A peptidomimetic modulator of the CaV2.2 N-type calcium channel for chronic pain.

Transmembrane Cav2.2 (N-type) voltage-gated calcium channels are genetically and pharmacologically validated, clinically relevant pain targets. Clinical block of Cav2.2 (e.g., with Prialt/Ziconotide) or indirect modulation [e.g., with gabapentinoids such as Gabapentin (GBP)] mitigates chronic pain but is encumbered by side effects and abuse liability. The cytosolic auxiliary subunit collapsin response mediator protein 2 (CRMP2) targets Cav2.2 to the sensory neuron membrane and regulates their function via an intrinsically disordered motif. A CRMP2-derived peptide (CBD3) uncouples the Cav2.2-CRMP2 interaction to inhibit calcium influx, transmitter release, and pain. We developed and applied a molecular dynamics approach to identify the A1R2 dipeptide in CBD3 as the anchoring Cav2.2 motif and designed pharmacophore models to screen 27 million compounds on the open-access server ZincPharmer. Of 200 curated hits, 77 compounds were assessed using depolarization-evoked calcium influx in rat dorsal root ganglion neurons. Nine small molecules were tested electrophysiologically, while one (CBD3063) was also evaluated biochemically and behaviorally. CBD3063 uncoupled Cav2.2 from CRMP2, reduced membrane Cav2.2 expression and Ca2+ currents, decreased neurotransmission, reduced fiber photometry-based calcium responses in response to mechanical stimulation, and reversed neuropathic and inflammatory pain across sexes in two different species without changes in sensory, sedative, depressive, and cognitive behaviors. CBD3063 is a selective, first-in-class, CRMP2-based peptidomimetic small molecule, which allosterically regulates Cav2.2 to achieve analgesia and pain relief without negative side effect profiles. In summary, CBD3063 could potentially be a more effective alternative to GBP for pain relief.

Distinct Role of Mono-2-ethylhexyl Phthalate in Neuronal Transmission in Rat CA3 Hippocampal Neurons: Involvement of Ion Channels.

Mono-(2-ethylhexyl) phthalate (MEHP) is one of the main active metabolites of di-(2-ethylhexyl) phthalate (DEHP). In our previous works, by using rat and Drosophila models, we showed a disruption of neural function due to DEHP. However, the exact neural effects of MEHP are still unclear. To explore the effects of MEHP on the central nervous system, the electrophysiological properties of spontaneous action potential (sAP), mini-excitatory postsynaptic currents (mEPSCs), ion channels, including Na+, Ca2+, and K+ channels from rat CA3 hippocampal neurons area were assessed. Our data showed that MEHP (at the concentrations of 100 or 300 µM) decreased the amplitude of sAP and the frequency of mEPSCs. Additionally, MEHP (100 or 300 µM) significantly reduced the peak current density of Ca2+ channels, whereas only the concentration of 300 µM decreased the peak current density of Na+ and K+ channels. Therefore, our results indicate that exposure to MEHP could affect the neuronal excitability and synaptic plasticity of rat CA3 hippocampal neurons by inhibiting ion channels' activity, implying the distinct role of MEHP in neural transmission.

Cdk5 phosphorylates CaV1.3 channels and regulates GABAA-mediated miniature inhibitory post-synaptic currents in striato-nigral terminals.

Neurotransmission is one of the most important processes in neuronal communication and depends largely on Ca2+ entering synaptic terminals through voltage-gated Ca2+ (CaV) channels. Although the contribution of L-type CaV channels in neurotransmission has not been unambiguously established, increasing evidence suggests a role for these proteins in noradrenaline, dopamine, and GABA release. Here we report the regulation of L-type channels by Cdk5, and its possible effect on GABA release in the substantia nigra pars reticulata (SNpr). Using patch-clamp electrophysiology, we show that Cdk5 inhibition by Olomoucine significantly increases current density through CaV1.3 (L-type) channels heterologously expressed in HEK293 cells. Likewise, in vitro phosphorylation showed that Cdk5 phosphorylates residue S1947 in the C-terminal region of the pore-forming subunit of CaV1.3 channels. Consistent with this, the mutation of serine into alanine (S1947A) prevented the regulation of Cdk5 on CaV1.3 channel activity. Our data also revealed that the inhibition of Cdk5 increased the frequency of high K+-evoked miniature inhibitory postsynaptic currents in rat SNpr neurons, acting on L-type channels. These results unveil a novel regulatory mechanism of GABA release in the SNpr that involves a direct action of Cdk5 on L-type channels.

Coexistence of D3 R typical and atypical signaling in striatonigral neurons during dopaminergic denervation. Correlation with D3 nf expression changes.

Dopamine D3 R are widely expressed in basal ganglia where interact with D1 R. D3 R potentiate cAMP accumulation and GABA release stimulated by D1 R in striatonigral neurons through "atypical" signaling. During dopaminergic denervation, D3 R signaling changes to a "typical" in which antagonizes the effects of D1 R, the mechanisms of this switching are unknown. D3 nf splice variant regulates membrane anchorage and function of D3 R and decreases in denervation; thus, it is possible that D3 R signaling switching correlates with changes in D3 nf expression and increases of membranal D3 R that mask D3 R atypical effects. We performed experiments in unilaterally 6-hydroxydopamine lesioned rats and found a decrease in mRNA and protein of D3 nf, but not of D3 R in the denervated striatum. Proximity ligation assay showed that D3 R-D3 nf interaction decreased after denervation, whereas binding revealed an increased Bmax in D3 R. The new D3 R antagonized cAMP accumulation and GABA release stimulated by D1 R; however, in the presence of N-Ethylmaleimide (NEM), to block Gi protein signaling, activation of D3 R produced its atypical signaling stimulating D1 R effects. Finally, we investigated if the typical and atypical effects of D3 R modulating GABA release are capable of influencing motor behavior. Injections of D3 R agonist into denervated nigra decreased D1 R agonist-induced turning behavior but potentiated it in the presence of NEM. Our data indicate the coexistence of D3 R typical and atypical signaling in striatonigral neurons during denervation that correlated with changes in the ratio of expression of D3 nf and D3 R isoforms. The coexistence of both atypical and typical signaling during denervation influences motor behavior.

Dopaminergic denervation switches dopamine D3 receptor signaling and disrupts its Ca(2+) dependent modulation by CaMKII and calmodulin in striatonigral projections of the rat.

In striatonigral projections activation of dopamine D3 receptors (D3Rs) potentiates the stimulation of GABA release and cAMP production caused by activation of dopamine D1 receptors (D1Rs). Cytoplasmic [Ca(2+)] in the terminals controls this response by modulating CaMKII, an enzyme that depresses D3R action. To examine the effects of dopamine deprivation on D3R signaling we investigated their function in striatonigral terminals of hemiparkinsonian rats. Denervation switched the signaling cascade initiated by D3R activation. In the non-lesioned side activation of D3R potentiated the stimulatory effects of D1R activation on cAMP production and K(+)-depolarization induced [(3)H] GABA release. In contrast, in the denervated side the stimulatory effects of both D1R activation and forskolin administration were blocked by D3R activation. In non-lesioned slices, D3R responses were inhibited by the activation of CaMKII produced by K(+)-depolarization (via increased Ca(2+) entry). The CaMKII-induced inhibition was blocked by the selective inhibitor KN-62. In denervated tissues the response to D3R stimulation was not modified either by K(+) depolarization or by blocking CaMKII with KN-62. Immunoblotting studies showed that depolarization-induced CaMKII binding to the D3 receptor and CaMKII phosphorylation were suppressed in denervated tissues. We also determined calmodulin expression with PCR and immunoblot techniques. Both techniques showed that calmodulin expression was depressed in the lesioned side. In sum, our studies show that dopaminergic denervation switches the D3R signaling cascade and depresses CaMKII signaling through a process that appears to involve reduced calmodulin levels. Since calmodulin is a major cytoplasmic Ca(2+) buffer our findings suggest that abnormal Ca(2+) buffering may be an important component of the abnormalities observed during dopaminergic denervation.

Mouse models of non-dystrophic and dystrophic myotonia exhibit nociplastic pain-like behaviors.

Pain is a prominent and debilitating symptom in myotonic disorders, yet its physiological mechanisms remain poorly understood. This study assessed preclinical pain-like behavior in murine models of pharmacologically induced myotonia and myotonic dystrophy type 1 (DM1). In both myotonia congenita and DM1, impairment of the CLCN1 gene, which encodes skeletal muscle voltage-gated CLC-1 chloride channels, reduces chloride ion conductance in skeletal muscle cells, leading to prolonged muscle excitability and delayed relaxation after contraction. We used the CLC-1 antagonist anthracene-9-carboxylic acid (9-AC) at intraperitoneal doses of 30 or 60 mg/kg and HSA LR20b DM1 mice to model CLC-1-induced myotonia. Our experimental approach included in vivo pain behavioral testing, ex vivo calcium imaging, and whole-cell current-clamp electrophysiology in mouse dorsal root ganglion (DRG) neurons. A single injection of 9-AC induced myotonia in mice, which persisted for several hours and resulted in long-lasting allodynic pain-like behavior. Similarly, HSA LR20b mice exhibited both allodynia and hyperalgesia. Despite these pain-like behaviors, DRG neurons did not show signs of hyperexcitability in either myotonic model. These findings suggest that myotonia induces nociplastic pain-like behavior in preclinical rodents, likely through central sensitization mechanisms rather than peripheral sensitization. This study provides insights into the pathophysiology of pain in myotonic disorders and highlights the potential of using myotonic mouse models to explore pain mechanisms and assess novel analgesics. Future research should focus on the central mechanisms involved in myotonia-induced pain and develop targeted therapies to alleviate this significant clinical burden.

Regulating neuronal excitability: The role of S-palmitoylation in NaV1.7 activity and voltage sensitivity.

S-palmitoylation, a reversible lipid post-translational modification, regulates the functions of numerous proteins. Voltage-gated sodium channels (NaVs), pivotal in action potential generation and propagation within cardiac cells and sensory neurons, can be directly or indirectly modulated by S-palmitoylation, impacting channel trafficking and function. However, the role of S-palmitoylation in modulating NaV1.7, a significant contributor to pain pathophysiology, has remained unexplored. Here, we addressed this knowledge gap by investigating if S-palmitoylation influences NaV1.7 channel function. Acyl-biotin exchange assays demonstrated that heterologously expressed NaV1.7 channels are modified by S-palmitoylation. Blocking S-palmitoylation with 2-bromopalmitate resulted in reduced NaV1.7 current density and hyperpolarized steady-state inactivation. We identified two S-palmitoylation sites within NaV1.7, both located in the second intracellular loop, which regulated different properties of the channel. Specifically, S-palmitoylation of cysteine 1126 enhanced NaV1.7 current density, while S-palmitoylation of cysteine 1152 modulated voltage-dependent inactivation. Blocking S-palmitoylation altered excitability of rat dorsal root ganglion neurons. Lastly, in human sensory neurons, NaV1.7 undergoes S-palmitoylation, and the attenuation of this post-translational modification results in alterations in the voltage-dependence of activation, leading to decreased neuronal excitability. Our data show, for the first time, that S-palmitoylation affects NaV1.7 channels, exerting regulatory control over their activity and, consequently, impacting rodent and human sensory neuron excitability. These findings provide a foundation for future pharmacological studies, potentially uncovering novel therapeutic avenues in the modulation of S-palmitoylation for NaV1.7 channels.

Targeted transcriptional upregulation of SENP1 by CRISPR activation enhances deSUMOylation pathways to elicit antinociception in the spinal nerve ligation model of neuropathic pain.

ABSTRACT: The voltage-gated sodium channel Na V 1.7 is an essential component of human pain signaling. Changes in Na V 1.7 trafficking are considered critical in the development of neuropathic pain. SUMOylation of collapsin response mediator protein 2 (CRMP2) regulates the membrane trafficking and function of Na V 1.7. Enhanced CRMP2 SUMOylation in neuropathic pain correlates with increased Na V 1.7 activity. Pharmacological and genetic interventions that interfere with CRMP2 SUMOylation in rodents with neuropathic pain have been shown to reverse mechanical allodynia. Sentrin or SUMO-specific proteases (SENPs) are vital for balancing SUMOylation and deSUMOylation of substrates. Overexpression of SENP1 and/or SENP2 in CRMP2-expressing cells results in increased deSUMOylation and decreased membrane expression and currents of Na V 1.7. Although SENP1 is present in the spinal cord and dorsal root ganglia, its role in regulating Na V 1.7 function and pain is not known. We hypothesized that favoring SENP1 expression can enhance CRMP2 deSUMOylation to modulate Na V 1.7 channels. In this study, we used a clustered regularly interspaced short palindromic repeats activation (CRISPRa) SENP1 lentivirus to overexpress SENP1 in dorsal root ganglia neurons. We found that SENP1 lentivirus reduced CRMP2 SUMOylation, Na V 1.7-CRMP2 interaction, and Na V 1.7 membrane expression. SENP1 overexpression decreased Na V 1.7 currents through clathrin-mediated endocytosis, directly linked to CRMP2 deSUMOylation. Moreover, enhancing SENP1 expression did not affect the activity of TRPV1 channels or voltage-gated calcium and potassium channels. Intrathecal injection of CRISPRa SENP1 lentivirus reversed mechanical allodynia in male and female rats with spinal nerve injury. These results provide evidence that the pain-regulating effects of SENP1 overexpression involve, in part, the modulation of Na V 1.7 channels through the indirect mechanism of CRMP2 deSUMOylation.

Intranasal CRMP2-Ubc9 inhibitor regulates Na V 1.7 to alleviate trigeminal neuropathic pain.

ABSTRACT: Dysregulation of voltage-gated sodium Na V 1.7 channels in sensory neurons contributes to chronic pain conditions, including trigeminal neuropathic pain. We previously reported that chronic pain results in part from increased SUMOylation of collapsin response mediator protein 2 (CRMP2), leading to an increased CRMP2/Na V 1.7 interaction and increased functional activity of Na V 1.7. Targeting this feed-forward regulation, we developed compound 194 , which inhibits CRMP2 SUMOylation mediated by the SUMO-conjugating enzyme Ubc9. We further demonstrated that 194 effectively reduces the functional activity of Na V 1.7 channels in dorsal root ganglia neurons and alleviated inflammatory and neuropathic pain. Here, we used a comprehensive array of approaches, encompassing biochemical, pharmacological, genetic, electrophysiological, and behavioral analyses, to assess the functional implications of Na V 1.7 regulation by CRMP2 in trigeminal ganglia (TG) neurons. We confirmed the expression of Scn9a , Dpysl2 , and UBE2I within TG neurons. Furthermore, we found an interaction between CRMP2 and Na V 1.7, with CRMP2 being SUMOylated in these sensory ganglia. Disrupting CRMP2 SUMOylation with compound 194 uncoupled the CRMP2/Na V 1.7 interaction, impeded Na V 1.7 diffusion on the plasma membrane, and subsequently diminished Na V 1.7 activity. Compound 194 also led to a reduction in TG neuron excitability. Finally, when intranasally administered to rats with chronic constriction injury of the infraorbital nerve, 194 significantly decreased nociceptive behaviors. Collectively, our findings underscore the critical role of CRMP2 in regulating Na V 1.7 within TG neurons, emphasizing the importance of this indirect modulation in trigeminal neuropathic pain.

Mechanism, and treatment of anti-CV2/CRMP5 autoimmune pain.

Paraneoplastic neurological syndromes arise from autoimmune reactions against nervous system antigens due to a maladaptive immune response to a peripheral cancer. Patients with small cell lung carcinoma or malignant thymoma can develop an autoimmune response against the CV2/collapsin response mediator protein 5 (CRMP5) antigen. For reasons that are not understood, approximately 80% of patients experience painful neuropathies. Here, we investigated the mechanisms underlying anti-CV2/CRMP5 autoantibodies (CV2/CRMP5-Abs)-related pain. We found that patient-derived CV2/CRMP5-Abs can bind to their target in rodent dorsal root ganglia (DRG) and superficial laminae of the spinal cord. CV2/CRMP5-Abs induced DRG neuron hyperexcitability and mechanical hypersensitivity in rats that were abolished by preventing binding to their cognate autoantigen CRMP5. The effect of CV2/CRMP5-Abs on sensory neuron hyperexcitability and mechanical hypersensitivity observed in patients was recapitulated in rats using genetic immunization providing an approach to rapidly identify possible therapeutic choices for treating autoantibody-induced pain including the repurposing of a monoclonal anti-CD20 antibody that selectively deplete B-lymphocytes. These data reveal a previously unknown neuronal mechanism of neuropathic pain in patients with paraneoplastic neurological syndromes resulting directly from CV2/CRMP5-Abs-induced nociceptor excitability. CV2/CRMP5-Abs directly sensitize pain responses by increasing sensory neuron excitability and strategies aiming at either blocking or reducing CV2/CRMP5-Abs can treat pain as a comorbidity in patients with paraneoplastic neurological syndromes.

Argentatin C Analogues with Potential Antinociceptive Activity and Other Triterpenoid Constituents from the Aerial Parts of Parthenium incanum.

Four new triterpenes, 25-dehydroxy-25-methoxyargentatin C (1), 20S-hydroxyargentatin C (2), 20S-hydroxyisoargentatin C (3), and 24-epi-argentatin C (4), together with 10 known triterpenes (5-14) were isolated from the aerial parts of Parthenium incanum. The structures of 1-4 were elucidated by detailed analysis of their spectroscopic data, and the known compounds 5-14 were identified by comparison of their spectroscopic data with those reported. Since argentatin C (11) was found to exhibit antinociceptive activity by decreasing the excitability of rat and macaque dorsal root ganglia (DRG) neurons, 11 and its new analogues 1-4 were evaluated for their ability to decrease the excitability of rat DRG neurons. Of the argentatin C analogues tested, 25-dehydroxy-25-methoxyargentatin C (1) and 24-epi-argentatin C (4) decreased neuronal excitability in a manner comparable to 11. Preliminary structure-activity relationships for the action potential-reducing effects of argentatin C (11) and its analogues 1-4, and their predicted binding sites in pain-relevant voltage-gated sodium and calcium channels (VGSCs and VGCCs) in DRG neurons are presented.

Betulinic acid analogs inhibit N- and T-type voltage-gated calcium channels to attenuate nerve-injury associated neuropathic and formalin models of pain.

Over the past three decades, there has been a significant growth in the use of natural products, with approximately 80% of individuals using them for some aspect of primary healthcare. Our laboratories have identified and studied natural compounds with analgesic effects from dry land plants or their associated fungus during the past ten years. Here, we isolated and characterized thirteen betulin analogs and fifteen betulinic acid analogs for their capacity to prevent calcium influx brought on by depolarization in sensory neurons. The in vitro inhibition of voltage-gated calcium channels by the top drugs was then assessed using whole cell patch clamp electrophysiology. In vivo experiments, conducted at two sites, evaluated the best compound in acute and tonic, neuropathic, inflammatory, post-operative and visceral models of pain. We found that the betulinic acid analog 8 inhibited calcium influx in rat dorsal root ganglion neurons by inhibiting N- (CaV2.2) and T- (CaV3) type voltage-gated calcium channels. Moreover, intrathecal delivery of analog 8 had analgesic activity in both spared nerve injury model of neuropathic pain and acute and tonic pain induced by formalin. The results presented herein highlight the potential antinociceptive properties of betulinic acid analog 8 and set the stage for the development of novel non-opioid pain therapeutics based on the triterpenoid scaffold of betulinic acid.

Intranasal CRMP2-Ubc9 Inhibitor Regulates Na V 1.7 to Alleviate Trigeminal Neuropathic Pain.

Dysregulation of voltage-gated sodium Na V 1.7 channels in sensory neurons contributes to chronic pain conditions, including trigeminal neuropathic pain. We previously reported that chronic pain results in part from increased SUMOylation of collapsin response mediator protein 2 (CRMP2), leading to an increased CRMP2/Na V 1.7 interaction and increased functional activity of Na V 1.7. Targeting this feed-forward regulation, we developed compound 194 , which inhibits CRMP2 SUMOylation mediated by the SUMO-conjugating enzyme Ubc9. We further demonstrated that 194 effectively reduces the functional activity of Na V 1.7 channels in dorsal root ganglia neurons and alleviated inflammatory and neuropathic pain. Here, we employed a comprehensive array of investigative approaches, encompassing biochemical, pharmacological, genetic, electrophysiological, and behavioral analyses, to assess the functional implications of Na V 1.7 regulation by CRMP2 in trigeminal ganglia (TG) neurons. We confirmed the expression of Scn9a , Dpysl2 , and UBE2I within TG neurons. Furthermore, we found an interaction between CRMP2 and Na V 1.7, with CRMP2 being SUMOylated in these sensory ganglia. Disrupting CRMP2 SUMOylation with compound 194 uncoupled the CRMP2/Na V 1.7 interaction, impeded Na V 1.7 diffusion on the plasma membrane, and subsequently diminished Na V 1.7 activity. Compound 194 also led to a reduction in TG neuron excitability. Finally, when intranasally administered to rats with chronic constriction injury of the infraorbital nerve (CCI-ION), 194 significantly decreased nociceptive behaviors. Collectively, our findings underscore the critical role of CRMP2 in regulating Na V 1.7 within TG neurons, emphasizing the importance of this indirect modulation in trigeminal neuropathic pain.

Neuropilin-1 is essential for vascular endothelial growth factor A-mediated increase of sensory neuron activity and development of pain-like behaviors.

ABSTRACT: Neuropilin-1 (NRP-1) is a transmembrane glycoprotein that binds numerous ligands including vascular endothelial growth factor A (VEGFA). Binding of this ligand to NRP-1 and the co-receptor, the tyrosine kinase receptor VEGFR2, elicits nociceptor sensitization resulting in pain through the enhancement of the activity of voltage-gated sodium and calcium channels. We previously reported that blocking the interaction between VEGFA and NRP-1 with the Spike protein of SARS-CoV-2 attenuates VEGFA-induced dorsal root ganglion (DRG) neuronal excitability and alleviates neuropathic pain, pointing to the VEGFA/NRP-1 signaling as a novel therapeutic target of pain. Here, we investigated whether peripheral sensory neurons and spinal cord hyperexcitability and pain behaviors were affected by the loss of NRP-1. Nrp-1 is expressed in both peptidergic and nonpeptidergic sensory neurons. A CRIPSR/Cas9 strategy targeting the second exon of nrp-1 gene was used to knockdown NRP-1. Neuropilin-1 editing in DRG neurons reduced VEGFA-mediated increases in CaV2.2 currents and sodium currents through NaV1.7. Neuropilin-1 editing had no impact on voltage-gated potassium channels. Following in vivo editing of NRP-1, lumbar dorsal horn slices showed a decrease in the frequency of VEGFA-mediated increases in spontaneous excitatory postsynaptic currents. Finally, intrathecal injection of a lentivirus packaged with an NRP-1 guide RNA and Cas9 enzyme prevented spinal nerve injury-induced mechanical allodynia and thermal hyperalgesia in both male and female rats. Collectively, our findings highlight a key role of NRP-1 in modulating pain pathways in the sensory nervous system.

The natural product argentatin C attenuates postoperative pain via inhibition of voltage-gated sodium and T-type voltage-gated calcium channels.

BACKGROUND AND PURPOSE: Postoperative pain occurs in as many as 70% of surgeries performed worldwide. Postoperative pain management still relies on opioids despite their negative consequences, resulting in a public health crisis. Therefore, it is important to develop alternative therapies to treat chronic pain. Natural products derived from medicinal plants are potential sources of novel biologically active compounds for development of safe analgesics. In this study, we screened a library of natural products to identify small molecules that target the activity of voltage-gated sodium and calcium channels that have important roles in nociceptive sensory processing. EXPERIMENTAL APPROACH: Fractions derived from the Native American medicinal plant, Parthenium incanum, were assessed using depolarization-evoked calcium influx in rat dorsal root ganglion (DRG) neurons. Further separation of these fractions yielded a cycloartane-type triterpene identified as argentatin C, which was additionally evaluated using whole-cell voltage and current-clamp electrophysiology, and behavioural analysis in a mouse model of postsurgical pain. KEY RESULTS: Argentatin C blocked the activity of both voltage-gated sodium and low-voltage-activated (LVA) calcium channels in calcium imaging assays. Docking analysis predicted that argentatin C may bind to NaV 1.7-1.9 and CaV 3.1-3.3 channels. Furthermore, argentatin C decreased Na+ and T-type Ca2+ currents as well as excitability in rat and macaque DRG neurons, and reversed mechanical allodynia in a mouse model of postsurgical pain. CONCLUSION AND IMPLICATIONS: These results suggest that the dual effect of argentatin C on voltage-gated sodium and calcium channels supports its potential as a novel treatment for painful conditions.

The Ca2+ channel ORAI1 is a regulator of oral cancer growth and nociceptive pain.

Oral cancer causes pain associated with cancer progression. We report here that the function of the Ca2+ channel ORAI1 is an important regulator of oral cancer pain. ORAI1 was highly expressed in tumor samples from patients with oral cancer, and ORAI1 activation caused sustained Ca2+ influx in human oral cancer cells. RNA-seq analysis showed that ORAI1 regulated many genes encoding oral cancer markers such as metalloproteases (MMPs) and pain modulators. Compared with control cells, oral cancer cells lacking ORAI1 formed smaller tumors that elicited decreased allodynia when inoculated into mouse paws. Exposure of trigeminal ganglia neurons to MMP1 evoked an increase in action potentials. These data demonstrate an important role of ORAI1 in oral cancer progression and pain, potentially by controlling MMP1 abundance.

Plant and fungi derived analgesic natural products targeting voltage-gated sodium and calcium channels.

Voltage-gated sodium and calcium channels (VGSCs and VGCCs) play an important role in the modulation of physiologically relevant processes in excitable cells that range from action potential generation to neurotransmission. Once their expression and/or function is altered in disease, specific pharmacological approaches become necessary to mitigate the negative consequences of such dysregulation. Several classes of small molecules have been developed with demonstrated effectiveness on VGSCs and VGCCs; however, off-target effects have also been described, limiting their use and spurring efforts to find more specific and safer molecules to target these channels. There are a great number of plants and herbal preparations that have been empirically used for the treatment of diseases in which VGSCs and VGCCs are involved. Some of these natural products have progressed to clinical trials, while others are under investigation for their action mechanisms on signaling pathways, including channels. In this review, we synthesize information from ~30 compounds derived from natural sources like plants and fungi and delineate their effects on VGSCs and VGCCs in human disease, particularly pain. [Figure: see text].

Cell specific regulation of NaV1.7 activity and trafficking in rat nodose ganglia neurons.

The voltage-gated sodium NaV1.7 channel sets the threshold for electrogenesis. Mutations in the gene encoding human NaV1.7 (SCN9A) cause painful neuropathies or pain insensitivity. In dorsal root ganglion (DRG) neurons, activity and trafficking of NaV1.7 are regulated by the auxiliary collapsin response mediator protein 2 (CRMP2). Specifically, preventing addition of a small ubiquitin-like modifier (SUMO), by the E2 SUMO-conjugating enzyme Ubc9, at lysine-374 (K374) of CRMP2 reduces NaV1.7 channel trafficking and activity. We previously identified a small molecule, designated 194, that prevented CRMP2 SUMOylation by Ubc9 to reduce NaV1.7 surface expression and currents, leading to a reduction in spinal nociceptive transmission, and culminating in normalization of mechanical allodynia in models of neuropathic pain. In this study, we investigated whether NaV1.7 control via CRMP2-SUMOylation is conserved in nodose ganglion (NG) neurons. This study was motivated by our desire to develop 194 as a safe, non-opioid substitute for persistent pain, which led us to wonder how 194 would impact NaV1.7 in NG neurons, which are responsible for driving the cough reflex. We found functioning NaV1.7 channels in NG neurons; however, they were resistant to downregulation via either CRMP2 knockdown or pharmacological inhibition of CRMP2 SUMOylation by 194. CRMP2 SUMOylation and interaction with NaV1.7 was consered in NG neurons but the endocytic machinery was deficient in the endocytic adaptor protein Numb. Overexpression of Numb rescued CRMP2-dependent regulation on NaV1.7, rendering NG neurons sensitive to 194. Altogether, these data point at the existence of cell-specific mechanisms regulating NaV1.7 trafficking.

Blockade of Intranigral and Systemic D3 Receptors Stimulates Motor Activity in the Rat Promoting a Reciprocal Interaction among Glutamate, Dopamine, and GABA.

In vivo activation of dopamine D3 receptors (D3Rs) depresses motor activity. D3Rs are widely expressed in subthalamic, striatal, and dendritic dopaminergic inputs into the substantia nigra pars reticulata (SNr). In vitro studies showed that nigral D3Rs modulate their neurotransmitter release; thus, it could be that these changes in neurotransmitter levels modify the discharge of nigro-thalamic neurons and, therefore, motor behavior. To determine how the in vitro responses correspond to the in vivo responses, we examined the effect of intra-nigral and systemic blockade of D3Rs in the interstitial content of glutamate, dopamine, and GABA within the SNr using microdialysis coupled to motor activity determinations in freely moving rats. Intranigral unilateral blockade of D3R with GR 103,691 increased glutamate, dopamine, and GABA. Increments correlated with increased ambulatory distance, non-ambulatory activity, and induced contralateral turning. Concomitant blockade of D3R with D1R by perfusion of SCH 23390 reduced the increase of glutamate; prevented the increment of GABA, but not of dopamine; and abolished behavioral effects. Glutamate stimulates dopamine release by NMDA receptors, while blockade with kynurenic acid prevented the increase in dopamine and, in turn, of GABA and glutamate. Finally, systemic administration of D3R selective antagonist U 99194A increased glutamate, dopamine, and GABA in SNr and stimulated motor activity. Blockade of intra-nigral D1R with SCH 23390 prior to systemic U 99194A diminished increases in neurotransmitter levels and locomotor activity. These data highlight the pivotal role of presynaptic nigral D3 and D1R in the control of motor activity and help to explain part of the effects of the in vivo administration of D3R agents.

Presynaptic CaMKIIα modulates dopamine D3 receptor activation in striatonigral terminals of the rat brain in a Ca²âº dependent manner.

CaMKIIα is expressed at high density in the nucleus accumbens where it binds to postsynaptic D3 receptors inhibiting their effects. In striatonigral projections, activation of presynaptic D3 receptors potentiates D1 receptor-induced stimulation of cAMP production and GABA release. In this study we examined whether the presynaptic effects of D3 receptor stimulation in the substantia nigra reticulata (SNr) are modulated by Ca²âº activation of CaMKIIα. In SNr synaptosomes two procedures that increase cytoplasmic Ca²âº, ionomycin and K⁺-depolarization, blocked the additional stimulation of cAMP accumulation produced by coactivating D3 and D1 dopamine receptors. The selective CaMKIIα inhibitor KN-62 reversed the blockade produced by ionomycin and K⁺-depolarization. Incubation in either Ca²-free solutions or with the selective Ca²âº blocker nifedipine, also reversed the blocking effects of K⁺-depolarization. Immunoblot studies showed that K⁺-depolarization increased CaMKIIα phosphorylation in a KN-62 sensitive manner and promoted CaMKIIα binding to D3 receptors. In K⁺-depolarized tissues, D3 receptors potentiated D1 receptor-induced stimulation of [³H]GABA release only when CaMKIIα was blocked with KN-62. In the presence of this inhibitor, the selective D3 agonist PD 128,907 reduced the ED50 for the D1 agonist SKF 38393 from 56 to 4 nM. KN-62 also enhanced the effects of dopamine on depolarization induced [³H]GABA release. KN-62 changed ED50 for dopamine from 584 to 56 nM. KN-62 did not affect D1 and D4 receptor responses. These experiments show that in striatonigral projections, CaMKIIα inhibits the action of D3 receptors in a Ca²âº dependent manner blocking their modulatory effects on GABA release. These findings suggest a mechanism through which the frequency of action potential discharge in presynaptic terminals regulates dopamine effects.