Faculty Bibliography

The N-type voltage-gated calcium channel (Cav 2.2) 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 Cav 2.2 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 Cav 2.2 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 Cav 2.2 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.

Neurological disabilities following traumatic brain injury (TBI) may be due to excitotoxic neuronal loss. The excitotoxic loss of neurons following TBI occurs largely due to hyperactivation of N-methyl-d-aspartate receptors (NMDARs), leading to toxic levels of intracellular Ca(2+). The axon guidance and outgrowth protein collapsin response mediator protein 2 (CRMP2) has been linked to NMDAR trafficking and may be involved in neuronal survival following excitotoxicity. Lentivirus-mediated CRMP2 knockdown or treatment with a CRMP2 peptide fused to HIV TAT protein (TAT-CBD3) blocked neuronal death following glutamate exposure probably via blunting toxicity from delayed calcium deregulation. Application of TAT-CBD3 attenuated postsynaptic NMDAR-mediated currents in cortical slices. In exploring modulation of NMDARs by TAT-CBD3, we found that TAT-CBD3 induced NR2B internalization in dendritic spines without altering somal NR2B surface expression. Furthermore, TAT-CBD3 reduced NMDA-mediated Ca(2+) influx and currents in cultured neurons. Systemic administration of TAT-CBD3 following a controlled cortical impact model of TBI decreased hippocampal neuronal death. These findings support TAT-CBD3 as a novel neuroprotective agent that may increase neuronal survival following injury by reducing surface expression of dendritic NR2B receptors.

We recently reported that merging key structural pharmacophores of the anticonvulsant drugs lacosamide (a functionalized amino acid) with safinamide (an α-aminoamide) resulted in novel compounds with anticonvulsant activities superior to that of either drug alone. Here, we examined the effects of six such chimeric compounds on Na(+)-channel function in central nervous system catecholaminergic (CAD) cells. Using whole-cell patch clamp electrophysiology, we demonstrated that these compounds affected Na(+) channel fast and slow inactivation processes. Detailed electrophysiological characterization of two of these chimeric compounds that contained either an oxymethylene ((R)-7) or a chemical bond ((R)-11) between the two aromatic rings showed comparable effects on slow inactivation, use-dependence of block, development of slow inactivation, and recovery of Na(+) channels from inactivation. Both compounds were equally effective at inducing slow inactivation; (R)-7 shifted the fast inactivation curve in the hyperpolarizing direction greater than (R)-11, suggesting that in the presence of (R)-7, a larger fraction of the channels are in an inactivated state. None of the chimeric compounds affected veratridine- or KCl-induced glutamate release in neonatal cortical neurons. There was modest inhibition of KCl-induced calcium influx in cortical neurons. Finally, a single intraperitoneal administration of (R)-7, but not (R)-11, completely reversed mechanical hypersensitivity in a tibial-nerve injury model of neuropathic pain. The strong effects of (R)-7 on slow and fast inactivation of Na(+) channels may contribute to its efficacy and provide a promising novel therapy for neuropathic pain, in addition to its antiepileptic potential.

The novel antiepileptic drug, (R)-N-benzyl 2-acetamido-3-methoxypropionamide ((R)-lacosamide, Vimpat(®) ((R)-1)), was recently approved in the US and Europe for adjuvant treatment of partial-onset seizures in adults. (R)-1 preferentially enhances slow inactivation of voltage-gated Na(+) currents, a pharmacological process relevant in the hyperexcitable neuron. We have advanced a strategy to identify lacosamide binding partners by attaching affinity bait (AB) and chemical reporter (CR) groups to (R)-1 to aid receptor detection and isolation. We showed that select lacosamide AB and AB&CR derivatives exhibited excellent activities similar to (R)-1 in the maximal electroshock seizure model in rodents. Here, we examined the effect of these lacosamide AB and AB&CR derivatives and compared them with (R)-1 on Na(+) channel function in CNS catecholaminergic (CAD) cells. Using whole-cell patch clamp electrophysiology, we demonstrated that the test compounds do not affect the Na(+) channel fast inactivation process, that they were far better modulators of slow inactivation than (R)-1, and that modulation of the slow inactivation process was stereospecific. The lacosamide AB agents that contained either an electrophilic isothiocyanate ((R)-5) or a photolabile azide ((R)-8) unit upon AB activation gave modest levels of permanent Na(+) channel slow inactivation, providing initial evidence that these compounds may have covalently reacted with their cognate receptor(s). Our findings support the further use of these agents to delineate the (R)-1-mediated Na(+) channel slow inactivation process.

Mutations of the neurofibromin gene (NF1) cause neurofibromatosis type 1 (NF1), a disease in which learning disabilities are common. Learning deficits also are observed in mice with a heterozygous mutation of Nf1 (Nf1(+/-)). Dysregulation of regulated neurotransmitter release has been observed in Nf1(+/-) mice. However, the role of presynaptic voltage-gated Ca(2+) channels mediating this release has not been investigated. We investigated whether Ca(2+) currents and transmitter release were affected by reduced neurofibromin in Nf1(+/-) mice. Hippocampal Ca(2+) current density was greater in neurons from Nf1(+/-) mice and a greater fraction of Ca(2+) currents was activated at less depolarized potentials. In addition, release of the excitatory neurotransmitter, glutamate, was increased in neuronal cortical cultures from Nf1(+/-) mice. Dendritic complexity and axonal length were also increased in neurons Nf1(+/-) mice compared to wild-type neurons, linking loss of neurofibromin to developmental changes in hippocampal axonal/cytoskeletal dynamics. Collectively, these results show that altered Ca(2+) channel density and transmitter release, along with increased axonal growth may account for the abnormal nervous system functioning in NF1.

Presynaptic N-type voltage-gated Ca(2+) channels (Cav2.2) form part of an extensive macromolecular complex in the presynaptic terminal. Regulation of Cav2.2 is achieved via protein-protein interactions within the terminal and can directly impact transmitter release which is dependent on Ca(2+) influx via these Cav2.2. We recently identified a novel Cav2.2 interacting partner-the collapsin response mediator protein (CRMP).1 CRMPs are a family of five proteins implicated in signal transduction of neurite outgrowth and axonal guidance. We showed that CRMP-2, a wellstudied member of this family, interacted with Cav2.2 via direct binding to cytoplasmic loops of Cav2.2. Depolarization enhanced the interaction. Further studies revealed that CRMP-2 facilitated an increase in Cav2.2 current density by inserting more Cav2.2 at the cell surface. As a consequence of CRMP-2-mediated increase in Ca(2+) influx, release of the excitatory neurotransmitter glutamate was also increased. CRMP-2 localized to synapses where, surprisingly, its overexpression increased synapse size. We hypothesize that the CRMP-2-calcium channel interaction represents a novel mechanism for modulation of Ca(2+) influx into nerve terminals and, hence, of synaptic strength. In this addendum, we further discuss the significance of this study and the possible implications to the field.

The anti-epileptic drug (R)-lacosamide ((2R)-2-(acetylamino)-N-benzyl-3-methoxypropanamide (LCM)) modulates voltage-gated sodium channels (VGSCs) by preferentially interacting with slow inactivated sodium channels, but the observation that LCM binds to collapsin response mediator protein 2 (CRMP-2) suggests additional mechanisms of action for LCM. We postulated that CRMP-2 levels affects the actions of LCM on VGSCs. CRMP-2 labeling by LCM analogs was competitively displaced by excess LCM in rat brain lysates. Manipulation of CRMP-2 levels in the neuronal model system CAD cells affected slow inactivation of VGSCs without any effects on other voltage-dependent properties. In silico docking was performed to identify putative binding sites in CRMP-2 that may modulate the effects of LCM on VGSCs. These studies identified five cavities in CRMP-2 that can accommodate LCM. CRMP-2 alanine mutants of key residues within these cavities were functionally similar to wild-type CRMP-2 as assessed by similar levels of enhancement in dendritic complexity of cortical neurons. Next, we examined the effects of expression of wild-type and mutant CRMP-2 constructs on voltage-sensitive properties of VGSCs in CAD cells: 1) steady-state voltage-dependent activation and fast-inactivation properties were not affected by LCM, 2) CRMP-2 single alanine mutants reduced the LCM-mediated effects on the ability of endogenous Na(+) channels to transition to a slow inactivated state, and 3) a quintuplicate CRMP-2 alanine mutant further decreased this slow inactivated fraction. Collectively, these results identify key CRMP-2 residues that can coordinate LCM binding thus making it more effective on its primary clinical target.

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.

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 Ca(2+) 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 Ca(2+) channel current density, whereas lentivirus-mediated CRMP-2 knockdown abolished this effect. Interestingly, the increase in Ca(2+) 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 Ca(2+) entry via CaV2.2 abolished this stimulated release. Thus, the CRMP-2-Ca(2+) channel interaction represents a novel mechanism for modulation of Ca(2+) influx into nerve terminals and, hence, of synaptic strength.

Munc18 is a presynaptic protein that is essential for transmitter release. Recent studies have indicated that this protein is involved in secretory vesicle docking but its binding partners in this role remain a mystery. We demonstrate using the isolated calyx-type presynaptic terminal of the chick ciliary ganglion that staining for Munc18 colocalizes and covaries with that for transmitter release site N type calcium channels (CaV2.2), consistent with elements of a common release site complex. Biochemical analysis demonstrated that the protein coprecipitates with CaV2.2 from lysates of rat or chick brain, including its synaptic, long-splice variant; presynaptic terminal surface membrane proteins, and a cell line coexpressing Munc18 and CaV2.2. Munc18 bound with high affinity to the CaV2.2 II-III intracellular loop, low affinity to the I-II loop but not to other channel intracellular regions. Over-expression of Munc18 in dorsal root ganglion neurons did not affect CaV2.2 current amplitude or fast kinetics but siRNA-knockdown resulted in a negative shift in the steady state inactivation curve, an effect attributed to an indirect action via syntaxin 1. Recombinant Munc18 also coprecipitated strongly with the v-SNARE synaptotagmin, but only weakly with other SNAREs. Thus, the calcium channel may serve as a surface membrane platform anchoring a Munc18-containing bridge to synaptotagmin and the synaptic vesicle.