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

• Published in: The Journal of biological chemistry Vol. 285; no. 33; pp. 25296 - 25307
• Main Authors: Wang, Yuying, Brittain, Joel M., Jarecki, Brian W., Park, Ki Duk, Wilson, Sarah M., Wang, Bo, Hale, Rachel, Meroueh, Samy O., Cummins, Theodore R., Khanna, Rajesh
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 13.08.2010; American Society for Biochemistry and Molecular Biology
• Subjects: Acetamides - metabolism; Acetamides - pharmacology; Acetamides - therapeutic use; Animals; Binding Sites - genetics; Binding Sites - physiology; Biophysics; Cells, Cultured; Collapsin Response Mediator Protein; Computation; Electrophysiology; Electrophysiology - methods; Epilepsy - drug therapy; Immunoblotting; in Silico Drug Docking; Intercellular Signaling Peptides and Proteins - chemistry; Intercellular Signaling Peptides and Proteins - genetics; Intercellular Signaling Peptides and Proteins - metabolism; Lacosamide; Male; Molecular Biophysics; Nerve Tissue Proteins - chemistry; Nerve Tissue Proteins - genetics; Nerve Tissue Proteins - metabolism; Neurobiology; Neuroscience; Protein Cross-linking; Rats; Rats, Sprague-Dawley; Site-directed Mutagenesis; Slow Inactivation; Sodium Channels; Sodium Channels - drug effects; Sodium Channels - metabolism; Collapsin Response Mediator Protein; Site-directed Mutagenesis; Neuroscience; in Silico Drug Docking; Computation; Sodium Channels; Electrophysiology; Slow Inactivation; Biophysics; Protein Cross-linking
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M110.128801

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.