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.
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.
Department of Pharmacology and Toxicology, Paul and Carole Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
Yuying
Y
Wang
Joel M
JM
Brittain
Brian W
BW
Jarecki
Ki Duk
KD
Park
Sarah M
SM
Wilson
Bo
B
Wang
Rachel
R
Hale
Samy O
SO
Meroueh
Theodore R
TR
Cummins
Rajesh
R
Khanna
D000081:Acetamides / Q000378:metabolism* / Q000494:pharmacology* / Q000627:therapeutic use; D000818:Animals; D001665:Binding Sites / Q000235:genetics / Q000502:physiology; D002478:Cells, Cultured; D004594:Electrophysiology / Q000379:methods*; D004827:Epilepsy / Q000188:drug therapy; D015151:Immunoblotting; D036341:Intercellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000078334:Lacosamide; D008297:Male; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015222:Sodium Channels / Q000187:drug effects* / Q000378:metabolism*
D000081:Acetamides; D036341:Intercellular Signaling Peptides and Proteins; D009419:Nerve Tissue Proteins; D015222:Sodium Channels; C095114:collapsin response mediator protein-2; D000078334:Lacosamide