Getting a Handle on Cav2.2 (N-Type) Voltage-Gated Ca2+ Channels

COMMENTARY COMMENTARY

Getting a handle on Ca 2.2 (N-type) voltage- + V gated Ca2 channels Jörg Striessniga,1

In PNAS, Nieto-Rostro et al. (1) report a mouse model no selective small-molecule channel blockers have 2+ expressing CaV2.2 (N-type) voltage-gated Ca channels so far been approved for clinical use (3). Clinical fail- + (VGCCs) with an extracellularly accessible hemagglutinin ures have been reported for inhibitors of NaV1.7 Na (HA) epitope tag engineered into their pore-forming channels and voltage-gated CaV3.2T-type(5)and KI/KI 2+ CaV2.2 α1 subunits (i.e., CaV2.2_HA mice). This CaV2.2N-typeCa channels (6). One possible model allowed the identification of endogenous CaV2.2 explanation is limitations in the usefulness of estab- channels in the plasma membrane of peripheral somato- lished rodent pain models to predict drug responses

sensory neurons and the role of accessory α2δ-1 subunits for certain types of pain in patients (7). In addition, for their plasma membrane targeting. These mice expand our understanding of the physiological function and our biochemical tools to reveal disease-associated pain-associated dysfunction of these ion channels is

changes in the subcellular distribution of CaV2.2 chan- still limited. This is also true for CaV2.2 channels. How- nels in the pain pathway and other neurons with high ever, evidence of a promising drug target for analge- sensitivity and specificity, which can eventually help sics comes from preclinical studies showing the crucial

to answer the still-unresolved question of whether role of CaV2.2 channels for pronociceptive neurotrans- 2+ CaV2.2 Ca channels are suitable targets for novel mitter release [calcitonin gene-related peptide analgesic therapies. (CGRP), substance P, and glutamate] from primary af- Chronic pain (defined as >3 mo of pain) is a major ferent terminals in laminae I and II in the dorsal horn medical issue impairing the physical and social well- (3). The specific pharmacological inhibition or genetic

being of patients and their overall quality of life. It is ablation of CaV2.2 channels decreases pain responses estimated to affect ∼1.5 billion people globally and in models of neuropathic and inflammatory pain (8). 50 million adults in the United States (2, 3). Neuro- Clinically used drugs also point toward an involve-

pathic pain especially causes a high disease burden, ment of CaV2.2 channels in pain signaling: morphine- and therapeutic options are limited. Neuropathic pain induced analgesia involves inhibition of N-type currents

is caused by damage of the somatosensory nerves (Fig. 1) (9, 10); the potent CaV2.2 blocking peptide due to direct lesions (e.g., peripheral nerve injury), toxin ziconotide (PRIALT) is approved as a last-line infections (e.g., postherpetic neuralgia), diabetic neu- treatment for the intrathecal treatment of severe ropathy, or neurotoxic substances (e.g., antitumor chronic pain; and the analgesic effects of GBPs seem,

drugs) (4). Current treatments, like antiepileptics [e.g., in part, mediated by CaV2.2-channel inhibition, which the gabapentinoids (GBPs) gabapentin and pregaba- requires binding to α2δ-1 subunits (11). α2δ-1 subunits lin], antidepressants (amitrytiline and duloxetine), and are strongly up-regulated after nerve damage (12). Rather

topical capsaicin, are often unsatisfactory. There is than directly blocking CaV2.2 channels, in vitro studies a high unmet medical need for new analgesics, which suggest that GBPs act indirectly by reducing α2δ-1– requires not only a better understanding of how currently mediated CaV2.2-channel trafficking (Fig. 1) to the used analgesics work but also the discovery of novel presynaptic plasma membrane in dorsal-horn syn- drug targets. apses of afferent fibers, thereby inhibiting synap- Progress in clinical development of new drugs to treat tic transmission of pain signals (8). Although never severe chronic pain has been slow despite exciting proved for endogenous channels in vivo, this mech-

reports of efficacy in animal models. Ion channels, which anism suggests that CaV2.2-channel function can are key determinants of neuronal excitability, are a be inhibited not only by reducing ion flux through majortargetforanalgesicdrugdiscovery(3).How- individual channels but also by interference with ever, despite promising preclinical drug candidates, plasma membrane targeting (Fig. 1). Other examples

aDepartment of Pharmacology and Toxicology, Institute of Pharmacy, Center for Molecular Biosciences, University of Innsbruck, 6020 Innsbruck, Austria Author contributions: J.S. wrote the paper. The author declares no conflict of interest. Published under the PNAS license. See companion article on page E12043. 1Email: [email protected]. Published online December 11, 2018.

12848–12850 | PNAS | December 18, 2018 | vol. 115 | no. 51 www.pnas.org/cgi/doi/10.1073/pnas.1818608115 Downloaded by guest on September 30, 2021 2+ Fig. 1. Signaling pathways regulating CaV2.2 Ca channel activity in peripheral somatosensory neurons and the role of α2δ-1 subunits. 2+ Presynaptic CaV2.2 (N-type) Ca channels play a key role in neurotransmitter release in neurons, where they are positioned close to synaptic vesicles (8). This includes nerve terminals of primary somatosensory afferents (15). Their central pore-forming α-1 subunit requires associated accessory β and, as shown by Nieto-Rostro et al. (1) in vivo, α2δ subunits for proper function and trafficking to the plasma membrane (8). G protein-dependent inhibition contributes to opioid-induced analgesia (8) of CaV2.2 channels, and their direct block by intrathecal administration of the peptide toxin ziconotide (“Z”) produces analgesia in humans and in rodent pain models. GBPs (“G”) are approved for the treatment of some forms of neuropathic pain. In rodents, their analgesic actions require binding to α2δ-1 subunits (11). In vitro data suggest that this interferes with α2δ-1–dependent targeting of CaV2.2 channels to the plasma membrane—a hypothesis that can now be tested in vivo with the KI/KI CaV2.2_HA mouse model. Other mechanisms for indirectly inhibiting CaV2.2-channel signaling have been explored and have shown efficacy in rodent pain models. This includes peptides (blue) interfering with the cytoplasmic interaction of collapsin response mediator protein 2 (CRMP-2) with the channel (23) and a small molecule (compound 6, “6”) inhibiting β-3 subunit association (13). Meanwhile, other α2δ-1 binding proteins have been identified. An extracellular N-terminal domain of big potassium (BK) channels binds α2δ-1 subunits (black bar) and thereby may also sequester them away from (and thus inhibit) CaV2.2 channels. A BK channel N-terminus peptide negatively regulates CaV2.2 plasma membrane expression when expressed as a membrane-anchored construct (red) and its intrathecal delivery as viral construct has analgesic effects in rodents (24). Note that GBPs may also exert their analgesic actions via CaV2.2 channels in other brain areas, including central neurons of ascending or descending pain pathways (6). Moreover, GBPs can inhibit interactions between α2δ-1 and synaptogenic thrombospondin-4, which is also up- regulated in primary afferents after peripheral nerve damage and can induce aberrant excitatory synaptogenesis that may contribute to the development of chronic neuropathic pain (15). The α2δ-1 subunit also forms a complex with NMDA receptors (NMDA-Rs) in rodent and human spinal cords and promotes surface trafficking and synaptic targeting of NMDA-Rs upon nerve injury. This is inhibited by gabapentin or a peptide KI/KI interfering with complex formation (20). The CaV2.2_HA mice will be very useful for monitoring changes of CaV2.2 channel expression and plasma membrane targeting upon treatment with GBPs or novel mechanisms indirectly interfering with CaV2.2 trafficking and function.

for indirect CaV2.2 inhibition have been published and are illus- nociceptors. In the spinal cord, CaV2.2 channel expression was trated in Fig. 1. highest in the dorsal horn (in particular, in superficial laminae I To follow changes of the expression and plasma membrane and II) and very weak in the ventral horn. Staining colocalized with

targeting of CaV2.2 channels in the pain pathway induced by the presynaptic marker CGRP, with IB4, and with the postsynaptic these concepts, the sensitive and specific immunolabeling of marker of excitatory synapses, Homer. HA-tagged endogenous

CaV2.2 α-1 subunits is necessary. This has so far been hampered channels could also be labeled for superresolution and electron by the limited availability of suitable antibodies. Changes in sur- microscopy, providing evidence for presynaptic localization in ac- face expression of VGCCs are biochemically often monitored in- tive zones of glomerular synapses at primary afferent terminals. directly using, for example, surface biotinylation experiments (see As outlined above, quantitative high-resolution imaging of

ref. 13 for a recent example). CaV2.2 channels is a prerequisite to track changes of their expres- In the mouse model described in PNAS, Nieto-Rostro et al. (1) sion and trafficking in chronic pain and upon drug treatment. As a

elegantly solve this problem by taking advantage of their previous first step, the authors used these mice to test the role of α2δ- work, in which they succeeded in introducing a tandem HA anti- 1 subunits for CaV2.2 cell-surface expression. Such a role was body tag into an extracellularly accessible position (domain II S3– inferred from previous studies with epitope-tagged channel con-

S4 linker) of the CaV2.2 α-1 subunit, where it does not affect its structs expressed in HEK-293 cells or in transfected cultured neu- function (14). In their present work, they introduce this HA tag into rons (14), but it could now be tested in vivo by crossing KI/KI KI/KI the CaV2.2 α-1 subunit gene (Cacna1b) in their CaV2.2_HA CaV2.2_HA mice with α2δ-1 knockout mice. Genetic ablation mice and show that this allows specific labeling of endogenous of α2δ-1 eliminated the cell-surface staining of CaV2.2 in intact CaV2.2 channels in primary somatosensory afferents and in the DRG neurons and strongly reduced CaV2.2 staining in the dorsal spinal cord. Specific surface staining was detected in intact dorsal horn. This indicates a prominent role of α2δ-1 for CaV2.2 trafficking root ganglion (DRG) neurons in vivo in CaV2.2 mice, which was to the primary afferent presynaptic terminals. Interestingly, de- preferentially associated with small CGRP-positive nociceptors spite this reduction, expression of pre- and postsynaptic markers and much less with isolectin-B4 (IB4)-positive nonpeptidergic was unchanged, although findings from other laboratories suggest

Striessnig PNAS | December 18, 2018 | vol. 115 | no. 51 | 12849 Downloaded by guest on September 30, 2021 a key role of α2δ-1 for mediating synaptogenic effects of thrombo- to have strong effects on the physiological trafficking and func- + spondins (15) (Fig. 1). tion of VGCCs in vivo, even in cells in which Ca2 channel ex-

However, the work of Nieto-Rostro et al. (1) leaves important pression requires α2δ-1 for normal function (18, 19). In vivo, questions unanswered. How does endogenous CaV2.2 expression GBPs may only reduce or prevent excess plasmalemmal expres- and membrane targeting change in the different populations of sion (and activity) after nerve injury without affecting physiolog-

DRG neurons and in their dorsal-horn terminals after peripheral ical levels of presynaptic CaV2.2. This can be nicely tested using KI/KI nerve injury? Can the previously reported up-regulation of CaV2.2 the CaV2.2_HA mouse model. be confirmed (16, 17)? How do the analgesic effects of GBP treat- Despite their central role in regulating VGCC activity, α2δ- ment correlate with changes in CaV2.2-channel expression and 1 subunits have channel-independent functions that could also trafficking? Does gabapentin reduce CaV2.2 surface expression mediate therapeutic effects of GBPs in neuropathic pain. These similar to α2δ-1 knockout? Such experiments in pain models are functions include regulation of NMDA receptor expression (20) time-consuming, and chronic drug administration to achieve hu- and a role for synaptogenesis through their interaction with extra- man therapeutic plasma levels in mice can be challenging. How- cellular matrix thrombospondins (21, 22) (Fig. 1). ever, the authors have previously shown that gabapentin reduced Although many of the findings reported in the paper by Nieto-

the surface expression of their CaV2.2-HA construct transfected Rostro et al. (1) are expected from previous in vitro studies, the KI/KI into cultured DRG neurons (14). It would have been nice to see if CaV2.2_HA mouse will provide an opportunity to better un- this is also the case with the tagged endogenous CaV2.2 chan- derstand the aberrant function of CaV2.2 channels in human dis- nels. However, we should also keep in mind that GBPs seem not ease and their potential as drug targets.

1 Nieto-Rostro M, Ramgoolam K, Pratt WS, Kulik A, Dolphin AC (2018) Ablation of α2δ-1 inhibits cell-surface trafficking of endogenous N-type calcium channels in the pain pathway in vivo. Proc Natl Acad Sci USA 115:E12043–E12052. 2 Dahlhamer J, et al. (2018) Prevalence of chronic pain and high-impact chronic pain among adults - United States, 2016. MMWR Morb Mortal Wkly Rep 67:1001–1006. 3 Stevens EB, Stephens GJ (2018) Recent advances in targeting ion channels to treat chronic pain. Br J Pharmacol 175:2133–2137. 4 Colloca L, et al. (2017) Neuropathic pain. Nat Rev Dis Primers 3:17002. 5 Snutch TP, Zamponi GW (2018) Recent advances in the development of T-type calcium channel blockers for pain intervention. Br J Pharmacol 175:2375–2383. 6 Patel R, Montagut-Bordas C, Dickenson AH (2018) Calcium channel modulation as a target in chronic pain control. Br J Pharmacol 175:2173–2184. 7 Rice ASC, Finnerup NB, Kemp HI, Currie GL, Baron R (2018) Sensory profiling in animal models of neuropathic pain: A call for back-translation. Pain 159:819–824. 8 Zamponi GW, Striessnig J, Koschak A, Dolphin AC (2015) The physiology, pathology, and pharmacology of voltage-gated calcium channels and their future therapeutic potential. Pharmacol Rev 67:821–870. 9 Lipscombe D, Raingo J (2007) Alternative splicing matters: N-type calcium channels in nociceptors. Channels (Austin) 1:225–227. 10 Zamponi GW, Currie KP (2013) Regulation of Ca(V)2 calcium channels by G protein coupled receptors. Biochim Biophys Acta 1828:1629–1643. 11 Field MJ, et al. (2006) Identification of the alpha2-delta-1 subunit of voltage-dependent calcium channels as a molecular target for pain mediating the analgesic actions of pregabalin. Proc Natl Acad Sci USA 103:17537–17542. 12 Patel R, et al. (2013) α2δ-1 gene deletion affects somatosensory neuron function and delays mechanical hypersensitivity in response to peripheral nerve damage. J Neurosci 33:16412–16426.

13 Chen X, et al. (2018) Small-molecule CaVα1·CaVβ antagonist suppresses neuronal voltage-gated calcium-channel trafficking. Proc Natl Acad Sci USA 115:E10566–E10575. 14 Cassidy JS, Ferron L, Kadurin I, Pratt WS, Dolphin AC (2014) Functional exofacially tagged N-type calcium channels elucidate the interaction with auxiliary α2δ- 1 subunits. Proc Natl Acad Sci USA 111:8979–8984.

15 Gong N, Park J, Luo ZD (2018) Injury-induced maladaptation and dysregulation of calcium channel α2 δ subunit proteins and its contribution to neuropathic pain development. Br J Pharmacol 175:2231–2243. 16 Costigan M, Scholz J, Woolf CJ (2009) Neuropathic pain: A maladaptive response of the nervous system to damage. Annu Rev Neurosci 32:1–32. 17 Cizkova D, et al. (2002) Localization of N-type Ca2+ channels in the rat spinal cord following chronic constrictive nerve injury. Exp Brain Res 147:456–463. 18 Fuller-Bicer GA, et al. (2009) Targeted disruption of the voltage-dependent calcium channel alpha2/delta-1-subunit. Am J Physiol Heart Circ Physiol 297:H117–H124.

19 Mastrolia V, et al. (2017) Loss of α2δ-1 calcium channel subunit function increases the susceptibility for diabetes. Diabetes 66:897–907. 20 Chen J, et al. (2018) The α2δ-1-NMDA receptor complex is critically involved in neuropathic pain development and gabapentin therapeutic actions. Cell Reports 22:2307–2321. 21 Pan B, et al. (2016) Thrombospondin-4 divergently regulates voltage-gated Ca2+ channel subtypes in sensory neurons after nerve injury. Pain 157:2068–2080. 22 Yu YP, Gong N, Kweon TD, Vo B, Luo ZD (2018) Gabapentin prevents synaptogenesis between sensory and spinal cord neurons induced by thrombospondin- 2+ 4 acting on pre-synaptic CaV α2 δ1 subunits and involving T-type Ca channels. Br J Pharmacol 175:2348–2361. 23 Chew LA, Khanna R (2018) CRMP2 and voltage-gated ion channels: Potential roles in neuropathic pain. Neuronal Signaling 2, 10.1042/NS20170220. 24 Zhang F-X, Gadotti VM, Souza IA, Chen L, Zamponi GW (2018) BK potassium channels suppress Cavα2δ subunit function to reduce inflammatory and neuropathic pain. Cell Reports 22:1956–1964.

12850 | www.pnas.org/cgi/doi/10.1073/pnas.1818608115 Striessnig Downloaded by guest on September 30, 2021