Abstract
N-Methyl-d-aspartate (NMDA) receptors play key roles in both physiological processes, particularly synaptic plasticity, and in neuropathological states such as epilepsy and acute neurodegeneration. R-(R*,S*)-α-(4-Hydroxyphenyl)-β-methyl-4-(phenyl-methyl)-1-piperidine propanol (RO 25-6981), is a high-affinity and selective blocker of NMDA receptors containing the NR2B subunit. Using site-directed mutagenesis, [3H]RO 25-6981 binding, Xenopus oocyte voltage-clamp recordings, and molecular modeling, we have identified several critical residues involved in the RO 25-6981 binding site within the N-terminal LIVBP-like domain of the human NR2B subunit. Two mutations, NR2B(D101A) and NR2B(F176A), resulted in a complete loss of [3H]RO 25-6981 binding and also abolished the high-affinity RO 25-6981-mediated inhibition of NMDA-induced currents. The mutation NR2B(T233A) led to a marked reduction in binding affinity by 13-fold. Mutations F182A, D104A, or K234A had a more moderate influence on the binding affinity (KD values increased by 8-, 7-, and 6-fold, respectively). In a three-dimensional model of the NR2B LIVBP-like domain based on the X-ray crystal structure of the amino-terminal domain of the mGlu1 receptor, the critical residues are located in the central cleft where interaction with RO 25-6981 may stabilize the closed structure of the domain. Our results suggest that the three amino acids Asp-101, Phe-176, and Thr-233 are important molecular determinants for the high-affinity binding of RO 25-6981 to the LIVBP-like domain of human NR2B. A possible binding mode for RO 25-6981 is proposed.
Footnotes
-
ABBREVIATIONS: NMDA, N-methyl-d-aspartate; iGlu, ionotropic glutamate; TM, transmembrane; LIVBP, leucine/isoleucine/valine-binding protein; mGlu, metabotropic glutamate; GlnBP, glutamine binding protein; DTG, 1,3-di-o-tolylguanidine; PCR, polymerase chain reaction; nt, nucleotide; HEK, human embryonic kidney; RO 25-6981, R-(R*,S*)-α-(4-hydroxyphenyl)-β-methyl-4-(phenyl-methyl)-1-piperidine propanol; RO 04-5595, 1-(4-chlorophenyl)-2-methyl-6-methoxy-7-hydroxy-1,2,3,4-tetrahydroisoquinolin; 3D, three-dimensional; WT, wild-type; ATD, amino-terminal domain; CP 101,606, (1S,2S)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propanol; SAR, structure-activity relationship.
-
DOI: 10.1124/jpet.103.056291.
-
↵1 Current address: Addex Pharmaceuticals SA, 12 Chemin Des Aulx, CH-1228 Plan Les Ouates, Switzerland.
-
↵2 Current address: Evotec Neurosciences GmbH, Schnackenburgallee 114, D-22525 Hamburg, Germany.
-
↵3 Current address: Psychiatry Centre of Excellence for Drug Discovery, GlaxoSmithKline, New Frontiers Science Park, Third Ave., Harlow, Essex CM19 5AW, UK.
- Received June 30, 2003.
- Accepted August 22, 2003.
- The American Society for Pharmacology and Experimental Therapeutics
JPET articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|