In Silico Docking and Electrophysiological Characterization of Lacosamide Binding Sites on Collapsin Response Mediator Protein-2 Identifies a Pocket Important in Modulating Sodium Channel Slow Inactivation

Abstract

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 wildtype 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 fastinactivation 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.

A functional coupling between CRMP1 and Nav1.7 for retrograde propagation of Semaphorin3A signaling

“…It has been reported that CRMP2 is involved in kinesin-1-dependent transport of the Sra1-WAVE1 complex (Arimura et al, 2009) and in regulation of trafficking by linking endocytic regulatory proteins to dynein motors (Rahajeng et al, 2010). CRMP2 has been reported to be able to directly bind to voltage-dependent Na + channels, modulating the channel's slow inactivation (Dustrude et al, 2013;Wang et al, 2010). CRMP2 also modulates Na v 1.7 trafficking via SUMOylation (Dustrude et al, 2013).…”

Inhibition of Transmitter Release and Attenuation of Anti-retroviral-associated and Tibial Nerve Injury-related Painful Peripheral Neuropathy by Novel Synthetic Ca2+ Channel Peptides

“…Catecholamine A-differentiated (CAD) Cell Culture-CAD cells were cultured exactly as described (20,21).…”

Cdk5‐mediated phosphorylation of CRMP‐2 enhances its interaction with CaV2.2

“…All mutations were made as previously described using Quikchange II XL (Agilent Technologies, Santa Clara, CA) and verified by DNA sequencing [30]. Constructs for wildtype and dominant negative CdK5 were purchased from Addgene (Cambridge, MA).…”

“…The neuronally derived CAD cells were grown at 37°C and in 5% CO 2 as previously described [30,31]. CAD cells were transfected as described above.…”