
==== Front
Curr Neuropharmacol
Curr Neuropharmacol
CN
Current Neuropharmacology
1570-159X
1875-6190
Bentham Science Publishers

37581322
CN-22-1909
10.2174/1570159X21666230811102700
Medicine, Neurology, Pharmacology, Neuroscience
Role of Cav2.3 (R-type) Calcium Channel in Pain and Analgesia: A Scoping Review
de Amorim Ferreira Marcella 1
Ferreira Juliano 1*
1 Graduate Program of Pharmacology, Universidade Federal de Santa Catarina, Florianopolis, SC, Brazil
* Address correspondence to this author at the Department of Pharmacology, Biological Science Center, Block “D”/CCB, Federal University of Santa Catarina, Trindade, 88040-900, Florianopolis, SC, Brazil; Fax: +55 48 3337 5479; E-mail: ferreiraj99@gmail.com
15 8 2023
2024
22 11 19091922
21 11 2022
22 12 2022
15 2 2023
© 2024 The Author(s). Published by Bentham Science Publishers
2024
The Author(s)
https://creativecommons.org/licenses/by/4.0/ © 2024 The Author(s). Published by Bentham Science Publishers. This is an open access article published under CC BY 4.0 https://creativecommons.org/licenses/by/4.0/legalcode.
Background

Voltage-gated calcium channels (VGCCs) play an important role in pain development and maintenance. As Cav2.2 and Cav3.2 channels have been identified as potential drug targets for analgesics, the participation of Cav2.3 (that gives rise to R-type calcium currents) in pain and analgesia remains incompletely understood.

Objective

Identify the participation of Cav2.3 in pain and analgesia.

Methods

To map research in this area as well as to identify any existing gaps in knowledge on the potential role of Cav2.3 in pain signalling, we conducted this scoping review. We searched PubMed and SCOPUS databases, and 40 articles were included in this study. Besides, we organized the studies into 5 types of categories within the broader context of the role of Cav2.3 in pain and analgesia.

Results

Some studies revealed the expression of Cav2.3 in pain pathways, especially in nociceptive neurons at the sensory ganglia. Other studies demonstrated that Cav2.3-mediated currents could be inhibited by analgesic/antinociceptive drugs either indirectly or directly. Some articles indicated that Cav2.3 modulates nociceptive transmission, especially at the pre-synaptic level at spinal sites. There are studies using different rodent pain models and approaches to reduce Cav2.3 activity or expression and mostly demonstrated a pro-nociceptive role of Cav2.3, despite some contradictory findings and deficiencies in the description of study design quality. There are three studies that reported the association of single-nucleotide polymorphisms in the Cav2.3 gene (CACNA1E) with postoperative pain and opioid consumption as well as with the prevalence of migraine in patients.

Conclusion

Cav2.3 is a target for some analgesic drugs and has a pro-nociceptive role in pain.

Keywords

VGCC
Cav2.3
calcium channel r-type
CACNA1E
pain
analgesia
==== Body
pmc1 INTRODUCTION

Noxious stimuli are detected in the periphery by primary afferent nerve fibers, called nociceptors, and then this stimulus is transmitted to CNS. There are two main classes of nociceptors: Aδ myelinated primary afferent fibers and C unmyelinated primary afferent fibers. The cell bodies of nociceptors are located in DRG. Primary afferent nerve fibers project to the dorsal horn of the spinal cord and stimulate the depolarization and release of neurotransmitters, such as glutamate, substance P and CGRP. These projection neurons are at the origin of multiple ascending pathways, including the spinothalamic and spinoreticulothalamic tracts, which carry pain messages to the thalamus and brainstem, respectively. In situations of persistent nociception or neuroplasticity after lesion, there are changes in transmission and modulation of pain. In chronic pain conditions, the activity of nociceptors is increased, nociceptive transmission is modified, there are changes in action potential firing, increase in the activation of voltage-gated channels (sodium and calcium), increased release of neurotransmitters, and aberrant activation of kinases and glial activities [1-3].

Chronic pain can be a debilitating condition that affects approximately 20 percent of adults and is a major cause of demand for health services. In 2013, pain conditions were the third cause of health spending, and in 2016, Americans spent 380 billion dollars on pain treatments [4]. Between 2007 to 2017 in USA, two of the ten most debilitating health problems were related to pain conditions (Global Burden of Disease, 2017). The International Association for the Study of Pain (IASP) defines pain as “An unpleasant sensory and emotional experience associated with, or resembling that associated with, actual or potential tissue damage” [5]. Thus, pain is not just the neural process of encoding noxious stimuli (nociception), but is always a personal experience that is influenced to varying degrees by biological, psychological, and social factors [6].

Pain can range widely in intensity, quality, and duration and arises from diverse pathophysiological mechanisms. The IASP classifies pain as nociceptive pain (pain arising from activation of nociceptors, including inflammatory pain), nociplastic pain (pain arising from altered nociception despite no clear evidence of actual or threatened tissue damage or evidence for disease or lesion of the somatosensory system) and neuropathic pain (pain caused by a lesion or disease of the somatosensory nervous system) [5]. There are also mixed forms of pain, with patients demonstrating nociceptive and neuropathic pain characteristics, such as cancer pain and post-operative pain [7]. Some patients may develop allodynia (pain in response to a non-nociceptive stimulus) and hyperalgesia (increased pain sensitivity to nociceptive stimulus), in addition to spontaneous pain [8].

Pain may also be classified according to anatomical localization, as somatic pain (superficial, well localized, reaching subcutaneous tissues, muscles and joints) or visceral pain (deep, diffuse and reach viscera) [9]. Based on its duration, pain can be described as acute and chronic. Acute pain starts immediately after injury, has limited duration, and has a temporal and causal relationship with the lesion or disease. In some individuals, there can be a transition from acute to chronic pain that is defined by persisting for at least 3 months [3], with underlying molecular mechanisms that are still not fully understood.

Analgesia can be defined as the absence of pain in response to stimulation which would normally be painful and may be achieved by non-pharmacological and pharmacological (analgesic drugs) management. Different types of pain may respond better to different analgesic drugs. For example, neuropathic pain may respond best to tricyclic antidepressants, serotonin and norepinephrine reuptake inhibitors (SNRIs) or anticonvulsants than to opioids [10]. In addition, the use of opioids is limited by side effects, including nausea, constipation, and respiratory depression, as well as the potential for analgesic tolerance and dependence with chronic use [11]. The use of nonsteroidal anti-inflammatory agents (NSAIDs) is indicated for acute inflammatory pain. However, NSAIDs do not work well for neuropathic pain treatment, and careful dosage compliance is essential to avoid gastric ulcer formation and harmful effects on renal function [12]. Accordingly, novel, effective, safe treatment approaches to pain treatment are urgently needed.

Various animal models of pain have been developed that recapitulate the diverse symptoms of different pain pathologies [13]. Such animal models have aided in our understanding of the pathophysiological mechanisms responsible for the generation of pain and allowed us to identify and validate effective and safe analgesic drug candidates [14]. However, animal pain models have limitations, including study design quality, reproducibility, predictive validity as well as detection of nociception instead pain [15].

Voltage-gated calcium channels (VGCCs) are important mediators of calcium influx into electrically excitable cells such as neurons. These channels are expressed in most plasma membrane compartments, and they are required for a plethora of cellular functions, including regulation of neurotransmitter release, hormone secretion, genic transcription, and others [16]. VGCCs are activated by membrane depolarization, and are classified according to their electrophysiological and pharmacological characteristics into high-voltage-activated channels (HVAs) that require large membrane depolarizations for opening and low-voltage activated (LVA) that open at more negative voltages. The HVA channels consist of L-type (long-lasting currents), P/Q-type (first described in Purkinje cells), N-type (found mostly in neuronal tissues), and R-type channels (which produce pharmacologically resistant currents). LVA channels encompass the T-type (transitory currents) channels [17, 18]. HVA channels are comprised of a pore-forming (α1) subunit and auxiliary subunits (α2-δ, β, and γ), whereas LVA channels only require the α1 subunit to be functional [17]. The mammalian genome encodes 10 different α1 subunits, which are named and subdivided into families based on their homology: family 1 (Cav1.1-1.4) generates L-type calcium currents, members of the Cav2 family mediate P/Q-type (Cav2.1), N-type (Cav2.2), and R-type (Cav2.3) currents, and family 3 (Cav3.1-3.3) generates T-type currents (Dolphin 2016). The members of the Cav2 family are found mostly in neuronal tissue, especially in pain pathways, such as the small diameter sensory neurons and the dorsal horn of the spinal cord, suggesting that they play an important role in nociception [17].

There are several examples of clinically active analgesics that target N-type calcium channel activity. Gabapentinoids (e.g., gabapentin and pregabalin) are analgesic drugs that target the auxiliary α2δ subunits of VGCCs thereby decreasing N-type calcium current amplitude as a result of altered channel trafficking [19]. There are four subtypes of α2δ’ subunits (α2δ-1 to 4), and gabapentinoids target both α2δ-1 and α2δ-2 subtypes which are known to associate with HVA channels [17]. Ziconotide (Prialt®) is a synthetic version of ω-conotoxin MVIIA that provides selective and potent Cav2.2 inhibition [20]. Intrathecal ziconotide was found to produce strong antinociceptive effects in a broad array of animal models and, after three positive randomized control trials, is now approved by FDA for the treatment of severe chronic pain [21, 22]. Finally, the activation of µ-opioid receptors is known to lead to inhibition of Cav2.2 channel activity via direct binding of G protein βγ subunits, which along with activation of G protein-coupled inward rectifier potassium channels, triggers analgesia [23]. Collectively, these examples highlight the clinical utility of N-type calcium channels as targets for analgesics. In addition to Cav2.2, there is now a large body of evidence for a key role of Cav3.2 T-type calcium channels in pain signalling, and numerous studies have highlighted these channels as a potential target for analgesics in preclinical models (reviewed recently by Snutch and Zamponi, 2018, Harding and Zamponi, 2022).

Unlike Cav2.2 and Cav3.2, the role of Cav2.3 in painful signalling is less clear. Cav2.3 channels are distributed in tissues associated with pain detection, transmission, and modulation, including sensory neurons, DRG, trigeminal ganglia and periaqueductal grey [24-26]. In the CNS, Cav2.3 participates in synaptic plasticity and neurotransmitter release [27]. Despite the evidence of the expression of Cav2.3 in nociceptive pathways, the literature that relates Cav2.3 and pain and analgesia remains sparse. Therefore, we conducted a scoping review to map research in this area, as well as to identify any existing gaps in knowledge.

2 MATERIALS AND METHODS

We conducted our literature review using the PRISMA Extension for Scoping Review (PRISMA-ScR) [28]. Inclusion criteria were all years of publication, written in English, and work conducted in human individuals, laboratory animals or cell culture. Studies with any pain outcome or using any analgesic/antinociceptive drugs were included, and other outcomes were excluded [29] (See below).

To identify potentially relevant documents, we searched PubMed and SCOPUS databases with the aid of librarians. We performed searches up to May 2022. The final search results were exported into Mendeley, and the search terms used are listed in Table 1.

Two reviewers (M.A.F and J.F.) working sequentially evaluated the titles, abstracts and then the full text of all publications identified by our searches for relevance. If needed, we resolved disagreements on study selection and data extraction by consensus and discussion with other reviewers.

Original papers, clinical and non-clinical, are included.

All populations are included.

Studies using Cav2.3 in any pain outcome are included.

Review articles are excluded (as they contain no primary data).

Studies not written in the English language are excluded.

Studies on other calcium channels that do not address Cav2.3 are excluded.

Studies on Cav2.3 that are not relevant to pain behavior or pain pathways are excluded.

We identified 66 studies in PubMed and 54 in SCOPUS, with 5 studies being duplicated in both data sets (Fig. 1). For these 115 studies, we applied exclusion criteria based on the title and the abstract, leading to the elimination of 15 studies because they dealt with channels other than Cav2.3. In addition, 50 studies were excluded because the outcome was not associated with any pain behavior or pathways. We excluded 17 further studies as they did not constitute original research. Further, we excluded one study written in Chinese. Thus, 32 studies remained for deeper analysis. After carefully reading the whole text, 8 additional studies were identified by analysis of cited literature. Thus, we identified 40 studies that are eligible for this scoping review (Fig. 1).

To enhance the organization of our hits, we grouped the studies as follows; these 40 articles were organized into 5 types of categories within the broader context of the role of Cav2.3 in pain and analgesia: 1) Expression of Cav2.3 in pain pathways and its alteration by painful conditions; 2) Direct or indirect inhibition of R-type currents by analgesic/antinociceptive drugs; 3) Cav2.3 modulation of nociceptive transmission; 4) Cav2.3 modulators on non-clinical (animal) models of pain and 5) Cav2.3 polymorphism association with clinical pain conditions. Additional groupings were done according to criteria such as type of population, pain outcomes, Cav2.3 focus, pain models used, intervention and some quality criteria (n, randomized, blinded, conflict of interest, sex, temperature, species) [30].

3 RESULTS AND DISCUSSION

3.1 Expression of Cav2.3 in Pain Pathways and its Alteration by Painful Conditions

The expression of Cav2.3 in pain pathways was predominantly reported in sensory ganglia (especially DRG of rodents, but also in humans), both at the mRNA and protein levels, typically in neuronal cell membranes (and occasionally in satellite cells) (Table 2). At the sensory ganglia level, Cav2.3 is co-expressed with different somatosensory neuron markers, especially but not exclusively, in the cell bodies of nonpeptidergic (IB4+) and peptidergic (IB4- and SP or TrkA+) unmyelinated C-nociceptive neurons. Moreover, Cav2.3 was co-expressed with several receptors and channels that are targets for analgesic drugs, including TRPV1, GABABR, Cav2.2 and NTSR2. Both down and up-regulation of Cav2.3 expression were reported in rodent models of cancer-related pain or inflammatory visceral hypersensitivity, respectively. Cav2.3 expression was also detected at the superficial laminae of the dorsal horn of the spinal cord in presynaptic membrane fractions, an anatomical region known to be critical for the first stage processing of noxious and thermal stimuli and that contains the synaptic terminals of nociceptive neurons [31]. A strong upregulation of Cav2.3 mRNA in the spinal cord after peripheral nerve injury in rats has been reported, an effect that was curiously not observed after injury in mice at the protein level (Table 2). Cav2.3 was also detected in supraspinal structures that are very important in the processing of nociceptive information, including the thalamus, hypothalamus, amygdala and periaqueductal gray where sensory-discriminative, endocrine and affective dimensions of pain are processed [32-35]. We were not able to find any alteration of Cav2.3 expression in supraspinal structures in painful conditions (Table 2) [36-46]. Finally, the expression of Cav2.3 protein in intrinsic primary afferent neurons of guinea pig intestine was reported. These are the first neurons of the intrinsic reflexes and may be involved in the analgesic effect of gabapentinoids in inflammatory bowel diseases (Table 2).

3.2 Direct or Indirect Inhibition of R-type Currents by Analgesic/Antinociceptive Drugs

Several drugs with analgesic/antinociceptive effects have been reported to inhibit depolarization evoked R-type currents directly or indirectly in cultured cells, both in primary cultures of neurons with native Cav2.3 expression (especially for the assessment of direct inhibition) or in immortalized cells heterologous expressing Cav2.3 without or with other receptors (specially to assess indirect inhibition) (Table 3 and Fig. 2). The plant phenol Eugenol is a unique drug that is able to produce analgesia in human patients (usually in dentistry) [47] and was reported to directly block R-type currents, independent of TRPV1 interactions (Table 3) [48]. Moreover, certain peptides purified from spider venoms (such as the toxins SNX-482 and PnTx3-3) [49], endogenous molecules (such as L-cysteine) [50], peptides (such as TAT-CBD3A6K) [51] as well as the plant steroid Phylasin F [52] were reported to produce antinociception in rodent models of pain (Table 5) and to directly block R-type currents, with toxins typically being the more selective blockers (Table 3). Of note, clinical studies indicate that the anticonvulsant drugs lamotrigine and topiramate may reduce neuropathic pain after spinal cord injury and prevent migraine headache attacks, respectively [53, 54]. Although both drugs are able to directly block R-type currents [55, 56], they also block several other calcium and sodium currents [57], making it difficult to determine the role of Cav2.3 on its analgesic effects.

Several drugs, such as opioids, capsaicin, zolmitriptan, baclophen somatostati, and contulakin-G, presented analgesic effects in patients with pain and can indirectly inhibit R-type currents, usually through the activation of G-protein related receptors, G-protein subunits, and protein kinase pathways [58-60] (Table 3 and Fig. 2). Opioids are classical analgesic drugs considered the gold standard for many types of pain, and they can indirectly inhibit R-type currents [61] along with their known potent action on Cav2.2. Baclophen is clinically indicated for the treatment of spasticity and can inhibit R-type currents via second messenger activation [62]. The activation of the receptor for the pronociceptive neuropeptide substance (NK1) produced a dual effect: weak activation stimulated Cav2.3, whereas strong activation inhibited these channels [63]. Clinically, antagonism of NK1 receptors effectively diminishes post-operative nausea and vomiting while increasing analgesic tolerance in laparoscopic gynecological procedures [64]. Capsaicin, which is used as a topical analgesic to treat neuropathic pain in certain patients, can block R-type currents in HEK293 cells expressing Cav2.3 and TRPV1 receptors, however, the underlying mechanism is not clear [65]. Triptans, which are the gold standard to treat migraine [66], indirectly inhibit R-type currents via 5-HT1B receptors [67]. Somatostatin inhibits R-type currents in trigeminal rat neurons through G-protein pathways [68]. In patients with cancer pain, intrathecal somatostatin showed an analgesic effect [69]. The toxin contulakin-G can indirectly block R-type currents in DRG rat neurons [70] and showed an analgesic effect in patients with central neuropathic pain [71]. Although these various compounds and drugs do produce inhibition of Cav2.3 calcium channels, they often have multiple other targets, and thus, it remains unclear to what extent their analgesic actions can be attributed to the inhibition of Cav2.3 channels.

3.3 Cav2.3 Modulation of Nociceptive Transmission

Another category for the involvement of Cav2.3 in pain is the effect of Cav2.3 inhibition on nociceptive transmission. All but one out of eight findings in this regard were obtained using the selective blocker SNX-482 (Table 4). Four studies applied SNX-482 to DRG neurons, causing excitability of intact type-A (highly myelinated and touch-pressure-vibration sensitive) neurons and reducing depolarization-activated Ca++ transients and R-currents in small (unmyelinated C nociceptive) neurons obtained from naïve rats (Table 4) [72, 73]. Nerve injury reduced the SNX-482 effect on calcium transients, but not its effect on whole-cell currents [74]. There is a subpopulation of DRG neurons that communicate with their immediate neighbors via trans glia, (neuron-glial cell-neuron) transmission (a.k.a. sandwich synapses) [75]. This transmission was unaffected by SNX-482 (Table 4). On the other hand, SNX-482 application in spinal cord slices reduced the frequency of spontaneous postsynaptic currents, including excitatory currents, indicating a pre-synaptic site of action at glutamatergic synapses [38]. Moreover, PKC activation increased sPSC frequency, an effect reduced by SNX-482 and by Cav2.3 global gene deletion [76]. Spinal SNX-482 and PnTx3-3 application in anesthetized rats exerted dose-dependent inhibition of noxious C-fiber- and Aδ-fiber-mediated neuronal responses in conditions of neuropathy, but not in sham-operated animals [77, 78]. Moreover, responses to innocuous mechanical and thermal stimuli were more sensitive to SNX-482 in nerve injury than in sham-operated animals. Finally, intrathecal SNX-482 inhibited the depolarization of rat colonic afferent neurons, especially in animals with colon inflammation induced by TNBS [36]. In summary, there is clear evidence that Cav2.3 channels are important mediators of the function of neurons within the primary afferent pain pathway, and this fits with the idea that these channels are important for nociceptive signaling.

3.4 Cav2.3 Modulators on Non-clinical (Animal) Models of Pain

Mice and rats were the only experiment animals used in non-clinical models of pain, including models of physiological nociceptive pain (e.g., using tail-flick, hot plate- and von Frey in naïve animals), inflammatory nociceptive pain (formalin, TNBS, CFA) and traumatic or drug-induced neuropathic pain (partial sciatic nerve or spinal nerve ligation traumatic models, streptozotocin, stavudine, paclitaxel and capsaicin) (Table 5).

Regarding physiological nociceptive pain models in naïve mice, mechanical pain threshold (withdrawal threshold to von Frey filaments) was unchanged by Cav2.3 global knockout as well as by spinal (i.t.) knockdown (ASO) or block (SNX-482 and PnTx3-3) (Table 5). On the other hand, knocking down Cav2.3 in the DRG of naïve mice by intra-ganglion injection of a specific AAV-shRNA reduced such thresholds (e.g., caused hyperalgesia) as well as reduced Cav2.3 protein in DRG.

Three studies investigated the role of Cav2.3 in models of inflammatory somatic pain induced by formalin. Cav2.3 KO or block with i.t. SNX-482 reduced spontaneous nocifensive responses in the 2nd phase of formalin-induced acute nociception in both mice and rats, an effect related to reduced SP release (NK1 internalization) and neuronal activation (c-fos) in superficial laminae of the spinal cord dorsal horn. The second phase of the formalin test is considered a model of central sensitization, demonstrating that Cav2.3 may participate in the induction of central sensitization [3]. Regarding inflammatory visceral pain, one study indicated that i.t. SNX-482 reduced mechanical hyperalgesia induced by intracolonic TNBS [42]. Finally, there are conflicting findings concerning the effect of Cav2.3 global gene deletion in the model of somatic-visceral nociception induced by intraperitoneal acetic acid. Whereas initial results demonstrated no alteration of nociception compared to wild-type mice [41], a recent study indicated increased nociception [61], suggesting that Cav2.3 activation usually produces a nociceptive effect in somatic and visceral inflammatory pain.

The role of Cav2.3 was also addressed in neuropathic pain models. Systemic, but especially intrathecal, Cav2.3 blockers (SNX-482, PnTx3-3, TAT-CBD3A6K, physalin F) were able to reduce mechanical hyperalgesia in rodent models of neuropathy related to diabetes (streptozotocin), to nerve trauma (sciatic or spinal nerve ligation) and to HIV or cancer treatment (stavudine and paclitaxel). On the other hand, Cav2.3 global or local (spinal cord and DRG) knockout were not capable of altering mechanical hyperalgesia in a model of traumatic neuropathic pain in mice, an event that may be related to a compensatory increase in N- and L-type currents [79]. Additionally, local Cav2.3 (spinal cord and DRG) knockout reversed the anti-hyperalgesic effect of neurotensin, but not morphine, in the traumatic model of neuropathy. It is known that members of the Cav2 family are indirectly regulated by GPCRs, including opioid receptors. There is some evidence that morphine can have a better analgesic effect when combined with a blocker of Cav2.3 [23, 80]. However, Cav2.3 block and knockdown (ASO) are capable to inhibit, with greater efficacy in female mice, the secondary hyperalgesia induced by capsaicin, a model of central sensitization [81].

The tools used in the study of Cav2.3 in pain pathways are quite limited. There is just one selective blocker of Cav2.3 channels SNX-482, however, it also acts on other ion channels at higher doses [82-84]. Global knockout for Cav2.3 is a good tool, but this type of ablation causes a compensatory effect in other channels [79]. An alternative method is the use of local knockouts for Cav2.3 using antisense, CRISPR/Cas9 or siRNA systems, although the use of these systems requires some knowledge in the development of stable sequences, with degradation resistance and a good delivered system [85, 86]. Due to these, new more selective Cav2.3 blockers and more effective tools for the downregulation of this channel are needed to better probe the role of this channel in pain.

Methodological quality and transparent reporting improve the standards by which animal research is conducted [90]. Considering animals’ studies and pain models to measure the participation of Cav2.3, we used some criteria of methodological quality to analyse the animal studies [30]. Of the 13 animal studies, 92% indicated the experimental sample size (N) used, 62% of these studies reported that they blinded the experiments, and just 31% of studies indicated that they randomized the experiments. One parameter that is considered a good indication of the quality of the study is the description of animal welfare. However, many details regarding housing conditions and timing outcome assessment are often unreported, and the current report based on the quality of animal studies is poor [91]. Just 46% of studies described the temperature of the cages of the animals, and 54% did not mention this parameter. Besides, 69% of the studies described a conflict of interest by the authors. When we analysed the type of animals used, 46% of these studies used mice and 54% used rats. Regarding the sexes used in these studies, 23% of the studies did not mention the sex of the animals used; among those that described the sex, 31% used male, 8% used female, and 38% used both sexes. Just 23% of these studies reported all parameters (Table 6).

These animals’ studies are important to understand the participation of Cav2.3 in different types of pain and the mechanisms involved in their development and maintenance. However, most of these studies demonstrated deficiencies in the description of study design quality which is a limitation of these studies.

3.5 Cav2.3 Polymorphism Association with Clinical Painful Conditions

We found 3 studies involving humans. Two of them showed that there is an association between a polymorphism in CACNA1E gene and phenotypes of postoperative pain. The rs3845446 single-nucleotide polymorphism (SNP) in the CACANA1E gene is an intronic tag SNP in the linkage disequilibrium block from intron 46 to exon 47, a region that contains a stop codon. Patients with the minor G allele of the rs3845446 single-nucleotide polymorphism in CACNA1E gene required less opioid for pain control, in a study with 355 Japanese patients who underwent painful orthognathic cosmetic surgery [92]. On the other hand, after gastrointestinal surgery, the same polymorphism in CACNA1E led to higher opioid requirements and increased pain scores [93]. These suggest that a single polymorphism in the CACNA1E gene can influence the analgesic effects of opioids, with opposite associations depending on the source of pain (somatic vs. visceral).

A third clinical study with healthy individuals and migraine patients demonstrated a single polymorphism rs35737760 in CACNA1E is more prevalent in hemiplegic and brain stem aura migraine. This variant causes a change from aspartate to glutamate at position 859 of Cav2.3 protein, and this polymorphism can modulate the function of R-type calcium [94].

The polymorphisms seen in these clinical studies are ambiguous because evidence shows that the increase in the activity of Cav2.3 cause nociception, and in another way, the decrease/block in Cav2.3 activity causes nociception. It appears that Cav2.3 can modulate pain in different ways depending on the type of pain and underlying mechanisms that are involved.

CONCLUSION

The role of Cav2.2 channels in pain pathways has been extensively studied, but the role of Cav2.3 channels has remained under-explored. Our analysis of the available literature suggests that there is dysregulation of Cav2.3 channels in the pain pathway of certain preclinical pain models in rodents. There is clear evidence that inhibition of Cav2.3 channels activity affects the electrophysiological properties of neurons in the afferent pain pathway in a manner that is largely consistent with a pronociceptive role of these channels, and this is to some extent backed up by studies involving deletion of Cav2.3 channels. There is also a good correlation between the analgesic effects of a number of different compounds and drug molecules and their ability to either directly r indirectly inhibit Cav2.3 channel activity. However, except for SNX-482, there is no selective inhibitor of Cav2.3 channels, and hence it remains unclear whether the observed analgesic effects of direct or indirect Cav2.3 channel inhibitors are indeed mediated by an action on these channels or other targets. Finally, there is emerging evidence of genetic mutations in CACNA1E that may be consistent with the role of Cav2.3 in pain signalling in humans. However, unlike in the case of seizure disorders [95], there is no gain of function mutations identified in patients with chronic pain conditions. In this context, it is interesting to note that gain of function mutations in CACNA1E that lead to epilepsy do not apparently cause persistent pain, but this can perhaps be explained by the existence of different splice variants of Cav2.3 with different susceptibility to functional alterations that may be differentially expressed in the brain versus the peripheral nervous system. Overall, we conclude that still more work is needed that will allow a clear-cut identification of the role of Cav2.3 channels in the transmission and processing of peripheral pain signals. The development of small organic selective inhibitors of Cav2.3 would be of tremendous utility in addressing this issue.

ACKNOWLEDGEMENTS

Declared None.

LIST OF ABBREVIATIONS

HVAs High-voltage-activated Channels

LVA Low-voltage Activated

NSAIDs Nonsteroidal Anti-inflammatory Agents

SNP Single-nucleotide Polymorphism

SNRIs Serotonin and Norepinephrine Reuptake Inhibitors

VGCCs Voltage-gated Calcium Channels

CONSENT FOR PUBLICATION

Not applicable.

STANDARDS OF REPORTING

PRISMA guidelines and methodology were followed.

FUNDING

None.

CONFLICT OF INTEREST

The authors declares no conflict of interest, financial or otherwise.

SUPPLEMENTARY MATERIAL

PRISMA checklist is available as supplementary material on the publisher’s website along with the published article.

Fig. (1) Prisma flow diagram.

Fig. (2) Direct or indirect inhibition of Cav2.3 by analgesic/antinociceptive drugs in cell culture. Abbreviations: NTS2R = Neurotensin 2 receptor; SSTR2 = Anti-somatostatin receptor type 2; HT1B/1D = 5-hidroxitriptamin receptors; GABABR = gamma-aminobutyric acid B receptor; TRPV1 = transient receptor potential cation channel subfamily V member 1; NK1 = Neurokinin 1; GRK2 = G protein-coupled receptor kinase 2; PKC = Protein kinase C; Src = Proto-oncogene tyrosine-protein kinase Src; P = Phosphorilation. Figure created in the Mind the Graph platform.

Table 1 Final search strategy used.

Search Strategy for PubMed	Descriptors	
For Cav2.3 intervention:	(“Calcium Channels, R-Type”[Mesh] OR “cav2.3” OR “CACNA1E” OR “α1e” OR “r-type channels” OR “R-type calcium channel” OR “class E calcium channel” OR “α1e” OR “alpha1e” OR “R-type Ca2+ channels” OR “R-type channel” OR “R-channel” OR “R-type calcium channels” OR “CaV2.3” OR “CaV2.3 calcium channels” OR “Cav2.3 calcium channels” OR “Cav2.3” OR “calcium channel alpha 1e” OR “R-type calcium channel”)	
For pain outcome:	(“pain” [MeSH Terms] OR analgesic OR analgesia OR nociception OR nociceptive OR antinociceptive OR antinociception OR antihyperalgesic OR hyperalgesia OR hypersensitivity OR hyposensitivity OR allodynia OR “neuropathic pain” OR “neuropathic” OR “Von-Frey filament” OR “vonFrey” OR “von frey”)	
Search Strategy for Scopus	Descriptors	
For Cav2.3 intervention:	cav2.3 OR voltage-gated calcium channel r-type OR voltage-dependent calcium channel r-type OR voltage-gated calcium channel Cav2.3 OR voltage-dependent calcium channel Cav2.3 OR VGCC r-type OR calcium channel r-type OR VGCC Cav2.3 OR voltage-sensitive calcium channel r-type OR VSCC r-type OR CACNA1E OR α1e OR r-type channels OR R-type calcium channel OR class E calcium channel OR R-type channel OR R-type calcium channels OR CaV2.3 calcium channels OR Cav2.3 calcium channels OR Cav2.3 R-type Ca2+ channel OR calcium channel alpha 1e OR R-type calcium channel	
For pain outcome:	pain OR analgesic OR analgesia OR nociception OR nociceptive OR antinociceptive OR antinociception OR antihyperalgesic OR hyperalgesia OR hypersensitivity OR hyposensitivity OR allodynia	

Table 2 Cav2.3 expression in pain pathways and its alteration by painful conditions.

Localization	Study	Critical Findings	Painful Conditions	
Sensory Ganglia	(Castro et al. 2017) [36]	Co-expression of Cav2.3 (mRNA and protein) with GABABR and Cav2.2 in human and mouse colonic DRG neurons	SNX-482 or Vc1.1 inhibited afferent depolarization in naïve and chronic visceral hypersensitivity mice (see table 4)	
(Fang et al. 2007; 2010) [24, 25]	Co-expression of Cav2.3 (mRNA) with TrkA and TRPV1 in small rat trigeminal and DRG neurons	Not studied	
(Gandla et al. 2017) [37]	Cav2.3 protein was expressed in most of the neurons (PGP+) and a few satellite cells (GFAP+) of mouse DRG. Co-expression of Cav2.3 (protein) was seen with IB4, SP and NF200 in mouse DRG neuron	Expression of Cav2.3 (protein) was reduced by the pronociceptive microRNA-34c-5p in the DRGs isolated from tumor-bearing mice (see table 5)	
(Martin et al. 2021) [38]	Co-expression of Cav2.3 (mRNA) with NTSR2 in mouse DRG neurons	Cav2.3 gene deletion did not alter mechanical hyperalgesia, but reversed the anti-hyperalgesic effect of somatostatin without altering morphine effects in the PSNL model (see table 5)	
(Murakami et al. 2001) [39]	Cav2.3 mRNA was detected in mouse DRG	SNX-482 caused antinociception in rodent models of pain [40] (see table 5)	
(Saegusa et al. 2000) [41]	Cav2.3 reporting gene (Gal) was expressed in DRG neurons, co-expressed with SP or IB4	Cav2.3 knockout did not alter nociception induced by mechanical or heat stimuli in naïve animals and by acetic-acid, but reduced both phases of formalin-induced spontaneous nociception (see table 5)	
(Qian et al. 2013) [42]	Expression of Cav2.3 (mRNA and protein) and R-type currents were increased in the DRGs isolated from rats with TNBS-induced inflammatory visceral hypersensitivity	SNX-482 intrathecal injection attenuates visceral pain in TNBS-induced inflammatory visceral hypersensitivity (see table 5)	
Spinal cord	(Martin et al. 2021) [38]	Cav2.3 protein was especially detected in the presynaptic fraction of the spinal cord	Expression or distribution of Cav2.3 protein was unchanged in the spinal dorsal horn of mice with partial sciatic nerve ligation	
(Murakami et al. 2001) [39]	Cav2.3 protein was expressed in mouse dorsal horn and column	SNX-482 caused antinociception in rodent models of pain (Murakami et al. 2004) (see table 4)	
(Saegusa et al. 2000) [41]	Cav2.3 gene expression was seen in mouse dorsal horn (laminae I-III) along the entire cord, co-expressed in IB4+ and IB4- terminals	Cav2.3 global gene deletion did not alter nociception induced by mechanical or heat stimuli in naïve animals and by acetic-acid, but reduced both phases of formalin-induced spontaneous nociception (see table 5)	
(Westenbroek et al. 1998) [43]	Cav2.3 protein was mainly expressed in the soma of neurons in the dorsal horn of rats	Not studied	
(Yang et al. 2004) [44]	Cav2.3 mRNA was detected in the dorsal horn of the spinal cord of naïve rats	Expression of Cav2.3 mRNA was the most strongly up-regulated ion channel in rat dorsal horn spinal cord after axotomy	
Supraespinal structures	(Saegusa et al. 2000) [41]	Cav2.3 reporting gene expressed in the periaqueductal gray, but not in nucleus raphe magnus of mice	Not studied	
(Yokoyama et al. 1995) [45]	Cav2.3 protein exhibited the most prominent staining in thalamus, hypothalamus, and amygdala of rats	Not studied	
Enteric nervous system	(Needham et al. 2010) [46]	Cav2.3 protein expressed in intrinsic primary afferent neurons of guinea pigs	Not studied	
Abbreviations: mRNA = messenger RNA; GABABR = gamma-aminobutyric acid B receptor; DRG = dorsal root ganglion; TrkA = Tropomyosin receptor kinase A; TRPV1 = transient receptor potential cation channel subfamily V member 1; PGP = Protein gene product; GFAP = Glial fibrillary acidic protein; IB4 = Isolectin B4; SP = Substance P; NF200 = Neurofilament 200; NTSR2 = Neurotensin receptor 2; TNBS = 2,4,6-Trinitrobenzenesulfonic acid.

Table 3 Direct or indirect inhibition of depolarization evoked R-type currents in cultured cells by analgesic/antinociceptive drugs.

Cell Type	Study	Critical Findings	Implication to Pain	
Direct Interactions	
HEK293 cells expressing Cav2.3 without or with TRPV1	(Chung et al. 2008) [60] (Mohammadreza et al. 2022) [61]	The plant phenol eugenol inhibits and blocks R-type currents, an effect unaltered by TRPV1 expression
L-cysteine increased the amplitudes of recombinant Cav2.3 currents	Topical eugenol is used as an anesthetic/analgesic in dentistry
L-cysteine lowered visceral pain in WT mice, and the effect was abolished in KO mice (see table 5)	
DRG and TG Rat and mouse neurons	(Fang et al. 2007; Murakami et al. 2004) [24, 40]	Toxin SNX-482 selectively inhibited R-type current with variable efficacy among different neurons	SNX-482 caused antinociception in rodent models of pain (see table 5)	
Cerebellar granule rat neurons	(Leão et al. 2000) [62]	The toxin PnTx3-3 unselectively inhibited R-type > N-type currents	PnTx3-3 caused antinociception in rodent models of pain (see table 5)	
DRG rat neurons	(Piekarz et al. 2012) [63]	The synthetic peptide TAT-CBD3A6K inhibited
R-type and T-type currents in small-diameter neurons	TAT-CBD3A6K caused antinociception in rodent models of pain (see table 5)	
(Shan et al. 2019) [64]	The plant steroid Physalin F inhibited N- > R-type currents	Phylasin caused antinociception in rodent models of pain (see table 5)	
Indirect Interactions	
Xenopus oocytes co-expressing Cav2.3 and μ--opioid receptor (rat)	(Bourinet et al. 1996) [65]	A selective agonist of μ-opioid receptor did not alter Cav2.3- currents	Opioids are gold-standard analgesic drugs to treat several types of pain	
Xenopus oocytes co-expressing Cav2.3 and μ-opioid receptor (murine)	(Ottolia et al. 1998) [66]	A selective agonist of μ-opioid receptors inhibited Cav2.3 current through the voltage- and G protein-dependent pathway	Opioids are gold-standard analgesic drugs to treat several types of pain	
HEK293 cells co-expressing human Cav2.3, μ, δ-, or κ-opioid receptors	(Berecki et al. 2016) [67]	Selective agonists of μ-, δ-, and κ-opioid receptors inhibited Cav2.3 currents through voltage-independent as well as G protein βγ subunit and GRK2 kinase-dependent pathways	Opioids are gold-standard analgesic drugs to treat several types of pain	
HEK293 cells co-expressing human Cav2.3 and GABAB receptor	Berecki et al. 2014) [68]	Baclofen or alpha-conotoxin Vc1.1 inhibited Cav2.3 channels through voltage-independent pathways by acting on GABAB receptors; the modulation involves G protein- and Src kinase-dependent pathways	Baclofen was analgesic in patients with severe spasticity [54]
Vc1.1 caused antinociception in a rodent pain model (see table 5)	
HEK293 cells co-expressing human Cav2.3 channels and NK1 receptor	(Meza et al. 2007) [69]	Weak NK1 receptor activation stimulated Cav2.3 through a PKC-dependent pathway, whereas strong activation elicited Cav2.3 inhibition	Neurokinin-1 receptor antagonism reduced post-operative pain [55]	
HEK293 cells expressing Cav2.3 and TRPV1	(Chung et al. 2008) [60]	The plant alkylamide capsaicin inhibited R-type current through a TRPV1-dependent pathway	Topical capsaicin is analgesic in patients with neuropathic pain [56]	
HEK293 cells expressing Cav2.3	(Morikawa et al. 2006) [70]	Zolmitriptan indirectly inhibited P/Q-type > R-type currents through 5-HT1B/1D receptor- and
Gi/o-dependent pathways	Triptans are gold-standard analgesic drugs to treat migraine [57]	
Trigeminal rat neurons	(Mehrke et al. 1997) [71]	Somatostatin inhibited Cav2.3-gated currents through a G protein-dependent pathway	Intrathecal and epidural somatostatin produced analgesia in humans with cancer or postoperative pain [58]	
DRG rat neurons	(Martin et al. 2021) [38]	The toxin contulakin-G indirectly inhibited R-type > N-type currents through NTSR2	Contulakin-G was analgesic in patients with spinal cord injury [59]	
Abbreviations: GRK2 = G protein-coupled receptor kinase 2; PKC = Protein kinase C; NK1 = Neurokinin 1; 5-HT1B/1D = 5-hidroxitriptamin receptors; Src = Proto-oncogene tyrosine-protein kinase Src.

Table 4 Cav2.3 modulation of nociceptive transmission.

Cell or Tissue (Specie)	Study	Conditions	Main Findings	
DRG A-type neurons/cell membrane potential (Rat)	(Lirk 2008) [75]	Naïve	SNX-482 caused neuronal excitability	
DRG neurons/depolarization-activated neurotransmitter release (Chicken)	(Rozanski et al. 2013) [72]	Naïve	SNX-482 did not alter transmitter release at sandwich synapses	
DRG neurons/depolarization-activated Ca++ transients (Rat)	(Fuchs et al. 2007) [76]	Sham-operated and Spinal nerve ligated	SNX-482 decreased calcium transients in small neurons of naïve rats, with reduced sensitivity in neuropathic rats	
DRG neurons/depolarization-activated
R-type current (Rat)	(McCallum 2011) [77]	Sham-operated and Spinal nerve ligated	SNX-482 decreased current in small neurons of naïve rats, without altered sensitivity in neuropathic rats	
Spinal cord/spontaneous excitatory postsynaptic currents (sEPSCs) (Mice)	(Martin et al. 2021) [38]	Naïve	SNX-482 reduced sEPSCs, especially their frequency	
Spinal cord/spontaneous postsynaptic currents (sPSCs) (Rat)	(Yang et al. 2013) [73]	Naïve	SNX-482 reduced sEPSC frequency in dorsal horn neurons. PKC activation increased sEPSC frequency, an effect reduced by SNX-482 and by Cav2.3 global gene deletion	
Spinal cord/spontaneous excitatory postsynaptic currents (sEPSCs) (Rat)	(Shan et al., 2019) [64]	Naïve	Physalin F reduced the frequency of sEPSCs	
Dorsal horn neuronal electrophysiological measurements (Rat)	(Matthews et al. 2007) [78]	Sham-operated and Spinal nerve ligated	Spinal SNX-482 inhibited noxious C-fibre- and Aδ-fiber-mediated neuronal responses in conditions of neuropathy, but not in sham-operated animals	
(Dalmolin et al. 2017) [74]	Sham-operated and Spinal nerve ligated	Spinal PnTx3-3 inhibited noxious C-fiber- and Aδ-fiber-mediated neuronal responses in conditions of neuropathy, but not in sham-operated animals	
Colonic afferents depolarization electrophysiological measurements (Mice)	(Castro et al. 2017) [36]	Naïve and TNBS-treated rat	SNX-482 or Vc1.1 inhibited afferents depolarization	
Abbreviations: sPSCs = spontaneous postsynaptic currents; sPSCs = spontaneous excitatory postsynaptic currents.

Table 5 Cav2.3 modulators on non-clinical (animal) models of pain.

Specie/Models	Study	Critical Behavior Finding	Supporting Ex vivo Findings	
Mice/von Frey, hot-plate, tail- and paw-flick-, formalin- and acetic acid	(Saegusa et al. 2000) [41]	Cav2.3 global gene deletion did not alter nociception induced by mechanical or heat stimuli in naïve animals and by acetic acid, but reduced both phases of formalin-induced spontaneous nociception	None	
Mice/Von Frey	(Gandla et al. 2017) [37]	Intraganglion injection of Cav2.3 knocking down (AAVs-shRNA Cav2.3) caused hyperalgesia	Cav2.3 AAVs-shRNA reduced the expression of Cav2.3 protein in DRG	
Mice/Tail-flick	(Yokoyama et al. 2004) [80]	Cav2.3 global gene deletion in mice resulted in greater analgesia to heat stimuli by morphine or by swim-stress (and resistance to morphine tolerance) than in controls	None	
Mice/Formalin	(Murakami et al. 2004) [40]	Intrathecal SNX-482 inhibited the second phase, but increased the first phase of nociception	None	
Rat/Formalin	(Terashima et al. 2013) [87]	Intrathecal SNX-482 inhibited both phases of nociception	Neurokinin 1 receptor internalization and c-Fos expression in the ipsilateral dorsal horn	
Rat/TNBS	(Qian et al. 2013) [42]	Intrathecal SNX-482 reduced the abdominal withdrawal reflex in response to colorectal distention	None	
Mice/Acetic acid	(Mohammadreza et al. 2022) [61]	Cav2.3 global gene deletion increased acetic acid-induced nociception; an effect reduced by systemic L-cysteine	L-cysteine increases R-type currents	
Mice/Partial sciatic nerve ligation (PSNL)	(Yang et al. 2009) [88]	Cav2.3 global gene deletion in mice did not alter the development of mechanical hyperalgesia	PSNL reduced the R-type current in DRG neurons and Cav2.3 global gene deletion increased N- and L-type currents	
(Martin et al. 2021) [38]	Cav2.3 local (DRG and spinal cord) gene deletion did not alter the development of mechanical hyperalgesia, but reversed the anti-hyperalgesic effect of neurotensin without altering morphine’s effect	None	
Rat and mice/PSNL and streptozotocin	(Dalmolin et al. 2011) [89]	Intrathecal PnTx3-3 reduced mechanical hyperalgesia in neuropathic rodents, but not in naïve animals	None	
Rat/Stavudine	(Piekarz et al. 2012) [63]	Intraperitoneal TAT-CBD3A6K produced antinociception in a rodent model of pain	None	
Rat/Paclitaxel and spinal nerve ligation	(Shan et al. 2019) [64]	Intrathecal phylasin F produced antinociception in rodent models of neuropathy	None	
Mice/ Capsaicin	(Ferreira et al. 2021) [81]	Intrathecal SNX-482 or Cav2.3 ASO caused antinociception in the secondary hyperalgesia trigged by capsaicin in a sex-dimorphic way and SNX-482 or Cav2.3 ASO did not alter the nociception induced by mechanical or heat stimuli in naïve animals	Cav2.3 ASO reduced Cav2.3 mRNA expression in the spinal cord	
Abbreviations: AAVs-shRNA = Adeno-Associated Virus short hairpin RNA; CFA = Complete Freund’s Adjuvant; PSNL = Partial sciatic nerve ligation; TNBS = 2,4,6-trinitrobenzenesulfonic acid.

Table 6 Quality analyses of the animal studies.

Study	N	Blind	Random	Temper.	Conflict	Specie	Sex	
Dalmolin 2011	OK	OK	-	OK	OK	Rats	M and F	
Ferreira 2021	OK	OK	OK	OK	OK	Mice	M and F	
Gandla 2017	OK	-	-	-	-	Mice	-	
Martin 2021	OK	OK	OK	OK	OK	Rats	M and F	
Mohammadreza 2022	OK	OK	-	-	OK	Mice	Male	
Murakami 2004	-	-	-	OK	-	Mice	-	
Piekarz 2012	OK	-	OK	OK	OK	Rats	Female	
Qian 2013	OK	OK	-	-	OK	Rats	Male	
Saegusa 2000	OK	OK	-	-	-	Mice	M and F	
Shan 2019	OK	OK	OK	OK	OK	Rats	Male	
Terashima 2013	OK	OK	-	-	OK	Rats	Male	
Yang 2009	OK	-	-	-	OK	Mice	-	
Yokoyama 2004	OK	-	-	-	-	Mice	M and F	
Abbreviations: N = number per experimental group, OK = the information described in the paper, - = no information found in the paper.
==== Refs
REFERENCES

1 Basbaum A.I. Bautista D.M. Scherrer G. Julius D. Cellular and molecular mechanisms of pain. Cell 2009 139 2 267 284 10.1016/j.cell.2009.09.028 19837031
2 Wang H. Woolf C.J. Pain TRPs. Neuron 2005 46 1 9 12 10.1016/j.neuron.2005.03.011 15820689
3 Latremoliere A. Woolf C.J. Central sensitization: A generator of pain hypersensitivity by central neural plasticity. J. Pain 2009 10 9 895 926 10.1016/j.jpain.2009.06.012 19712899
4 Dieleman J.L. Baral R. Birger M. Bui A.L. Bulchis A. Chapin A. Hamavid H. Horst C. Johnson E.K. Joseph J. Lavado R. Lomsadze L. Reynolds A. Squires E. Campbell M. DeCenso B. Dicker D. Flaxman A.D. Gabert R. Highfill T. Naghavi M. Nightingale N. Templin T. Tobias M.I. Vos T. Murray, C.J.L. m.fl. US spending on personal health care and public health, 1996-2013. JAMA -. JAMA 2016 316 24 2627 2646 10.1001/jama.2016.16885 28027366
5 Raja S.N. Carr D.B. Cohen M. Finnerup N.B. Flor H. Gibson S. Keefe F.J. Mogil J.S. Ringkamp M. Sluka K.A. Song X.J. Stevens B. Sullivan M.D. Tutelman P.R. Ushida T. Vader K. The revised International Association for the Study of Pain definition of pain: Concepts, challenges, and compromises. Pain 2020 161 9 1976 1982 10.1097/j.pain.0000000000001939 32694387
6 Loeser J.D. Treede R.D. The Kyoto protocol of IASP Basic Pain Terminology. Pain 2008 137 3 473 477 10.1016/j.pain.2008.04.025 18583048
7 Freynhagen R. Parada H.A. Calderon-Ospina C.A. Chen J. Rakhmawati E.D. Fernández-Villacorta F.J. Current understanding of the mixed pain concept: A brief narrative review. Curr. Med. Res. Opin. 2019 35 1011 1018 1117
8 Cruccu G. Sommer C. Anand P. Attal N. Baron R. Garcia-Larrea L. Haanpaa M. Jensen T.S. Serra J. Treede R.D. EFNS guidelines on neuropathic pain assessment: revised 2009. Eur. J. Neurol. 2010 17 8 1010 1018 10.1111/j.1468-1331.2010.02969.x 20298428
9 Boezaart A.P. Smith C.R. Chembrovich S. Zasimovich Y. Server A. Morgan G. Visceral versus somatic pain: An educational review of anatomy and clinical implications. Reg. Anesth. Pain Med. 2021 46 629 636 34145074
10 Armitage P. Berry G. The planning os statistical investigations: Statistical methods in medical research. 2nd ed Oxford Blackwell 1987 179 185
11 Khademi H. Kamangar F. Brennan P. Malekzadeh R. Opioid therapy and its side effects: A review. Arch. Iran Med. 2016 19 870 876 27998163
12 Greenwood-Van Meerveld B. Johnson A.C. Grundy D. Gastrointestinal physiology and function. Handb. Exp. Pharmacol. 2017 239 1 16 10.1007/164_2016_118 28176047
13 Abboud C. Duveau A. Bouali-Benazzouz R. Massé K. Mattar J. Brochoire L. Animal models of pain: Diversity and benefits. J. Neurosci. Methods 2021 348 108997 33188801
14 Muley M.M. Krustev E. McDougall J.J. Preclinical assessment of inflammatory pain. CNS Neurosci. Ther. 2016 22 2 88 101 10.1111/cns.12486 26663896
15 Klinck M.P. Mogil J.S. Moreau M. Lascelles B.D.X. Flecknell P.A. Poitte T. Troncy E. Translational pain assessment: Could natural animal models be the missing link? Pain 2017 158 9 1633 1646 10.1097/j.pain.0000000000000978 28614187
16 Simms B.A. Zamponi G.W. Neuronal voltage-gated calcium channels: Structure, function, and dysfunction. Neuron 2014 82 1 24 45 10.1016/j.neuron.2014.03.016 24698266
17 Zamponi G.W. Striessnig J. Koschak A. Dolphin A.C. The physiology, pathology, and pharmacology of voltage-gated calcium channels and their future therapeutic potential. Pharmacol. Rev. 2015 67 4 821 870 10.1124/pr.114.009654 26362469
18 Catterall W.A. Voltage-gated calcium channels. Cold Spring Harb. Perspect. Biol. 2011 3 8 a003947 10.1101/cshperspect.a003947 21746798
19 Dolphin A.C. Voltage-gated calcium channels and their auxiliary subunits: physiology and pathophysiology and pharmacology. J. Physiol. 2016 594 19 5369 5390 10.1113/JP272262 27273705
20 Eldabe S. Batterham A. Ziconotide monotherapy: A systematic review of randomised controlled trials. Curr. Neuropharmacol. 2016 15 217 231
21 Wallace M.S. Rauck R. Fisher R. Charapata S.G. Ellis D. Dissanayake S. Intrathecal ziconotide for severe chronic pain: Safety and tolerability results of an open-label, long-term trial. Anesth. Analg. 2008 106 2 628 637 10.1213/ane.0b013e3181606fad 18227325
22 Mogil J.S. Davis K.D. Derbyshire S.W. The necessity of animal models in pain research. Pain 2010 151 1 12 17 10.1016/j.pain.2010.07.015 20696526
23 Weiss N. Zamponi G.W. Opioid receptor regulation of neuronal voltage-gated calcium channels. Cell. Mol. Neurobiol. 2021 41 5 839 847 10.1007/s10571-020-00894-3 32514826
24 Fang Z. Park C.K. Li H.Y. Kim H.Y. Park S.H. Jung S.J. Kim J.S. Monteil A. Oh S.B. Miller R.J. Molecular basis of Ca(v)2.3 calcium channels in rat nociceptive neurons. J. Biol. Chem. 2007 282 7 4757 4764 10.1074/jbc.M605248200 17145762
25 Fang Z. Hwang J.H. Kim J.S. Jung S.J. Oh S.B. R-type calcium channel isoform in rat dorsal root ganglion neurons. Korean J. Physiol. Pharmacol. 2010 14 1 45 49 10.4196/kjpp.2010.14.1.45 20221279
26 Schneider T. Dibué M. Hescheler J. How “pharmacoresistant” is cav2.3, the major component of voltage-gated R-type Ca2+ channels? Pharmaceuticals 2013 6 6 759 776 10.3390/ph6060759 24276260
27 Wormuth C. Lundt A. Henseler C. Müller R. Broich K. Papazoglou A. Weiergräber, M. m.fl. Review: Cav2.3 R-type voltage-gated Ca2+ channels - Functional implications in convulsive and non-convulsive seizure activity. Open Neurol. J. 2016 10 1 99 126 10.2174/1874205X01610010099 27843503
28 Tricco A.C. Lillie E. Zarin W. O’Brien K.K. Colquhoun H. Levac D. PRISMA extension for scoping reviews (PRISMA-ScR): Checklist and explanation. Ann. Intern. Med. 2018 169 467 473 30178033
29 Pham M.T. Rajić A. Greig J.D. Sargeant J.M. Papadopoulos A. McEwen S.A. A scoping review of scoping reviews: Advancing the approach and enhancing the consistency. Res. Synth. Methods 2014 5 4 371 385 10.1002/jrsm.1123 26052958
30 Sena E. van der Worp H.B. Howells D. Macleod M. How can we improve the pre-clinical development of drugs for stroke? Trends Neurosci. 2007 30 9 433 439 10.1016/j.tins.2007.06.009 17765332
31 Le Pichon C.E. Chesler A.T. The functional and anatomical dissection of somatosensory subpopulations using mouse genetics. Front. Neuroanat. 2014 8 21 10.3389/fnana.2014.00021 24795573
32 Martella G. Costa C. Pisani A. Cupini L.M. Bernardi G. Calabresi P. Antiepileptic drugs on calcium currents recorded from cortical and PAG neurons: Therapeutic implications for migraine. Cephalalgia 2008 28 12 1315 1326 10.1111/j.1468-2982.2008.01682.x 18771493
33 Kortus S. Srinivasan C. Forostyak O. Zapotocky M. Ueta Y. Sykova E. Sodium-calcium exchanger and R-type Ca2+ channels mediate spontaneous [Ca2+]i oscillations in magnocellular neurones of the rat supraoptic nucleus. Cell Calcium 2016 59 289 298 27052156
34 Siwek M.E. Müller R. Henseler C. Broich K. Papazoglou A. Weiergräber M. The CaV2.3 R-type voltage-gated Ca2+ channel in mouse sleep architecture. Sleep 2014 37 5 881 892 10.5665/sleep.3652 24790266
35 Lee S.C. Choi S. Lee T. Kim H.L. Chin H. Shin H.S. Molecular basis of R-type calcium channels in central amygdala neurons of the mouse. Proc. Natl. Acad. Sci. USA 2002 99 5 3276 3281 10.1073/pnas.052697799 11854466
36 Castro J. Harrington A.M. Garcia-Caraballo S. Maddern J. Grundy L. Zhang J. Page G. Miller P.E. Craik D.J. Adams D.J. Brierley S.M. α-Conotoxin Vc1.1 inhibits human dorsal root ganglion neuroexcitability and mouse colonic nociception via GABAB receptors. Gut 2017 66 6 1083 1094 10.1136/gutjnl-2015-310971 26887818
37 Gandla J. Lomada S.K. Lu J. Kuner R. Bali K.K. miR-34c-5p functions as pronociceptive microRNA in cancer pain by targeting Cav2.3 containing calcium channels. Pain 2017 158 9 1765 1779 10.1097/j.pain.0000000000000971 28614186
38 Martin L. Ibrahim M. Gomez K. Yu J. Cai S. Chew L.A. Conotoxin contulakin-G engages a neurotensin receptor 2/R-type calcium channel (Cav2.3) pathway to mediate spinal antinociception. Pain 2021 163 9 1751 1762 35050960
39 Murakami M. Suzuki T. Nakagawasai O. Murakami H. Murakami S. Esashi A. Taniguchi R. Yanagisawa T. Tan-No K. Miyoshi I. Sasano H. Tadano T. Distribution of various calcium channel α1 subunits in murine DRG neurons and antinociceptive effect of ω-conotoxin SVIB in mice. Brain Res. 2001 903 1-2 231 236 10.1016/S0006-8993(01)02427-1 11382408
40 Murakami M. Nakagawasai O. Suzuki T. Mobarakeh I.I. Sakurada Y. Murata A. Yamadera F. Miyoshi I. Yanai K. Tan-No K. Sasano H. Tadano T. Iijima T. Antinociceptive effect of different types of calcium channel inhibitors and the distribution of various calcium channel α1 subunits in the dorsal horn of spinal cord in mice. Brain Res. 2004 1024 1-2 122 129 10.1016/j.brainres.2004.07.066 15451373
41 Saegusa H. Kurihara T. Zong S. Minowa O. Kazuno A. Han W. Matsuda Y. Yamanaka H. Osanai M. Noda T. Tanabe T. Altered pain responses in mice lacking α1E subunit of the voltage-dependent Ca2+ channel. Proc. Natl. Acad. Sci. USA 2000 97 11 6132 6137 10.1073/pnas.100124197 10801976
42 Qian A. Song D. Li Y. Liu X. Tang D. Yao W. Yuan Y. Role of voltage gated Ca2+ channels in rat visceral hypersensitivity change induced by 2,4,6-trinitrobenzene sulfonic acid. Mol. Pain 2013 9 1744-8069-9 15 10.1186/1744-8069-9-15 23537331
43 Westenbroek R.E. Hoskins L. Catterall W.A. Localization of Ca2+ channel subtypes on rat spinal motor neurons, interneurons, and nerve terminals. J. Neurosci. 1998 18 16 6319 6330 10.1523/JNEUROSCI.18-16-06319.1998 9698323
44 Yang L. Zhang F-X. Huang F. Lu Y-J. Li G-D. Bao L. Peripheral nerve injury induces trans-synaptic modification of channels, receptors and signal pathways in rat dorsal spinal cord. Eur. J. Neurosci. 2004 19 871 883 15009134
45 Yokoyama T. Westenbroek I.R.E. Hell W. Snutch P. Biochemical properties and subcellular distribution neuronal class E E calcium channel alpha 1 subunit. J. Neurosci. 1995 15 10 6419 6432 7472405
46 Needham K. Bron R. Hunne B. Nguyen T.V. Turner K. Nash M. Identification of subunits of voltage-gated calcium channels and actions of pregabalin on intrinsic primary afferent neurons in the guinea-pig ileum. Neurogastroenterol. Motil. 2010 22 e301 e308 20618837
47 Nkambeu B. Ben Salem J. Beaudry F. Eugenol and other vanilloids hamper Caenorhabditis elegans response to noxious heat. Neurochem. Res. 2021 46 2 252 264 10.1007/s11064-020-03159-z 33123873
48 He A. Song D. Zhang L. Li C. Unveiling the relative efficacy, safety and tolerability of prophylactic medications for migraine: Pairwise and network-meta analysis. J. Headache Pain 2017 18 26 28220376
49 Ling H-Q. Chen Z-H. He L. Feng F. Weng C-G. Cheng S-J. Comparative efficacy and safety of 11 drugs as therapies for adults with neuropathic pain after spinal cord injury: a bayesian network analysis based on 20 randomized controlled trials. Front. Neurol. 2022 13 818522 35386408
50 Hainsworth A.H. McNaughton N.C.L. Pereverzev A. Schneider T. Randall A.D. Actions of sipatrigine, 202W92 and lamotrigine on R-type and T-type Ca2+ channel currents. Eur. J. Pharmacol. 2003 467 77 80 12706458
51 Kuzmiski J.B. Barr W. Zamponi G.W. MacVicar B.A. Topiramate inhibits the initiation of plateau potentials in CA1 neurons by depressing R-type calcium channels. Epilepsia 2005 46 4 481 489 10.1111/j.0013-9580.2005.35304.x 15816941
52 Wormuth C. Lundt A. Henseler C. Müller R. Broich K. Papazoglou A. Weiergräber, M. m.fl. Review: Cav2.3 R-type voltage-gated Ca2+ channels - Functional implications in convulsive and non-convulsive seizure activity. Open Neurol. J. 2016 10 1 99 126 10.2174/1874205X01610010099 27843503
53 Sng B.L. Sia A.T.H. Quek K. Woo D. Lim Y. Incidence and risk factors for chronic pain after caesarean section under spinal anaesthesia. Anaesth. Intensive Care 2009 37 5 748 752 10.1177/0310057X0903700513 19775038
54 Marathe A. Allahabadi S. Abd-Elsayed A. Saulino M. Hagedorn J.M. Orhurhu V. Karri J. Intrathecal baclofen monotherapy and polyanalgesia for treating chronic pain in patients with severe spasticity. Curr. Pain Headache Rep. 2021 25 12 79 10.1007/s11916-021-00994-9 34894303
55 Kakuta N. Tsutsumi Y.M. Horikawa Y.T. Kawano H. Kinoshita M. Tanaka K. Neurokinin-1 receptor antagonism, aprepitant, effectively diminishes post-operative nausea and vomiting while increasing analgesic tolerance in laparoscopic gynecological procedures. J. Med. Invest. 2011 58 246 251 21921426
56 Gaskell H. Derry S. Moore R.A. Treating chronic non-cancer pain in older people - more questions than answers? Maturitas 2014 79 1 34 40 10.1016/j.maturitas.2014.06.013 25048719
57 Ferrari M.D. Goadsby P.J. Roon K.I. Lipton R.B. Triptans (serotonin, 5-HT1B/1D agonists) in migraine: Detailed results and methods of a meta-analysis of 53 trials. Cephalalgia 2002 22 8 633 658 10.1046/j.1468-2982.2002.00404.x 12383060
58 Mollenholt P. Rawal N. Gordh T. Jr Olsson Y. Intrathecal and epidural somatostatin for patients with cancer. Analgesic effects and postmortem neuropathologic investigations of spinal cord and nerve roots. Anesthesiology 1994 81 3 534 542 10.1097/00000542-199409000-00004 7916546
59 Sang C.N. Barnabe K.J. Kern S.E. Phase IA clinical trial evaluating the tolerability, pharmacokinetics, and analgesic efficacy of an intrathecally administered neurotensin a analogue in central neuropathic pain following spinal cord injury. Clin. Pharmacol. Drug Dev. 2016 5 250 258 27310326
60 Chung G. Rhee J.N. Jung S.J. Kim J.S. Oh S.B. Modulation of CaV2.3 calcium channel currents by eugenol. J. Dent. Res. 2008 87 2 137 141 10.1177/154405910808700201 18218839
61 Mohammadreza S. Mackenzie G. Toni W. Slobodan S. L- cysteine modulates visceral nociception mediated by the CaV2.3 R-type calcium channels. Pflugers Arch. 2022 474 4 435 445 35267086
62 Leão R.M. Cruz J.S. Diniz C.R. Cordeiro M.N. Beirão P.S.L. Inhibition of neuronal high-voltage activated calcium channels by the ω-Phoneutria nigriventer Tx3-3 peptide toxin. Neuropharmacology 2000 39 10 1756 1767 10.1016/S0028-3908(99)00267-1 10884557
63 Piekarz A.D. Due M.R. Khanna M. Wang B. Ripsch M.S. Wang R. Meroueh S.O. Vasko M.R. White F.A. Khanna R. CRMP-2 peptide mediated decrease of high and low voltageactivated calcium channels, attenuation of nociceptor excitability, and anti-nociception in a model of AIDS therapy-induced painful peripheral neuropathy. Mol. Pain 2012 8 1744-8069-8 54 10.1186/1744-8069-8-54 22828369
64 Shan Z. Cai S. Yu J. Zhang Z. Vallecillo T.G. Serafini M.J. Thomas A.M. Pham N.Y.N. Bellampalli S.S. Moutal A. Zhou Y. Xu G.B. Xu Y.M. Luo S. Patek M. Streicher J.M. Gunatilaka A.A.L. Khanna R. Reversal of peripheral neuropathic pain by the small-molecule natural product physalin F via block of CaV2.3 (R-type) and CaV2.2 (N-type) voltage-gated calcium channels. ACS Chem. Neurosci. 2019 10 6 2939 2955 10.1021/acschemneuro.9b00166 30946560
65 Bourinet E. Soong T.W. Stea A. Snutch T.P. Determinants of the G protein-dependent opioid modulation of neuronal calcium channels. Proc. Natl. Acad. Sci. USA 1996 93 4 1486 1491 10.1073/pnas.93.4.1486 8643659
66 Ottolia M. Platano D. Qin N. Noceti F. Birnbaumer M. Toro L. Birnbaumer L. Stefani E. Olcese R. Functional coupling between human E-type Ca2+ channels and μ opioid receptors expressed in Xenopus oocytes. FEBS Lett. 1998 427 1 96 102 10.1016/S0014-5793(98)00401-3 9613607
67 Berecki G. Motin L. Adams D.J. Voltage-Gated R-Type Calcium Channel Inhibition via Human μ -, δ -, and κ -opioid Receptors Is Voltage-Independently Mediated by G βγ Protein Subunits. Mol. Pharmacol. 2016 89 1 187 196 10.1124/mol.115.101154 26490245
68 Berecki G. McArthur J.R. Cuny H. Clark R.J. Adams D.J. Differential Cav2.1 and Cav2.3 channel inhibition by baclofen and alpha-conotoxin Vc1.1 via GABAB receptor activation. J. Gen. Physiol. 2014 143 465 479 24688019
69 Meza U. Thapliyal A. Bannister R.A. Adams B.A. Neurokinin 1 receptors trigger overlapping stimulation and inhibition of CaV2.3 (R-type) calcium channels. Mol. Pharmacol. 2007 71 1 284 293 10.1124/mol.106.028530 17050807
70 Morikawa T. Matsuzawa Y. Makita K. Katayama Y. Antimigraine drug, zolmitriptan, inhibits high-voltage activated calcium currents in a population of acutely dissociated rat trigeminal sensory neurons. Mol. Pain 2006 2 10 16549032
71 Mehrke G. Pereverzev A. Grabsch H. Hescheler J. Schneider T. Receptor-mediated modulation of recombinant neuronal class E calcium channels. FEBS Lett. 1997 408 3 261 270 10.1016/S0014-5793(97)00437-7 9188773
72 Rozanski G.M. Nath A.R. Adams M.E. Stanley E.F. Low voltage-activated calcium channels gate transmitter release at the dorsal root ganglion sandwich synapse. J. Physiol. 2013 591 22 5575 5583 10.1113/jphysiol.2013.260281 24000176
73 Yang L. Topia I. Schneider T. Stephens G.J. Phorbol ester modulation of Ca2+ channels mediates nociceptive transmission in dorsal horn neurones. Pharmaceuticals 2013 6 777 787 24276261
74 Dalmolin G.D. Bannister K. Gonçalves L. Sikandar S. Patel R. Cordeiro M. Effect of the spider toxin Tx3-3 on spinal processing of sensory information in naive and neuropathic rats. Pain Rep. 2017 2 e610 10.1097/PR9.0000000000000610 29392225
75 Lirk P. Modulators of calcium influx regulate membrane excitability in rat dorsal root ganglion neurons. Anesth. Analg. 2008 107 673 685 18633052
76 Fuchs A. Rigaud M. Sarantopoulos C.D. Filip P. Hogan Q.H. Contribution of calcium channel subtypes to the intracellular calcium signal in sensory neurons: The effect of injury. Anesthesiology 2007 107 1 117 127 10.1097/01.anes.0000267511.21864.93 17585223
77 McCallum J.B. Subtype-specific reduction of voltage-gated calcium current in medium-sized dorsal root ganglion neurons after painful peripheral nerve injury. Neuroscience 2011 179 1 244 255 10.1016/j.neuroscience.2011.01.049 21277351
78 Matthews E.A. Bee L.A. Stephens G.J. Dickenson A.H. The Cav2.3 calcium channel antagonist SNX-482 reduces dorsal horn neuronal responses in a rat model of chronic neuropathic pain. Eur. J. Neurosci. 2007 25 3561 3569 17610575
79 Weiergräber M. Henry M. Südkamp M. de Vivie E.R. Hescheler J. Schneider T. Ablation of Ca(v)2.3/E-type voltage-gated calcium channel results in cardiac arrhythmia and altered autonomic control within the murine cardiovascular system. Basic Res. Cardiol. 2005 100 1 1 13 10.1007/s00395-004-0488-1 15490203
80 Yokoyama K. Kurihara T. Saegusa H. Zong S. Makita K. Tanabe T. Blocking the R-type (Cav2.3) Ca2+ channel enhanced morphine analgesia and reduced morphine tolerance. Eur. J. Neurosci. 2004 20 3516 3519 15610184
81 Ferreira M.A. Lückemeyer D.D. Macedo-Júnior S.J. Schran R.G. Silva A.M. Prudente A.S. Tonello R. Ferreira J. Sex-dependent Cav2.3 channel contribution to the secondary hyperalgesia in a mice model of central sensitization. Brain Res. 2021 1764 147438 10.1016/j.brainres.2021.147438 33753067
82 Newcomb R. Szoke B. Palma A. Wang G. Chen X. Hopkins W. Cong R. Miller J. Urge L. Tarczy-Hornoch K. Loo J.A. Dooley D.J. Nadasdi L. Tsien R.W. Lemos J. Miljanich G. Selective peptide antagonist of the class E calcium channel from the venom of the tarantula Hysterocrates gigas. Biochemistry 1998 37 44 15353 15362 10.1021/bi981255g 9799496
83 Newcomb R. Chen X. Dean R. Dayanithi G. SNX-482: A novel class E calcium channel antagonist from tarantula venom. Biochemistry 2000 37 15353 15362
84 Bourinet E. Stotz S.C. Spaetgens R.L. Dayanithi G. Lemos J. Nargeot J. Zamponi G.W. Interaction of SNX482 with domains III and IV inhibits activation gating of alpha(1E) (Ca(V)2.3) calcium channels. Biophys. J. 2001 81 1 79 88 10.1016/S0006-3495(01)75681-0 11423396
85 Bishop K.M. Progress and promise of antisense oligonucleotide therapeutics for central nervous system diseases. Neuropharmacology 2017 120 56 62 10.1016/j.neuropharm.2016.12.015 27998711
86 Schoch K.M. Miller T.M. Antisense oligonucleotides: Translation from mouse models to human neurodegenerative diseases. Neuron 2017 94 6 1056 1070 10.1016/j.neuron.2017.04.010 28641106
87 Terashima T. Xu Q. Yamaguchi S. Yaksh T.L. Intrathecal P/Q- and R-type calcium channel blockade of spinal substance P release and c-Fos expression. Neuropharmacology 2013 75 1 8 10.1016/j.neuropharm.2013.06.018 23810829
88 Yang L. Stephens G.J. Effects of neuropathy on high-voltage-activated Ca2+ current in sensory neurones. Cell Calcium 2009 46 4 248 256 10.1016/j.ceca.2009.08.001 19726083
89 Dalmolin G.D. Silva C.R. Rigo F.K. Gomes G.M. do Nascimento Cordeiro M. Richardson M. Silva M.A.R. Prado M.A.M. Gomez M.V. Ferreira J. Antinociceptive effect of Brazilian armed spider venom toxin Tx3-3 in animal models of neuropathic pain. Pain 2011 152 10 2224 2232 10.1016/j.pain.2011.04.015 21570770
90 Percie du Sert N. Rice A.S.C. Improving the translation of analgesic drugs to the clinic: Animal models of neuropathic pain. Br. J. Pharmacol. 2014 171 12 2951 2963 10.1111/bph.12645 24527763
91 Hooijmans C.R. Rovers M.M. de Vries R.B.M. Leenaars M. Ritskes-Hoitinga M. Langendam M.W. SYRCLE’s risk of bias tool for animal studies. BMC Med. Res. Methodol. 2014 14 1 43 10.1186/1471-2288-14-43 24667063
92 Ide S. Nishizawa D. Fukuda K. Kasai S. Hasegawa J. Hayashida M. Minami M. Ikeda K. Association between genetic polymorphisms in Cav2.3 (R-type) Ca2+ channels and fentanyl sensitivity in patients undergoing painful cosmetic surgery. PLoS One 2013 8 8 e70694 10.1371/journal.pone.0070694 23940630
93 Amano K. Nishizawa D. Mieda T. Tsujita M. Kitamura A. Hasegawa J. Inada E. Hayashida M. Ikeda K. Opposite associations between the rs3845446 single-nucleotide polymorphism of the CACNA1E gene and postoperative pain-related phenotypes in gastrointestinal surgery versus previously reported orthognathic surgery. J. Pain 2016 17 10 1126 1134 10.1016/j.jpain.2016.07.001 27480382
94 Ambrosini A. D’Onofrio M. Buzzi M.G. Arisi I. Grieco G.S. Pierelli F. Santorelli F.M. Schoenen J. Possible involvement of the CACNA1E gene in migraine: A search for single nucleotide polymorphism in different clinical phenotypes. Headache 2017 57 7 1136 1144 10.1111/head.13107 28573794
95 Helbig K.L. Lauerer R.J. Bahr J.C. Souza I.A. Myers C.T. Schwarz N. De novo pathogenic variants in CACNA1E cause developmental and epileptic encephalopathy with contractures, macrocephaly, and dyskinesias. Am. J. Hum. Genet. 2018 103 5 666 678 30343943
