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 var iants thereof, using the whole-cell patch clamp technique, was undertaken t o evaluate the contribution of various residues towards the onset and recov ery of N-methyl-D-aspartate (NMDA) receptor inhibition in cultured embryoni c murine hippocampal neurons. The results obtained indicate that the two mo st C-terminal gamma -carboxyglutamic acid (Gla) residues of the conantokins , while not essential for activity. provided for more tenacious binding to the receptor. Specifically, con-T[gamma 10K/gamma 14K] and con-R[gamma 11A/ gamma 15A] displayed 5.6- and 8.4-fold decreases in tau (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], t he tau (off) was over 80- and 40-fold faster, respectively, compared to con -T. For the latter peptide, the coapplication of 300 muM spermine enhanced the onset rate constant from 3.1x10(3) M-1 s(-1) to 12.6x10(3) M-1 s(-1). F rom analysis of equilibrium dose-inhibition curves using the Cheng-Prusoff equation, a K-i value of 1.1 muM for the peptide was obtained. Con-T[1-9/G1 Src/Q6G] demonstrated an apparent competitive mode of inhibition relative t o NMDA. Schild analysis of the data yielded an equilibrium dissociation con stant of 2.4 muM for the interaction of con-T[1-9/G1Src/Q6G] with the recep tor. (C) 2001 Elsevier Science Ltd. All rights reserved.