R and S enantiomers of CBD3063, a CaV2.2N-type calcium channel modulator, alleviate capsaicin-induced inflammatory pain.

Mechanisms of conotoxin inhibition of N-type (Cav2.2) calcium channels

Regulation of N-type Voltage-Gated Calcium Channels and Presynaptic Function by Cyclin-Dependent Kinase 5

A Role for Runx Transcription Factor Signaling in Dorsal Root Ganglion Sensory Neuron Diversification

Intrathecal drug therapy for chronic pain: from basic science to clinical practice.

ABSTRACTAmongst the regulators of voltage-gated ion channels is the collapsin response mediator protein 2 (CRMP2). CRMP2 regulation of the activity and trafficking of NaV1.7 voltage-gated sodium channels as well as the N-type (CaV2.2) voltage-gated calcium channel (VGCC) has been reported. On the other hand, CRMP2 does not appear to regulate L- (CaV1.x), P/Q- (CaV2.1), and R- (CaV2.3) type high VGCCs. Whether CRMP2 regulates low VGCCs remains an open question. Here, we asked if CRMP2 could regulate the low voltage-gated (T-type/CaV3.x) channels in sensory neurons. Reducing CRMP2 protein levels with short interfering RNAs yielded no change in macroscopic currents carried by T-type channels. No change in biophysical properties of the T-type currents was noted. Future studies pursuing CRMP2 druggability in neuropathic pain will benefit from the findings that CRMP2 regulates only the N-type (CaV2.2) calcium channels.

Collapsin response mediator proteins (CRMPs) mediate signal transduction of neurite outgrowth and axonal guidance during neuronal development. Voltage-gated Ca(2+) channels and interacting proteins are essential in neuronal signaling and synaptic transmission during this period. We recently identified the presynaptic N-type voltage-gated Ca(2+) channel (Cav2.2) as a CRMP-2-interacting partner. Here, we investigated the effects of a functional association of CRMP-2 with Cav2.2 in sensory neurons. Cav2.2 colocalized with CRMP-2 at immature synapses and growth cones, in mature synapses and in cell bodies of dorsal root ganglion (DRG) neurons. Co-immunoprecipitation experiments showed that CRMP-2 associates with Cav2.2 from DRG lysates. Overexpression of CRMP-2 fused to enhanced green fluorescent protein (EGFP) in DRG neurons, via nucleofection, resulted in a significant increase in Cav2.2 current density compared with cells expressing EGFP. CRMP-2 manipulation changed the surface levels of Cav2.2. Because CRMP-2 is localized to synaptophysin-positive puncta in dense DRG cultures, we tested whether this CRMP-2-mediated alteration of Ca(2+) currents culminated in changes in synaptic transmission. Following a brief high-K(+)-induced stimulation, these puncta became loaded with FM4-64 dye. In EGFP and neurons expressing CRMP-2-EGFP, similar densities of FM-loaded puncta were observed. Finally, CRMP-2 overexpression in DRG increased release of the immunoreactive neurotransmitter calcitonin gene-related peptide (iCGRP) by approximately 70%, whereas siRNA targeting CRMP-2 significantly reduced release of iCGRP by approximately 54% compared with control cultures. These findings support a novel role for CRMP-2 in the regulation of N-type Ca(2+) channels and in transmitter release.

Uncoupling the protein-protein interaction between collapsin response mediator protein 2 (CRMP2) and N-type voltage-gated calcium channel (CaV2.2) with an allosteric CRMP2-derived peptide (CBD3) is antinociceptive in rodent models of inflammatory and neuropathic pain. We investigated the efficacy, duration of action, abuse potential, and neurobehavioral toxicity of an improved mutant CRMP2 peptide. A homopolyarginine (R9)-conjugated CBD3-A6K (R9-CBD3-A6K) peptide inhibited the CaV2.2-CRMP2 interaction in a concentration-dependent fashion and diminished surface expression of CaV2.2 and depolarization-evoked Ca influx in rat dorsal root ganglia neurons. In vitro studies demonstrated suppression of excitability of small-to-medium diameter dorsal root ganglion and inhibition of subtypes of voltage-gated Ca channels. Sprague-Dawley rats with tibial nerve injury had profound and long-lasting tactile allodynia and ongoing pain. Immediate administration of R9-CBD3-A6K produced enhanced dopamine release from the nucleus accumbens shell selectively in injured animals, consistent with relief of ongoing pain. R9-CBD3-A6K, when administered repeatedly into the central nervous system ventricles of naive rats, did not result in a positive conditioned place preference demonstrating a lack of abusive liability. Continuous subcutaneous infusion of R9-CBD3-A6K over a 24- to 72-hour period reversed tactile allodynia and ongoing pain, demonstrating a lack of tolerance over this time course. Importantly, continuous infusion of R9-CBD3-A6K did not affect motor activity, anxiety, depression, or memory and learning. Collectively, these results validate the potential therapeutic significance of targeting the CaV-CRMP2 axis for treatment of neuropathic pain.

Inflammatory pain is a common type of pathological pain. Although the dorsal root ganglion (DRG) is key to pathogenesis of inflammatory pain, the underlying specific molecular and cellular mechanisms remain unclear. In this study, we used mouse models of acute or chronic inflammatory pain, induced by formalin or complete Freund’ s adjuvant (CFA), respectively, to explore whether tyrosine kinase receptor ErbB4 participates in the pathogenesis of inflammatory pain. Firstly, we found that both the expression of Neuregulin 1 (Nrg1) and phosphorylation of ErbB4 receptor were upregulated in DRG after inflammatory pain, implying the activation of ErbB4 in DRG. Using ErbB4-mutant mice, we found reduced pain sensitivity of mice when ErbB4 gene expression was largely ablated; furthermore, ErbB4 deletion decreased the inflammatory pain hypersensitivity of either formalin- or CFA-induced mouse models. Moreover, the pain sensitivity was reduced in mice with specific deletion of ErbB4 on advillin-positive neurons within DRG. Importantly, pain hypersensitivity also decreased in Advillin-Cre;ErbB4-/- cKO mice after formalin- or CFA-induced inflammatory pain. Finally, gene quantification differential expression analysis, using RNAseq technology in combination with GO and KEGG enrichment analysis, suggested that calcium signaling pathway possibly mediated the roles of ErbB4 on DRG sensory neurons in inflammatory pain models. Together, these results indicate that ErbB4 on advillin-positive sensory neurons enhances inflammatory pain sensitivity, providing new clues towards the pathogenic mechanisms of inflammatory pain.

N-type voltage-gated calcium (Cav 2.2) channels are critical determinants of increased neuronal excitability and neurotransmission accompanying persistent neuropathic pain. Although Cav 2.2 channel antagonists are recommended as first-line treatment for neuropathic pain, calcium-current blocking gabapentinoids inadequately alleviate chronic pain symptoms and often exhibit numerous side effects. Collapsin response mediator protein 2 (CRMP2) targets Cav 2.2 channels to the sensory neuron membrane and allosterically modulates their function. A 15-amino-acid peptide (CBD3), derived from CRMP2, disrupts the functional protein-protein interaction between CRMP2 and Cav 2.2 channels to inhibit calcium influx, transmitter release and acute, inflammatory and neuropathic pain. Here, we have mapped the minimal domain of CBD3 necessary for its antinociceptive potential. Truncated as well as homology-guided mutant versions of CBD3 were generated and assessed using depolarization-evoked calcium influx in rat dorsal root ganglion neurons, binding between CRMP2 and Cav 2.2 channels, whole-cell voltage clamp electrophysiology and behavioural effects in two models of experimental pain: post-surgical pain and HIV-induced sensory neuropathy induced by the viral glycoprotein 120. The first six amino acids within CBD3 accounted for all in vitro activity and antinociception. Spinal administration of a prototypical peptide (TAT-CBD3-L5M) reversed pain behaviours. Homology-guided mutational analyses of these six amino acids identified at least two residues, Ala1 and Arg4, as being critical for antinociception in two pain models. These results identify an antinociceptive scaffold core in CBD3 that can be used for development of low MW mimetics of CBD3. This article is part of a themed section on Recent Advances in Targeting Ion Channels to Treat Chronic Pain. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.12/issuetoc.

Graded Activity of Transcription Factor Runx3 Specifies the Laminar Termination Pattern of Sensory Axons in the Developing Spinal Cord

ABSTRACTWe have previously reported that the microtubule-associated collapsin response mediator protein 2 (CRMP2) is necessary for the expression of chronic pain. CRMP2 achieves this control of nociceptive signaling by virtue of its ability to regulate voltage-gated calcium and sodium channels. To date, however, no drugs exist that target CRMP2. Recently, the small molecule edonerpic maleate (1 -{3-[2-(1-benzothiophen-5-yl)ethoxy]propyl}azetidin-3-ol maleate), a candidate therapeutic for Alzheimer’s disease was reported to be a novel CRMP2 binding compound with the potential to decrease its phosphorylation level in cortical tissues in vivo. Here we sought to determine the mechanism of action of edonerpic maleate and test its possible effect in a rodent model of chronic pain. We observed: (i) no binding between human CRMP2 and edonerpic maleate; (ii) edonerpic maleate had no effect on CRMP2 expression and phosphorylation in dorsal root ganglion (DRG) neurons; (iii) edonerpic maleate-decreased calcium but increased sodium current density in DRG neurons; and (iv) edonerpic maleate was ineffective in reversing post-surgical allodynia in male and female mice. Thus, while CRMP2 inhibiting compounds remain a viable strategy for developing new mechanism-based pain inhibitors, edonerpic maleate is an unlikely candidate.

Neurofibromatosis type 1 (NF1), a genetic disorder linked to inactivating mutations or a homozygous deletion of the Nf1 gene, is characterized by tumorigenesis, cognitive dysfunction, seizures, migraine, and pain. Omic studies on human NF1 tissues identified an increase in the expression of collapsin response mediator protein 2 (CRMP2), a cytosolic protein reported to regulate the trafficking and activity of presynaptic N-type voltage-gated calcium (Cav2.2) channels. Because neurofibromin, the protein product of the Nf1 gene, binds to and inhibits CRMP2, the neurofibromin-CRMP2 signaling cascade will likely affect Ca channel activity and regulate nociceptive neurotransmission and in vivo responses to noxious stimulation. Here, we investigated the function of neurofibromin-CRMP2 interaction on Cav2.2. Mapping of >275 peptides between neurofibromin and CRMP2 identified a 15-amino acid CRMP2-derived peptide that, when fused to the tat transduction domain of HIV-1, inhibited Ca influx in dorsal root ganglion neurons. This peptide mimics the negative regulation of CRMP2 activity by neurofibromin. Neurons treated with tat-CRMP2/neurofibromin regulating peptide 1 (t-CNRP1) exhibited a decreased Cav2.2 membrane localization, and uncoupling of neurofibromin-CRMP2 and CRMP2-Cav2.2 interactions. Proteomic analysis of a nanodisc-solubilized membrane protein library identified syntaxin 1A as a novel CRMP2-binding protein whose interaction with CRMP2 was strengthened in neurofibromin-depleted cells and reduced by t-CNRP1. Stimulus-evoked release of calcitonin gene-related peptide from lumbar spinal cord slices was inhibited by t-CNRP1. Intrathecal administration of t-CNRP1 was antinociceptive in experimental models of inflammatory, postsurgical, and neuropathic pain. Our results demonstrate the utility of t-CNRP1 to inhibit CRMP2 protein-protein interactions for the potential treatment of pain.

The naked mole-rat (NMR, Heterocephalus glaber) is a subterranean rodent that exhibits a range of unusual physiological traits, including diminished inflammatory pain. For example, nerve growth factor (NGF), a key inflammatory mediator, fails to induce sensitization of sensory neurons and thermal hyperalgesia in NMRs. This lack of NGF-induced neuronal sensitization and thermal hyperalgesia results from hypofunctional signaling of the NGF receptor, tropomyosin receptor kinase A (TrkA). Like NGF-TrkA signaling, the neurotrophic factor artemin, a member of the glial cell line-derived neurotrophic factor (GDNF) family, is implicated in mediating inflammatory pain through its receptor, GDNF family receptor α3 (GFRα3), which is expressed by a subset of dorsal root ganglia (DRG) sensory neurons. Here we investigated GFRα3 expression in DRG neurons of mice and NMRs, as well as measuring the impact of artemin on DRG sensory neuron function in both species in vitro. Using immunohistochemistry, we observed a similar abundance of GFRα3 in mouse and NMR DRG sensory neurons, high coexpression with the transient receptor potential vanilloid 1 (TRPV1) ion channel suggesting that these neurons are nociceptive neurons. Using in vitro electrophysiology to record from cultured DRG sensory neurons, we observed that artemin induced depolarization of the resting membrane potential and decreased the rheobase in both species, as well as diminishing the degree of TRPV1 desensitization to multiple capsaicin stimuli. Overall, results indicate that artemin similarly sensitizes sensory neurons in both mice and NMRs, future in vivo studies being required to confirm if the conserved in vitro sensitization also occurs in vivo.

In addition to many important roles for Cdk5 in brain development and synaptic function, we reported previously that Cdk5 regulates inflammatory pain signaling, partly through phosphorylation of transient receptor potential vanilloid 1 (TRPV1), an important Na(+)/Ca(2+) channel expressed in primary nociceptive afferent nerves. Because TGF-β regulates inflammatory processes and its receptor is expressed in TRPV1-positive afferents, we studied the cross-talk between these two pathways in sensory neurons during experimental peripheral inflammation. We demonstrate that TGF-β1 increases transcription and protein levels of the Cdk5 co-activator p35 through ERK1/2, resulting in an increase in Cdk5 activity in rat B104 neuroblastoma cells. Additionally, TGF-β1 enhances the capsaicin-induced Ca(2+) influx in cultured primary neurons from dorsal root ganglia (DRG). Importantly, Cdk5 activity was reduced in the trigeminal ganglia and DRG of 14-day-old TGF-β1 knock-out mice, resulting in reduced Cdk5-dependent phosphorylation of TRPV1. The decreased Cdk5 activity is associated with attenuated thermal hyperalgesia in TGF-β1 receptor conditional knock-out mice, where TGF-β signaling is significantly reduced in trigeminal ganglia and DRG. Collectively, our results indicate that active cross-talk between the TGF-β and Cdk5 pathways contributes to inflammatory pain signaling.

Targeting proteins within the N-type voltage-gated calcium channel (CaV2.2) complex has proven to be an effective strategy for developing novel pain therapeutics. We describe a novel peptide aptamer derived from the collapsin response mediator protein 2 (CRMP2), a CaV2.2-regulatory protein. Addition of a 14-carbon myristate group to the peptide (myr-tat-CBD3) tethered it to the membrane of primary sensory neurons near surface CaV2.2. Pull-down studies demonstrated that myr-tat-CBD3 peptide interfered with the CRMP2-CaV2.2 interaction. Quantitative confocal immunofluorescence revealed a pronounced reduction of CaV2.2 trafficking after myr-tat-CBD3 treatment and increased efficiency in disrupting CRMP2-CaV2.2 colocalization compared with peptide tat-CBD3. Consequently, myr-tat-CBD3 inhibited depolarization-induced calcium influx in sensory neurons. Voltage clamp electrophysiology experiments revealed a reduction of Ca, but not Na, currents in sensory neurons after myr-tat-CBD3 exposure. Current clamp electrophysiology experiments demonstrated a reduction in excitability of small-diameter dorsal root ganglion neurons after exposure to myr-tat-CBD3. Myr-tat-CBD3 was effective in significantly attenuating carrageenan-induced thermal hypersensitivity and reversing thermal hypersensitivity induced by a surgical incision of the plantar surface of the rat hind paw, a model of postoperative pain. These effects are compared with those of tat-CBD3-the nonmyristoylated tat-conjugated CRMP2 peptide as well as scrambled versions of CBD3 and CBD3-lacking control peptides. Our results demonstrate that the myristoyl tag enhances intracellular delivery and local concentration of the CRMP2 peptide aptamer near membrane-delimited calcium channels resulting in pronounced interference with the calcium channel complex, superior suppression of calcium influx, and better antinociceptive potential.