title: Kinetic and mechanistic characterization of NMDA receptor antagonism by replacement and truncation variants of the conantokin peptides.
abstract: 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 gamma-carboxyglutamic acid (Gla) residues of the conantokins, while not essential for activity, provided for more tenacious binding to the receptor. Specifically, con-T[gamma10K/gamma14K] and con-R[gamma11A/gamma15A] 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], the tau(off) was over 80- and 40-fold faster, respectively, compared to con-T. For the latter peptide, the coapplication of 300 microM spermine enhanced the onset rate constant from 3.1x10(3)M(-1) x s(-1) to 12.6x10(3)M(-1) x s(-1). From analysis of equilibrium dose-inhibition curves using the Cheng-Prusoff equation, a K(i) value of 1.1 microM 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 microM for the interaction of con-T[1-9/G1Src/Q6G] with the receptor.
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA.
authors.forename: R C
authors.initials: RC
authors.lastname: Klein
authors.forename: S E
authors.initials: SE
authors.lastname: Warder
authors.forename: Z
authors.initials: Z
authors.lastname: Galdzicki
authors.forename: F J
authors.initials: FJ
authors.lastname: Castellino
authors.forename: M
authors.initials: M
authors.lastname: Prorok
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001667:Binding, Competitive; D002478:Cells, Cultured; D020916:Conotoxins / Q000737:chemistry / Q000378:metabolism*; D018690:Excitatory Amino Acid Agonists / Q000378:metabolism; D018691:Excitatory Amino Acid Antagonists / Q000378:metabolism*; D005333:Fetus; D006624:Hippocampus / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D008978:Mollusk Venoms / Q000737:chemistry / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D016194:Receptors, N-Methyl-D-Aspartate / Q000819:agonists / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D013096:Spermine / Q000494:pharmacology
chemical_list: D020916:Conotoxins; D018690:Excitatory Amino Acid Agonists; D018691:Excitatory Amino Acid Antagonists; D036341:Intercellular Signaling Peptides and Proteins; D008978:Mollusk Venoms; D010446:Peptide Fragments; D010455:Peptides; D016194:Receptors, N-Methyl-D-Aspartate; C403083:conantokin R; C063571:conantokin-T; D013096:Spermine