Kinetic and mechanistic characterization of NMDA receptor antagonism by replacement and truncation variants of the conantokin peptides

The characterization of conantokin-T (con-T), conantokin-R (con-R), and variants thereof, using the whole-cell patch clamp technique, was undertaken to evaluate the contribution of various residues towards the onset and recovery of N-methyl-D-aspartate (NMDA) receptor inhibition in cultured embryonic murine hippocampal neurons. The results obtained indicate that the two most C-terminal γ-carboxyglutamic acid (Gla) residues of the conantokins, while not essential for activity, provided for more tenacious binding to the receptor. Specifically, con-T[γ10K/γ14K] and con-R[γ11A/γ15A] displayed 5.6- and 8.4-fold decreases in τ_off, respectively, compared to the parent peptides. For the truncated con-T variants, con-T[1-9/Q6G], and a sarcosine (Src)-containing species, con-T[1-9/G1Src/Q6G], the τ_off was over 80- and 40-fold faster, respectively, compared to con-T. For the latter peptide, the coapplication of 300 μM spermine enhanced the onset rate constant from 3.1×10³ M^-1 s^-1 to 12.6×10³ M^-1 s^-1. From analysis of equilibrium dose-inhibition curves using the Cheng-Prusoff equation, a K_i value of 1.1 μM for the peptide was obtained. Con-T[1-9/G1Src/Q6G] demonstrated an apparent competitive mode of inhibition relative to NMDA. Schild analysis of the data yielded an equilibrium dissociation constant of 2.4 μM for the interaction of con-T[1-9/G1Src/Q6G] with the receptor.