Andrew D. Piekarz

An Atypical Role for Collapsin Response Mediator Protein 2 (CRMP-2) in Neurotransmitter Release via Interaction with Presynaptic Voltage-gated Calcium Channels

Collapsin response mediator proteins (CRMPs) specify axon/dendrite fate and axonal growth of neurons through protein-protein interactions. Their functions in presynaptic biology remain unknown. Here, we identify the presynaptic N-type Ca2+ channel (CaV2.2) as a CRMP-2-interacting protein. CRMP-2 binds directly to CaV2.2 in two regions: the channel domain I-II intracellular loop and the distal C terminus. Both proteins co-localize within presynaptic sites in hippocampal neurons. Overexpression in hippocampal neurons of a CRMP-2 protein fused to enhanced green fluorescent protein caused a significant increase in Ca2+ channel current density, whereas lentivirus-mediated CRMP-2 knockdown abolished this effect. Interestingly, the increase in Ca2+ current density was not due to a change in channel gating. Rather, cell surface biotinylation studies showed an increased number of CaV2.2 at the cell surface in CRMP-2-overexpressing neurons. These neurons also exhibited a significant increase in vesicular release in response to a depolarizing stimulus. Depolarization of CRMP-2-enhanced green fluorescent protein-overexpressing neurons elicited a significant increase in release of glutamate compared with control neurons. Toxin block of Ca2+ entry via CaV2.2 abolished this stimulated release. Thus, the CRMP-2-Ca2+ channel interaction represents a novel mechanism for modulation of Ca2+ influx into nerve terminals and, hence, of synaptic strength.

Human voltage-gated sodium channel mutations that cause inherited neuronal and muscle channelopathies increase resurgent sodium currents

Inherited mutations in voltage-gated sodium channels (VGSCs; or Nav) cause many disorders of excitability, including epilepsy, chronic pain, myotonia, and cardiac arrhythmias. Understanding the functional consequences of the disease-causing mutations is likely to provide invaluable insight into the roles that VGSCs play in normal and abnormal excitability. Here, we sought to test the hypothesis that disease-causing mutations lead to increased resurgent currents, unusual sodium currents that have not previously been implicated in disorders of excitability. We demonstrated that a paroxysmal extreme pain disorder (PEPD) mutation in the human peripheral neuronal sodium channel Nav1.7, a paramyotonia congenita (PMC) mutation in the human skeletal muscle sodium channel Nav1.4, and a long-QT3/SIDS mutation in the human cardiac sodium channel Nav1.5 all substantially increased the amplitude of resurgent sodium currents in an optimized adult rat-derived dorsal root ganglion neuronal expression system. Computer simulations indicated that resurgent currents associated with the Nav1.7 mutation could induce high-frequency action potential firing in nociceptive neurons and that resurgent currents associated with the Nav1.5 mutation could broaden the action potential in cardiac myocytes. These effects are consistent with the pathophysiology associated with the respective channelopathies. Our results indicate that resurgent currents are associated with multiple channelopathies and are likely to be important contributors to neuronal and muscle disorders of excitability.

Neuroexcitatory effects of morphine-3-glucuronide are dependent on Toll-like receptor 4 signaling

BackgroundMultiple adverse events are associated with the use of morphine for the treatment of chronic non-cancer pain, including opioid-induced hyperalgesia (OIH). Mechanisms of OIH are independent of opioid tolerance and may involve the morphine metabolite morphine-3-glucuronide (M3G). M3G exhibits limited affinity for opioid receptors and no analgesic effect. Previous reports suggest that M3G can act via the Toll-like receptor 4 (TLR4)/myeloid differentiation protein-2 (MD-2) heterodimer in the central nervous system to elicit pain.MethodsImmunoblot and immunocytochemistry methods were used to characterize the protein expression of TLR4 present in lumbar dorsal root ganglion (DRG). Using in vitro intracellular calcium and current clamp techniques, we determined whether TLR4 activation as elicited by the prototypical agonists of TLR4, lipopolysaccharide (LPS) and M3G, contributed to changes in intracellular calcium and increased excitation. Rodents were also injected with M3G to determine the degree to which M3G-induced tactile hyperalgesia could be diminished using either a small molecule inhibitor of the MD-2/TLR4 complex in rats or TLR4 knockout mice. Whole cell voltage-clamp recordings were made from small- and medium-diameter DRG neurons (25 μm < DRG diameter <45 μm) for both control and M3G-treated neurons to determine the potential influence on voltage-gated sodium channels (NaVs).ResultsWe observed that TLR4 immunoreactivity was present in peptidergic and non-peptidergic sensory neurons in the DRG. Non-neuronal cells in the DRG lacked evidence of TLR4 expression. Approximately 15% of assayed small- and medium-diameter sensory neurons exhibited a change in intracellular calcium following LPS administration. Both nociceptive and non-nociceptive neurons were observed to respond, and approximately 40% of these cells were capsaicin-insensitive. Increased excitability observed in sensory neurons following LPS or M3G could be eliminated using Compound 15, a small molecule inhibitor of the TLR4/MD-2 complex. Likewise, systemic injection of M3G induced rapid tactile, but not thermal, nociceptive behavioral changes in the rat, which were prevented by pre-treating animals with Compound 15. Unlike TLR4 wild-type mice, TLR4 knockout mice did not exhibit M3G-induced hyperalgesia. As abnormal pain sensitivity is often associated with NaVs, we predicted that M3G acting via the MD-2/TLR4 complex may affect the density and gating of NaVs in sensory neurons. We show that M3G increases tetrodotoxin-sensitive and tetrodotoxin-resistant (NaV1.9) current densities.ConclusionsThese outcomes provide evidence that M3G may play a role in OIH via the TLR4/MD-2 heterodimer complex and biophysical properties of tetrodotoxin-sensitive and tetrodotoxin-resistant NaV currents.

Nav1.7 mutations associated with paroxysmal extreme pain disorder, but not erythromelalgia, enhance Navβ4 peptide-mediated resurgent sodium currents

Abnormal pain sensitivity associated with inherited and acquired pain disorders occurs through increased excitability of peripheral sensory neurons in part due to changes in the properties of voltage‐gated sodium channels (Navs). Resurgent sodium currents (INaR) are atypical currents believed to be associated with increased excitability of neurons and may have implications in pain. Mutations in Nav1.7 (peripheral Nav isoform) associated with two genetic pain disorders, inherited erythromelalgia (IEM) and paroxysmal extreme pain disorder (PEPD), enhance Nav1.7 function via distinct mechanisms. We show that changes in Nav1.7 function due to mutations associated with PEPD, but not IEM, are important in INaR generation, suggesting that INaR may play a role in pain associated with PEPD. This knowledge provides us with a better understanding of the mechanism of INaR generation and may lead to the development of specialized treatment for pain disorders associated with INaR.

Further insights into the antinociceptive potential of a peptide disrupting the N-type calcium channel–CRMP-2 signaling complex

The N-type voltage-gated calcium channel ( Cav2.2) has gained immense prominence in the treatment of chronic pain. While decreased channel function is ultimately anti-nociceptive, directly targeting the channel can lead to multiple adverse side effects. Targeting modulators of channel activity may facilitate improved analgesic properties associated with channel block and a broader therapeutic window. A novel interaction between Cav2.2 and collapsin response mediator protein 2 (CRMP-2) positively regulates channel function by increasing surface trafficking. We recently identified a CRMP-2 peptide (TAT-CBD3), which effectively blocks this interaction, reduces or completely reverses pain behavior in a number of inflammatory and neuropathic models. Importantly, TAT-CBD3 did not produce many of the typical side effects often observed with Cav2.2 inhibitors. Notably chronic pain mechanisms offer unique challenges as they often encompass a mix of both neuropathic and inflammatory elements, whereby inflammation likely causes damage to the neuron leading to neuropathic pain, and neuronal injury may produce inflammatory reactions. To this end, we sought to further disseminate the ability of TAT-CBD3 to alter behavioral outcomes in two additional rodent pain models. While we observed that TAT-CBD3 reversed mechanical hypersensitivity associated with a model of chronic inflammatory pain due to lysophosphotidylcholine-induced sciatic nerve focal demyelination (LPC), injury to the tibial nerve (TNI) failed to respond to drug treatment. Moreover, a single amino acid mutation within the CBD3 sequence demonstrated amplified Cav2.2 binding and dramatically increased efficacy in an animal model of migraine. Taken together, TAT-CBD3 potentially represents a novel class of therapeutics targeting channel regulation as opposed to the channel itself.

Tetrodotoxin-resistant sodium channels in sensory neurons generate slow resurgent currents that are enhanced by inflammatory mediators.

Resurgent sodium currents contribute to the regeneration of action potentials and enhanced neuronal excitability. Tetrodotoxin-sensitive (TTX-S) resurgent currents have been described in many different neuron populations, including cerebellar and dorsal root ganglia (DRG) neurons. In most cases, sodium channel Nav1.6 is the major contributor to these TTX-S resurgent currents. Here we report a novel TTX-resistant (TTX-R) resurgent current recorded from rat DRG neurons. The TTX-R resurgent currents are similar to classic TTX-S resurgent currents in many respects, but not all. As with TTX-S resurgent currents, they are activated by membrane repolarization, inhibited by lidocaine, and enhanced by a peptide-mimetic of the β4 sodium channel subunit intracellular domain. However, the TTX-R resurgent currents exhibit much slower kinetics, occur at more depolarized voltages, and are sensitive to the Nav1.8 blocker A803467. Moreover, coimmunoprecipitation experiments from rat DRG lysates indicate the endogenous sodium channel β4 subunits associate with Nav1.8 in DRG neurons. These results suggest that slow TTX-R resurgent currents in DRG neurons are mediated by Nav1.8 and are generated by the same mechanism underlying TTX-S resurgent currents. We also show that both TTX-S and TTX-R resurgent currents in DRG neurons are enhanced by inflammatory mediators. Furthermore, the β4 peptide increased excitability of small DRG neurons in the presence of TTX. We propose that these slow TTX-R resurgent currents contribute to the membrane excitability of nociceptive DRG neurons under normal conditions and that enhancement of both types of resurgent currents by inflammatory mediators could contribute to sensory neuronal hyperexcitability associated with inflammatory pain.

The human Nav1.5 F1486 deletion associated with long QT syndrome leads to impaired sodium channel inactivation and reduced lidocaine sensitivity.

•  We investigated how the F1486 deletion LQT3 mutation impairs the functional properties of the human cardiac voltage‐gated sodium channel (hNav1.5) and alters action potential firing. •  Voltage‐clamp recordings from HEK 293 cells and cardiomyocytes expressing recombinant channels demonstrated that the F1486del mutation reduces peak current density but also impairs inactivation and increases late current density. •  Current‐clamp recordings from cardiomyocytes indicated that the increase in late current density would result in prolonged action potential duration and this was confirmed using computer simulations. •  The deletion of F1486 abolished the ability of lidocaine to stabilize the inactivated state and eliminated the high‐affinity binding of lidocaine to inactivated channels. •  Our data show that the hNav1.5‐F1486del mutation has complex functional consequences and indicate that knowledge of the specific molecular defect is critical when developing potential treatments for individuals with prolonged QT intervals.

Characterisation of Nav1.7 functional expression in rat dorsal root ganglia neurons by using an electrical field stimulation assay

Background The Nav1.7 subtype of voltage-gated sodium channels is specifically expressed in sensory and sympathetic ganglia neurons where it plays an important role in the generation and transmission of information related to pain sensation. Human loss or gain-of-function mutations in the gene encoding Nav1.7 channels (SCN9A) are associated with either absence of pain, as reported for congenital insensitivity to pain, or with exacerbation of pain, as reported for primary erythromelalgia and paroxysmal extreme pain disorder. Based on this important human genetic evidence, numerous drug discovery efforts are ongoing in search for Nav1.7 blockers as a novel therapeutic strategy to treat pain conditions. Results We are reporting here a novel approach to study Nav1.7 function in cultured rat sensory neurons. We used live cell imaging combined with electrical field stimulation to evoke and record action potential-driven calcium transients in the neurons. We have shown that the tarantula venom peptide Protoxin-II, a known Nav1.7 subtype selective blocker, inhibited electrical field stimulation-evoked calcium responses in dorsal root ganglia neurons with an IC50 of 72 nM, while it had no activity in embryonic hippocampal neurons. The results obtained in the live cell imaging assay were supported by patch-clamp studies as well as by quantitative PCR and Western blotting experiments that confirmed the presence of Nav1.7 mRNA and protein in dorsal root ganglia but not in embryonic hippocampal neurons. Conclusions The findings presented here point to a selective effect of Protoxin-II in sensory neurons and helped to validate a new method for investigating and comparing Nav1.7 pharmacology in sensory versus central nervous system neurons. This will help in the characterisation of the selectivity of novel Nav1.7 modulators using native ion channels and will provide the basis for the development of higher throughput models for enabling pain-relevant phenotypic screening.