"To develop a reagent to disrupt the interaction of CRMP-2 with the CaV2.2 complex in vivo , we synthesized a series of overlapping 15-amino-acid peptides covering the entire length of CRMP-2, including three CaV binding domains (CBDs1–3) we had previously identified in vitro as crucial for the CRMP-2–CaV2.2 interaction xref ."
"Second , despite being an indirect ‘blocker’ of Ca 2+ influx, ( S )-LCM provides improved efficacy compared to TROX-1, a small-molecule, state-dependent blocker of CaV2 channels [ xref ] as well as a membrane-tethered CRMP2 peptide that uncouples the CRMP2-CaV2.2 interaction that we recently reported [ xref ]."
"The CRMP2–CaV2.2 interaction was dynamic as potassium chloride-induced depolarization led to an increase in the interaction."
"We previously showed that tat-CBD3 inhibited the CaV2.2–CRMP2 interaction. xref Therefore, we next tested whether the myristoylated CBD3 peptide could interfere with the CaV2.2–CRMP2 interaction."
"We determined that CRMP-2 interacts with CaV2.2 and that overexpression of CRMP-2 leads to increased surface expression of CaV2.2 and enhanced Ca 2+ currents xref , xref ."
"Pull-down studies demonstrated that myr-tat-CBD3 peptide interfered with the CRMP2–CaV2.2 interaction."
"Although the control peptides did not alter this interaction, the tat-CBD3 peptide was able to inhibit CaV2.2–CRMP2 interaction by ~43% at 10 μM. Myr-tat-CBD3, in a concentration-dependent manner, attenuated this interaction with greater than ~81% inhibition at 10 μM ( xref ; top blot and xref )."
"ST1-104, which disrupts the interaction between CaV2.2 and CRMP2 interaction, reduces persistent mechanical hypersensitivity induced by systemic administration of ddC [ xref ]."
"Co-immunoprecipitation experiments showed that CRMP-2 associates with Cav2.2 from DRG lysates."
"As CBD3 disrupts the interaction between CRMP2 and CaV2.2, this supports the conclusion that the interaction between CRMP2 and CaV2.2 is likely responsible for the observed increase in Ca 2+ currents."
"Thus, in vitro , CBD3 disrupts the CRMP-2-CaV2.2 interaction and affects CaV2.2 trafficking and Ca 2+ current density."
"In addition, more recent studies have reported that CRMP-2 can bind to the voltage-gated calcium channel Cav2.2, and this interaction may play a crucial role in neurotransmitter release from the presynaptic terminals of hippocampal neurons ( xref )."
"Competition between syntaxin 1A and neurofibromin for a singular CRMP2 binding domain regulates CRMP2’s direct interaction with Cav2.2."
"The pharmacological action of peptides observed in our in vitro experiments is linked to uncoupling of the CaV2.2–CRMP2 interaction culminating in alleviation of inflammatory and neuropathic pain. xref , xref , xref , xref , xref Sustained, but incomplete, relief of neuropathic pain has been successfully demonstrated for a nonmyristoylated CRMP2 peptide. xref We postulate that the myristoylated version described herein may afford greater efficacy and clinical utility as a gene therapy strategy for chronic pain."
"The functional consequence of the disrupted CaV2.2–CRMP2 interaction was a significantly higher extent (i.e. efficacy) of inhibition of calcium influx in sensory neurons."
"Finally, to confirm the disruption of CRMP2 interaction with Cav2.2 in sensory neurons, we used a PLA."
"Here, we report ST2-104 –a peptide wherein the cell-penetrating TAT motif has been supplanted with a homopolyarginine motif, which dose-dependently inhibits the CaV2.2–CRMP2 interaction and inhibits depolarization-evoked Ca 2+ influx in sensory neurons."
"These findings suggest that the biochemical interaction between CRMP-2 and CaV2.2 is required for proper channel trafficking and function."
"These results demonstrate that by uncoupling the CaV2.2– CRMP2 interaction, both tat-CBD3 and myr-tat-CBD3 reduce surface trafficking of CaV2.2 (eg, 0.60 PCC and 0.65 MOC for the myr-tat-CBD3-treated DRG as noted by the lack of colocalization in the insets)."
"We previously showed that CRMP2 is a novel binding partner of CaV2.2. xref , xref A 15-amino acid peptide derived from CRMP2 (designated CBD3 for calcium channel binding domain) uncoupled the CaV2.2–CRMP2 interaction leading to a decrement in Ca 2+ current and neurotransmitter release and, consequently, suppressed persistent inflammatory and neuropathic hypersensitivity. xref , xref , xref Mutating single residues within the CBD3 peptide sequence or changing the cell-penetrating motif improved the efficacy of CBD3 in models of neuropathic pain. xref , xref Here, we tested the hypothesis that tethering the CBD3 peptide to the membrane with myristate would confine the myristoylated peptide’s action(s) to uncoupling membrane CaV2.2–CRMP2 interactions, block Ca 2+ influx in sensory neurons, and be effective in reducing pain-related behaviors in models of pain."
"Similar to TAT-conjugated CBD3 (designated ST1-104), the R9-conjugated CBD3 (designated ST2-104) peptide interfered with the CaV2.2–CRMP2 interaction."
"N-myristate-tat-CBD3 peptide inhibits the CaV2.2–CRMP2 interaction."
"We report that myristoylated tat-CBD3 is membrane tethered, restricts the CaV2.2–CRMP2 interaction, blocks CaV2.2 trafficking, and strongly inhibits Ca 2+ influx from primary sensory neurons."
"These results demonstrate that the myristoyl group improves the ability of the peptide to break the CaV2.2–CRMP2 interaction."
"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."
"These results demonstrate that the R9-conjugated mutant peptide retains its ability to efficiently uncouple the CRMP2–CaV2.2 interaction."
"Interference of the protein-protein interaction between the target collapsin response mediator protein 2 (CRMP2) and CaV2.2 in sensory neurons by unique peptides effectively diminishes trafficking of the ion channel to the plasma membrane and serves to attenuate peripheral sensitization in rodent models of inflammation or neuropathic pain ( xref , xref , xref , xref , xref )."
"We tested the hypothesis that replacement of the TAT motif with the presumably superior cell penetrating prowess of the homopolyarginine motif ( xref ) would prevent CRMP2–CaV2.2 interaction, block Ca 2+ influx in sensory neurons, and be effective in reducing pain-related behavior in various models of peripheral neuropathy."
"We reported that collapsin response mediator protein 2 (CRMP2) interacts with CaV2.2 and enhances its functional activity [ xref ; xref ; xref ; xref ]."
"Data here demonstrate a novel efficacious compound to inhibit pain without demonstrating any addiction or motor deficits ( xref , xref ) providing an instructive example of how designing peptides tailored to limit membrane CaV2.2–CRMP2 interactions can have a great utility in the treatment of post-surgical and inflammatory pain."
"In rat DRGs, only CBD3’s N-terminal fragment successfully inhibited depolarization-evoked calcium influx, reduced Ca 2+ currents, and disrupted the CRMP2-Cav2.2 interaction in situ [ xref ]."
"We first tested if the nona-arginine (R9) motif conjugated to the CBD3 peptide of CRMP2, designated ST2-104 peptide, could interfere with the CaV2.2–CRMP2 interaction."
"Over the last few years, we have described an interaction between CaV2.2 and tetrameric collapsin response mediator protein 2 (CRMP-2) that positively regulates channel function by increasing cell surface trafficking [ xref , xref , xref ]."
"The functional consequence of the disrupted CaV2.2– CRMP2 interaction was a significantly higher extent (i.e. efficacy) of inhibition of calcium influx in sensory neurons."
"Probing of the CRMP2-enriched fraction with a CaV2.2 antibody demonstrated a robust interaction between CaV2.2 and CRMP2 ( xref ; top blot, lanes 1, 2 ) whereas a glutathione beads only pull-down did not capture any CaV2.2 ( xref ; top blot, lane 7 )."
"Disruption of the interaction between CaV2.2 and CRMP2 by a short peptide (CBD3) corresponding to a 15 amino acid region of CRMP-2 produces a number of changes including pain signal transmission [ xref ]."
"A search for small molecules that could recapitulate uncoupling of the CaV2.2-CRMP2 interaction identified (S)-lacosamide [(S)-LCM], the inactive enantiomer of the Food and Drug Administration-approved antiepileptic drug (R)-lacosamide [(R)-LCM, Vimpat]."
"Importantly, disrupting the CRMP2-Cav2.2 interaction did not alter core sympathetic nervous system functions such as pulsatile arterial pressure, mean arterial pressure, heart rate, and core body temperature [ xref ]."
"We reported that Cdk5-mediated CRMP2 phosphorylation increases its binding to CaV2.2, which augments calcium influx xref ; thus, the increased interaction between CRMP2 and CaV2.2 may underlie the mechanism of antinociception in this particular model."
"Systemic administration of ( S )-LCM to mice, at three daily doses of 20 mg/kg over four days, successfully uncoupled the CRMP2-Cav2.2 interaction in brain lysates, with no detectable effects on locomotion, feeding, or behavior [ xref ]."
"Towards this end, we recently identified a short peptide, designated CBD3, derived from collapsin response mediator protein 2 (CRMP-2) that suppressed inflammatory and neuropathic hypersensitivity by inhibiting CRMP-2 binding to CaV2.2 [Brittain et al ., Nature Medicine 17:822–829 (2011)]."
"Reasoning that the reported superior cell penetrating ability of the nona-arginine CPP ( xref ) may allow for greater uptake of the peptide which may result in superior biochemical and functional uncoupling of the CaV2.2–CRMP2 interaction and suppression of transmitter release from nociceptive neurons culminating in efficacy in chronic pain models, we tested the R9 grafted CBD3 (i.e. ST2-104) peptide in the TNI model of traumatic neuropathy."
"These results suggest a coordinated mechanism involving interactions between CRMP2 and CaV2.2 to facilitate the release of pro-nociceptive CGRP that consequently leads to cephalic pain."
"This value is in agreement with an IC 50 value of ~2.8 µM for inhibition of Ca 2+ influx for a membrane-tethered CRMP2 peptide that uncouples the CRMP2-CaV2.2 interaction that we recently reported [ xref ]."
"These findings suggest that the biochemical interaction between CRMP-2 and CaV2.2 is required for proper channel trafficking and function."
"The interaction between CaV2.2 and CRMP2 can be disrupted by a short peptide (CBD3) corresponding to a 15 amino acid region of CRMP-2 ( xref )."
"The study of CaV2.2 trafficking modulators xref led us to identify axon/dendrite specification collapsin response mediator protein 2 (CRMP2) as a CaV2.2-binding partner, xref which enhances CaV2.2 membrane trafficking and facilitates synaptic transmission. xref , xref CaV2.2 membrane trafficking was effectively disrupted by a 15-amino-acid region of CRMP2, named Ca 2+ channel-binding domain 3 (CBD3). xref Systemic injection of CBD3 peptide conjugated to the cell-penetrating motif of the HIV transduction domain protein tat (tat-CBD3) reduced hypersensitive behavior in chemically induced inflammatory and neuropathic pain models. xref , xref , xref , xref tat-CBD3 reduced CaV2.2-mediated currents by inhibiting CaV2.2–CRMP2 interaction, xref which resulted in reduced nociceptor excitability, diminished neuropeptide release from primary sensory neurons, and lowered excitatory synaptic transmission. xref "
"In parallel, it was discovered that phosphorylation of CRMP2 by Cdk5 dynamically regulates and increases the association of CRMP2 with Cav2.2 [ xref ]."
"Here we report suppression of both inflammatory and neuropathic hypersensitivity by inhibiting the binding of the axonal collapsin response mediator protein 2 (CRMP-2) to CaV2.2, thus reducing channel function."
"Having established that both peptides can inhibit the CaV2.2– CRMP2 interaction in vitro, we next asked whether this translated into a disruption in cells."
"A screening protocol of >50 novel molecules unexpectedly identified the small molecule ( S )-lacosamide (( S )-LCM) xref as a novel inhibitor of CRMP2 interactions with CaV2.2."
"We previously showed that all cell penetrant forms of CBD3 inhibited the CRMP2–CaV2.2 interaction [ xref ; xref ; xref ; xref ]."
"We determined that CRMP-2 interacts with CaV2.2 and that overexpression of CRMP-2 leads to increased surface expression of CaV2.2 and enhanced Ca 2+ currents [ xref , xref , xref ]."
"Similar to TAT-CBD, the R9-conjugated CBD3 peptide interfered with the CaV2.2–CRMP2 interaction."
"Probing of the CRMP2-enriched fraction with a Cav2.2 antibody demonstrated a robust interaction between Cav2.2 and CRMP2 ( xref ; top blot, lane 2 )."
"In a series of studies examining targets of synthetic CBD peptides, conjugated to the HIV1 TAT (t−) domain for cell penetrance, only t-CBD3, comprised of CRMP2 residues 484-498, succeeded in biochemically inhibiting the CRMP2-Cav2.2 interaction [ xref ]."
"CRMP-2 binds directly to CaV2.2 in two regions: the channel domain I-II intracellular loop and the distal C terminus."
"Thus, we used immunofluorescent microscopy to determine the time course of CBD3-mediated disruption of the CaV2.2–CRMP2 interaction."
"The authors’ results demonstrated that a direct interaction between CRMP2 and CaV2.2 increases cell surface trafficking of CaV2.2 as well as calcium current density, which leads not only to an increased release of the neurotransmitter glutamate and synaptic vesicle recycling but also impacts axonal growth of hippocampal neurons."
"Although CBD3 has been previously demonstrated to interfere with the CaV2.2–CRMP2 interaction, the dynamics of the disruption over time by this peptide have never been studied."
"Probing of the CRMP2-enriched fraction with a CaV2.2 antibody demonstrated a robust interaction between CaV2.2 and CRMP2 ( xref ; top blot, lane 2 )."
"We show that ST2-104 interferes with the CaV2.2–CRMP2 interaction and inhibits Ca 2+ influx from sensory neurons."
"These findings suggested that the biochemical interaction between CRMP2 and CaV2.2 was required for proper channel trafficking and function."
"These findings are entirely consistent with our previous results demonstrating longer periods of pain-related behavior reversal in the TNI versus the ddC model following systemic injection of a TAT-conjugated peptide from calcium channels that breaks the CaV2.2–CRMP2 interaction ( xref ) as well as with the CRMP2-derived ST1-104 peptide ( xref )."
"While activation of Cdk5 and increased CRMP2 expression in rat DRGs contribute to several pain phenotypes, ( S )-LCM biochemically disrupts the CRMP2-Cav2.2 interaction while inhibiting depolarization-evoked Ca 2+ influx and Ca 2+ currents [ xref , xref , xref – xref ]."
"Thus, our results indicate that using TAT-CBD3 which interferes with CaV2.2 and CRMP-2 interactions can reduce inflammatory and neuropathic pain behaviors."
"Lastly, we utilized the recently developed peptide myr-TAT-CBD3 to disrupt the interaction between CRMP2 and CaV2.2 in vivo."
"Although the precise nature of NF1-linked pain syndromes remains unknown, patients clearly experience pain independent of their tumor burden, and these findings affirm the necessity of CRMP2 in NF1-related pain, while suggesting a physiological role for neurofibromin’s C-terminal domain-mediated inhibition of the CRMP2-Cav2.2 interaction [ xref ]."
"We tested the hypothesis ( xref ) that uncoupling the CRMP-2–CaV2.2 interaction would lead to a physiologically relevant decrease in Ca 2+ current and neurotransmitter release and, in turn, suppress persistent inflammatory and neuropathic hypersensitivity."
"The present findings make a compelling case for the use of a myristoylated CRMP2 peptide, which uncouples the CaV2.2–CRMP2 interaction, for the treatment of inflammatory and incision-induced pain."
"Immunoprecipitations from spinal cord lysates demonstrated that CBD3 peptide inhibited the interaction between CRMP-2 and CaV2.2 ( xref ; top, middle ) but did not affect the interaction between tubulin and CRMP-2 xref ( xref , bottom )."
"Peptides uncoupling CaV2.2 interactions with the axonal collapsin response mediator protein 2 (CRMP2) were antinociceptive without effects on memory, depression, and reward/addiction."
"Here, we investigated the effects of a functional association of CRMP-2 with Cav2.2 in sensory neurons."
"Interestingly, in this animal model of pain, the peptide is administered systemically after the development of chronic hypersensitivity and is still able to effectively attenuate nociceptive behaviors, suggesting a continued role for the interaction of CRMP-2 and CaV2.2 on neurotransmitter release."
"Here we report that inflammatory and neuropathic hypersensitivity can be suppressed by inhibiting the binding of collapsin response mediator protein 2 (CRMP-2) to CaV2.2 and thereby reducing channel function."
"Concomitant expression of CaV2.2, CRMP2, and CGRP was detected in a subset of neurons from the ophthalmic branch of TGs that receive input from the dura mater. xref These results suggest a coordinated mechanism involving interactions between CRMP2 and CaV2.2 to facilitate the release of pro-nociceptive CGRP that consequently leads to cephalic pain."
"We previously reported that CBD3, a 15 amino acid fragment of CRMP2, disrupts the CRMP2–CaV2.2 interaction [ xref ] irrespective of the cell penetrating motif it is appended to: the HIV-1 transactivator of transcription domain (TAT) [ xref ], a myristate (Myr) tag [ xref ], or homopolyarginine (R9) [ xref ]."
"Binding affinity of Ct-Cav2.2/CRMP2 was ~75 fold greater than between L1-Cav2.2/CRMP2, and only disruption of the Ct-Cav2.2/CRMP2 interaction inhibited evoked CGRP release in rat DRGs [ xref ]."
"As previously described, CaV2.2–CRMP2 disruption by tat-CBD3 decreased surface CaV2.2 in DRG. xref , xref Myr-tat-CBD3 peptide interfered with the CaV2.2–CRMP2 interaction more efficiently than tat-CBD3 as observed by a reduction in colocalization within minutes of incubation ( xref )."
"TAT-CBD3 peptide interfered with CRMP2-CaV2.2 interactions resulting in acute inhibition of CaV2.2 currents in sensory and hippocampal neurons; acute inhibition of frequency of spontaneous excitatory postsynaptic currents (sEPSCs) in spinal cord slices as well as layer V pyramidal neurons suggesting reduction in probability of glutamate release from stimulated presynaptic terminals; and inhibition of evoked calcitonin gene-related peptide (CGRP) in sensory neurons in culture (acute and long-term inhibition observed) and in spinal cord slices."
"We reasoned that by uncoupling the interaction between CaV2.2 and CRMP-2 within primary afferent sensory neurons, we would be able to mimic the inhibition of neurotransmitter release observed with siRNA knockdown of CRMP-2."
"Our initial mapping of the CaV2.2:CRMP2 interaction had identified a surface-exposed region in CRMP2, which we designated calcium channel binding domain 3 (CBD3) [ xref ]."
"We previously identified ST1-104, a short peptide from the collapsin response mediator protein 2 (CRMP2), which disrupted the CaV2.2–CRMP2 interaction and suppressed a model of HIV-related neuropathy induced by antiretroviral therapy but not traumatic neuropathy."
"From these findings, we propose that TAT-CBD3 allows suppression of pain hypersensitivity without directly blocking CaV2.2, but rather by inhibiting the binding of a regulator of CaV2.2 function, CRMP-2."
"Probing of the CRMP2-enriched fraction with a CaV2.2 antibody demonstrated a robust interaction between CaV2.2 and CRMP2 ( xref ; top blot, lane 2)."
"R9-CBD3-A6K disrupts the CRMP2–CaV2.2 interaction and inhibits surface trafficking of CaV2.2 in DRGs."