Molecular determinants of the anticonvulsant felbamate binding site in the N-methyl-D-aspartate receptor.
The antiepileptic effect of felbamate (FBM) is ascribable to gating modification of NMDA receptors. Using site-directed mutagenesis and electrophysiological studies, we found that single-point mutations of four pairs of homologous residues in the external vestibule of the receptor pore, namely V644(NR1)-L643(NR2B) (the two inner pairs) and T648(NR1)-T647(NR2B) (the two outer pairs), significantly decrease FBM binding. Moreover, double mutations involving either the inner or the outer pair always show cooperative (nonadditive) effects on FBM binding, whereas double mutations involving both inner and outer pairs always show additive (noncooperative) effects. Most interestingly, triple mutations of any three of the four critical residues essentially abolish the effect of FBM. These findings indicate that T648(NR1)/T647(NR2B) and V644(NR1)/L643(NR2B) act cooperatively to contribute directly to the "outer binding region" and "inner binding region" in the FBM binding site, respectively. The outer and inner binding regions, however, seem to contribute independently to FBM binding. We conclude that residues L643 and T647 in NR2B as well as homologous residues V644 and T648 in NR1 are the major, and very likely the exclusive, molecular determinants constituting the FBM binding site in the NMDA receptor.
Chang
Huai-Ren
HR
Department of Physiology, National Taiwan University College of Medicine, 1 Jen-Ai Road, First Section, Taipei 100, Taiwan.
Kuo
Chung-Chin
CC
D000818:Animals; D000927:Anticonvulsants / Q000737:chemistry / Q000494:pharmacology*; D001665:Binding Sites / Q000187:drug effects; D001667:Binding, Competitive / Q000187:drug effects / Q000235:genetics; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D000078328:Felbamate; D005786:Gene Expression Regulation / Q000187:drug effects; D015640:Ion Channel Gating / Q000187:drug effects / Q000235:genetics; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D048448:Phenylcarbamates / Q000737:chemistry / Q000494:pharmacology*; D011409:Propylene Glycols / Q000737:chemistry / Q000494:pharmacology*; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D013329:Structure-Activity Relationship; D014981:Xenopus
D000927:Anticonvulsants; D048448:Phenylcarbamates; D011409:Propylene Glycols; D016194:Receptors, N-Methyl-D-Aspartate; D000078328:Felbamate