Evidence for the involvement of a carboxyl group in the vicinity of the MK801 and magnesium ion binding site of the N-methyl-D-aspartate receptor.
A series of protein modifying reagents were tested for their effects on the specific binding of [3H]MK801 to adult rat brain membranes. N-Bromosuccinimide, acetyl imidazole, 2,3-butanedione, 5,5'-dithiobis-(2-nitrobenzoic acid) and dithiothreitol all had no significant effect on binding. The carboxylic acid residue modification reagent, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC), inhibited [3H]MK801 specific binding in a dose-dependent manner with an IC50 = 1.9 mM. The inhibition by EDAC was due to a decrease in the Bmax with no change in KD. The inhibition of [3H]MK801 binding by EDAC was not prevented by prior incubation with competitive antagonists. Protection against EDAC inactivation was obtained, however, in a dose-dependent manner by preincubation with the divalent cations, Ca2+ and Mg2+, but not Zn2+. These results suggest that EDAC modifies an important carboxyl group located within the voltage-dependent Mg2+ binding site of the N-methyl-D-aspartate receptor. This modification yields a decrease in the specific [3H]MK801 binding activity thus demonstrating a close association between the two allosteric regulatory sites.
Chazot
P L
PL
Department of Pharmaceutical Chemistry, School of Pharmacy, London, U.K.
Fotherby
A
A
Stephenson
F A
FA
D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D001923:Brain Chemistry; D002234:Carbodiimides / Q000494:pharmacology; D016291:Dizocilpine Maleate / Q000037:antagonists & inhibitors / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D015195:Drug Design; D008274:Magnesium / Q000737:chemistry*; D051381:Rats; D017208:Rats, Wistar; D016194:Receptors, N-Methyl-D-Aspartate / Q000737:chemistry*
D000596:Amino Acids; D002234:Carbodiimides; D016194:Receptors, N-Methyl-D-Aspartate; C074139:1-ethyl-3-(3-dimethylaminoethyl)carbodiimide; D016291:Dizocilpine Maleate; D008274:Magnesium