depolarizations. Neuronal output (top traces) and corresponding inward ICaT current trajectories (lower traces) are shown for the three Cav3 isoforms. (Fernandez et al., 2021)

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In rodents, Cav3.2 channels are expressed in specific subtypes of primary afferent fibers including fibers positive for peripherin and vesicular glutamate transporter 3 (vGlut3), which mark a subset of nociceptive C-fibers, and neurofilament protein 200 (NF200) and tropomyosin receptor kinase B (TRKB), which are found in D-hair mechanoreceptors (François et al., 2015). In the spinal cord, Cav3.2 has been shown to overlap with the markers for neuronal nuclear protein (NeuN), Tlx3, Pax2, calretinin, protein kinase C gamma (PKCγ), calbindin, nNos, and parvalbumin, indicating expression in both glutamatergic and GABAergic interneurons (Candelas et al., 2019). The precise function of Cav3.2 channel in spinal cord neurons needs to be explored further, but there is clear evidence that these channels are able to shape the firing properties of lamina II cells (Candelas et al., 2019).

There is also emerging evidence of Cav3.2 channel mutations that are linked to chronic pain. Two heterozygous mutations (P769L and A1059S) in different alleles were identified in a pediatric chronic pain patient (Souza et al., 2019). When introduced and expressed in tsA-201 cells, the mutations mediated slight gain-of-function effects; they were however dependent on the particular recording conditions used for electrophysiological measurements. A number of Cav3.2 channel mutations were also identified in patients with trigeminal neuralgia (Dong et al., 2020); however, to date, they have not been functionally characterized.

Dysregulation of Cav3.2 Channel Expression and Function in Chronic Pain States

An important role of Cav3.2 channels is further underscored by the observation that these channels are upregulated in sensory neurons and/or spinal cord in a number of chronic pain conditions in rodents. This includes peripheral nerve injury (Jagodic et al., 2008; Wen et al., 2010; Feng et al., 2019; Tomita et al., 2020), spinal cord injury (Lauzadis et al., 2020), chronic diabetic conditions (Jagodic et al., 2007), visceral and CFA-induced inflammation (Marger et al., 2011; García-Caballero et al., 2014), osteoarthritis (Shin et al., 2020), certain types of chemotherapy-induced neuropathies (Li et al., 2017; Tomita et al., 2019), and models of post-surgical pain (Joksimovic et al., 2018). Two different molecular mechanisms that may contribute to this enhanced Cav3.2 activity have been identified. First, N-linked glycosylation of a set of four different asparagine residues has been shown to lead to an increase in Cav3.2 channel surface expression (Weiss et al., 2013; Orestes et al., 2013). Importantly, exposing cells to high glucose levels promoted an increase in Cav3.2 surface expression, whereas neuraminidase inhibited Cav3.2 channel membrane expression (Orestes et al., 2013). Thus, glycosylation may at least in part contribute to the upregulation of T-type channel activity during diabetic pain states. Second, Cdk5- and PKC-mediated phosphorylation has been associated with an increase in Cav3.2 channel activity in different models of neuropathic pain (Gomez et al., 2020; Gaifullina et al., 2019). Third, Cav3.2 channels are regulated by ubiquitination by the ubiquitin ligase WWP1 and the deubiquitinase USP5 (García-Caballero et al., 2014). The intracellular linker region between domains III and IV of Cav3.2 contains a ubiquitin ligase consensus motif as well as two lysine residues that are ubiquitinated. Importantly, this region also associates with USP5, an enzyme that is upregulated in the dorsal root ganglia and spinal cord in several mouse models of chronic pain (Gadotti et al., 2015b). This includes models of inflammatory pain induced by intraplantar delivery of CFA, neuropathic pain induced by sciatic nerve injury, visceral pain, chronic diabetes (Gadotti et al., 2015b), and surgery (Joksimovic et al., 2018). Importantly, depletion of USP5 or preventing the association of USP5 with Cav3.2 by delivery of decoy peptides mediated analgesia in these conditions in both male and female mice (García-Caballero et al., 2014, 2016; Gadotti & Zamponi, 2018). Small organic molecules targeting the USP5–Cav3.2 interaction also mediated analgesia (Gadotti et al., 2015b), overall suggesting this molecular interaction as a potential drug target for treating pain. Interestingly, USP5 upregulation could also be observed in response to transcutaneous non-invasive optogenetic activation of C-fibers (Stemkowski et al., 2016). This led to a transient sensitization of the stimulated paw that subsided after 24 h along with a decrease in USP5 levels back to baseline. This then suggests that activity-dependent upregulation of USP5 and an associated increase in Cav3.2 channel activity may have originally evolved as an adaptive (i.e., protective) response that can become maladaptive under certain circumstances. Altogether, there are at least two molecular mechanisms that may contribute to the aberrant upregulation of Cav3.2 channels in chronic pain states, and it is possible that there are others. This could include the Cavα2δ subunit, which has been shown to promote cell surface expression of Cav3.2 channels in expression system even though this does not appear to occur via a physical interaction (Dubel et al., 2004).

We note that the trigeminal system appears to be different from other peripheral afferents in that trigeminal neuropathic pain has been associated with an upregulation of Cav3.3 rather than Cav3.2, and inhibiting these channels mediates analgesia (Montera et al., 2021) suggesting that trigeminal neuralgia may involve different T-type calcium channel signaling than peripheral nerve neuropathy. Finally, T-type calcium channels do not only contribute to nociceptive signaling in the afferent pain pathway, but also there is evidence that they do so in the brain. Cav3.1 null mice have been reported to show increased visceral pain sensitivity, and this was attributed to alterations in function of ventroposterolateral thalamic neurons (Kim et al., 2003). Along these lines, mice lacking Cav3.1 showed a reduction in trigeminal neuropathic pain, due to alterations in thalamic signaling (Choi et al., 2016). Direct delivery of the T-type channel inhibitor NCC-55-0396 into the anterior cingulate cortex was shown to inhibit neuropathic pain in rats (Shen et al., 2015). From a therapeutic point of view, it is thus important to consider all types of Cav3 channels, both peripherally and in the CNS.

T-Type Calcium Channel Blockers as Possible Pain Therapeutics

There is considerable evidence from preclinical models that inhibitors of T-type calcium channels mediate analgesia in a number of different chronic pain models, including diabetic, visceral, inflammatory, and neuropathic pain arising from physical nerve injury. Several detailed reviews of different classes of T-type calcium channel blockers with analgesic properties in rodent pain models were recently published (Snutch & Zamponi, 2018; Weiss & Zamponi, 2019a, b) and we refer the reader to these publications for additional detail. It has been known for some time that certain dihydropyridines can effectively block T-type calcium channels. For example, nimodipine blocks Cav3 channels in the micromolar range, although it should be noted that nimodipine has a much higher affinity for L-type calcium channels, especially at depolarized membrane potentials. Nonetheless, hexahydroquinoline–dihydropyridine derivatives have been shown to block T-type calcium channel preferentially over L-types (Bladen et al., 2014), with several derivatives of this compound series mediating analgesia in mouse models of both neuropathic and inflammatory pain (Bladen et al., 2015a; Gadotti et al., 2015a). Another class of T-type channel inhibitors with analgesic properties is derived from cannabinoids and endocannabinoids. The cannabinoid receptor ligands anandamide (Chemin et al., 2001), as well as N-arachnidonylyglycine (Barbara et al., 2009; Ross et al., 2009) were both shown to potently block Cav3 calcium channels. Importantly, the analgesic effects of these endocannabinoids were abolished in Cav3.2 null mice (Barbara et al., 2009), indicating that they inhibit pain via this calcium channel subtype. Subsequently, a number of compounds related to cannabinoid receptor agonists were developed and shown to not only inhibit Cav3.2 calcium channels but also mediate analgesia by blocking this channel subtype (You et al., 2011; Berger et al., 2014). Derivatives of this compound series that do not act on cannabinoid receptors were subsequently developed, and shown to mediate potent T-type channel inhibition along with analgesic effects (Bladen et al., 2015b), as well as the ability to inhibit acute itch in mice (Gadotti et al., 2020). Piperidine- and piperazine-based compounds such as penfluridol and flunarizine have been known to block Cav3-type calcium channels for some time (Santi et al., 2002). A number of related compounds have since been developed, including TTA-P2 and Z944, and shown to inhibit pain (Choe et al., 2011; Harding et al., 2021). Of note, Z944 has also been shown to mediate analgesic CNS effects by affecting thalamocortical connectivity in rats with neuropathic pain (LeBlanc et al., 2016).

Only a limited number of clinical pain studies with T-type calcium channels have been reported. ABT-639, a potent T-type calcium channel inhibitor, has failed multiple phase II clinical trials (Wallace et al., 2016), including for diabetic pain (Serra et al., 2015; Ziegler et al., 2015). Recently, ethosuximide was tested as a potential pain drug, but failed a randomized controlled trial for neuropathic pain (Kerckhove et al., 2018). Z944 on the other hand successfully completed a phase 1 trial; however, to date no data on efficacy in phase II are available.

The availability of a cryo-electron microscopy (EM) structure for Cav3.1 channels (Zhao et al., 2019) in complex with Z944 has opened new avenues for rational design of T-type calcium channel blockers. Homology models for Cav3.2 and Cav3.3 based on the Cav3.1 structure will aid the development of compounds with specificity for individual T-type calcium channel isoforms, opening new therapeutic options for pain as well as new means for probing the physiological roles of T-type calcium channels in a broader context.

Conclusions

In summary, Cav2.2 and Cav3.2 calcium channels are the predominant calcium channel isoforms involved in the afferent pain pathways, with a possible contribution from Cav2.3. The only clinically approved calcium channel inhibitors for treating chronic pain are the gabapentinoids, which target the ancillary Cavα2δ subunit of Cav2.2 channels; Prialt, which specifically target Cav2.2; and opioids, which inhibit Cav2.2 channel activity via activation of MORs. There remains a paucity of direct small organic inhibitors of Cav2.2 and Cav3.2 channels for clinical use in chronic pain.