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.
Wang
Yuying
Y
Department of Pharmacology and Toxicology, Paul and Carole Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
Brittain
Joel M
JM
Jarecki
Brian W
BW
Park
Ki Duk
KD
Wilson
Sarah M
SM
Wang
Bo
B
Hale
Rachel
R
Meroueh
Samy O
SO
Cummins
Theodore R
TR
Khanna
Rajesh
R
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