Direct binding properties of conantokins to native N-methyl-d-aspartate receptors.

Conantokin-G (con-G) is a small, gamma-carboxyglutamic acid (Gla)-containing peptide that functions neurophysiologically by inhibiting the N-methyl-d-aspartate receptor (NMDAR). In the current study, the receptor binding properties of an alanine-rich, Gla-deficient con-G variant, Ala-con-G, were assessed following tracer radioiodination with 125I. Direct binding experiments with [125I]Ala-con-G yielded a single site defined by a Kd value of 516 +/- 120 nm. Displacement of [125I]Ala-con-G binding by Ala-con-G resulted in 100% displacement with an IC50 value of 564 +/- 33 nm, while heterologous displacement by con-G[S16Y], con-G, con-T, and con-R[1-17] yielded IC50 values in the range of 15-45 microm. No displacement was observed with d-gamma-con-G or con-G[L5A], analogs that are inactive at NMDARs. Specific [125I]Ala-con-G binding was displaced by NMDA and 2-amino-5-phosphopentanoic acid in a dose-dependent manner, suggesting an interaction at the glutamate binding site. The direct binding of [125I]Ala-con-G to adult rat brain sections revealed an anatomical distribution of binding sites in all regions known to contain the NR2B subunit of the NMDAR. These results constitute the only known demonstration of the direct binding of a radiolabeled conantokin to the NMDARs present in rat brain membrane preparations and rat brain sections, and suggest that radiolabeled Ala-con-G, and similar conantokin derivatives, may find utility as probes of NMDARs in a variety of systems.