Voltage-gated sodium channel (NaV) trafficking is incompletely understood. Post-translational modifications of NaVs and/or auxiliary subunits and protein-protein interactions have been posited as NaV-trafficking mechanisms. Here, we tested if modification of the axonal collapsin response mediator protein 2 (CRMP2) by a small ubiquitin-like modifier (SUMO) could affect NaV trafficking; CRMP2 alters the extent of NaV slow inactivation conferred by the anti-epileptic (R)-lacosamide, implying NaV-CRMP2 functional coupling. Expression of a CRMP2 SUMOylation-incompetent mutant (CRMP2-K374A) in neuronal model catecholamine A differentiated (CAD) cells did not alter lacosamide-induced NaV slow inactivation compared with CAD cells expressing wild type CRMP2. Like wild type CRMP2, CRMP2-K374A expressed robustly in CAD cells. Neurite outgrowth, a canonical CRMP2 function, was moderately reduced by the mutation but was still significantly higher than enhanced GFP-transfected cortical neurons. Notably, huwentoxin-IV-sensitive NaV1.7 currents, which predominate in CAD cells, were significantly reduced in CAD cells expressing CRMP2-K374A. Increasing deSUMOylation with sentrin/SUMO-specific protease SENP1 or SENP2 in wild type CRMP2-expressing CAD cells decreased NaV1.7 currents. Consistent with a reduction in current density, biotinylation revealed a significant reduction in surface NaV1.7 levels in CAD cells expressing CRMP2-K374A; surface NaV1.7 expression was also decreased by SENP1 + SENP2 overexpression. Currents in HEK293 cells stably expressing NaV1.7 were reduced by CRMP2-K374A in a manner dependent on the E2-conjugating enzyme Ubc9. No decrement in current density was observed in HEK293 cells co-expressing CRMP2-K374A and NaV1.1 or NaV1.3. Diminution of sodium currents, largely NaV1.7, was recapitulated in sensory neurons expressing CRMP2-K374A. Our study elucidates a novel regulatory mechanism that utilizes CRMP2 SUMOylation to choreograph NaV1.7 trafficking.
Collapsin response mediator protein 2 (CRMP-2), traditionally viewed as an axon/dendrite specification and axonal growth protein, has emerged as nidus in regulation of both pre- and post-synaptic Ca ( 2+) channels. Building on our discovery of the interaction and regulation of Ca ( 2+) channels by CRMP-2, we recently identified a short sequence in CRMP-2 which, when appended to the transduction domain of HIV TAT protein, suppressed acute, inflammatory and neuropathic pain in vivo by functionally uncoupling CRMP-2 from the Ca ( 2+) channel. Remarkably, we also found that this region attenuated Ca ( 2+) influx via N-methylD-Aspartate receptors (NMDARs) and reduced neuronal death in a moderate controlled cortical impact model of traumatic brain injury (TBI). Here, we sought to extend these findings by examining additional neuroprotective effects of this peptide (TAT-CBD3) and exploring the biochemical mechanisms by which TAT-CBD3 targets NMDARs. We observed that an intraperitoneal injection of TAT-CBD3 peptide significantly reduced infarct volume in an animal model of focal cerebral ischemia. Neuroprotection was observed when TAT-CBD3 peptide was given either prior to or after occlusion but just prior to reperfusion. Surprisingly, a direct biochemical complex was not resolvable between the NMDAR subunit NR2B and CRMP-2. Intracellular application of TAT-CBD3 failed to inhibit NMDAR current. NR2B interactions with the post synaptic density protein 95 (PSD-95) remained intact and were not disrupted by TAT-CBD3. Peptide tiling of intracellular regions of NR2B revealed two 15-mer sequences, in the carboxyl-terminus of NR2B, that may confer binding between NR2B and CRMP-2 which supports CRMP-2's role in excitotoxicity and neuroprotection.
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.
The novel antiepileptic drug lacosamide (LCM; SPM927, Vimpat®) has been heralded as having a dual-mode of action through interactions with both the voltage-gated sodium channel and the neurite outgrowth-promoting collapsin response mediator protein 2 (CRMP2). Lacosamide's ability to dampen neuronal excitability through the voltage-gated sodium channel likely underlies its efficacy in attenuating the symptoms of epilepsy (i.e., seizures). While the role of CRMP2 in epilepsy has not been well studied, given the proposed involvement of circuit reorganization in epileptogenesis, the ability of lacosamide to alter CRMP2 function may prove disease modifying. Recently, however, the validity of lacosamide's interaction with CRMP2 has come under scrutiny. In this review, we address the contradictory reports concerning the binding of lacosamide to CRMP2 as well as the ability of lacosamide to directly impact CRMP2 function. Additionally, we address similarly the contradicting reports regarding the potential disease-modifying effect of lacosamide on the development and progression of epilepsy. As the vast majority of antiepileptic drugs influences only the symptoms of epilepsy, the ability to hinder disease progression would be a major breakthrough in efforts to cure or prevent this debilitating syndrome.
The integral synaptic vesicle protein and putative calcium sensor, synaptotagmin 1 (STG), has also been implicated in synaptic vesicle (SV) recovery. However, proteins with which STG interacts during SV endocytosis remain poorly understood. We have isolated an STG-associated endocytic complex (SAE) from presynaptic nerve terminals and have used a novel fractional recovery (FR) assay based on electrostatic dissociation to identify SAE components and map the complex structure. The location of SAE in the presynaptic terminal was determined by high-resolution quantitative immunocytochemistry at the chick ciliary ganglion giant calyx-type synapse.