The Amino Acid Residue at Sequence Position 5 in the Conantokin Peptides Partially Governs Subunit-selective Antagonism of RecombinantN-Methyl-d-aspartate Receptors

Abstract

Whole cell voltage clamp recordings were performed to assess the ability of conantokin-G (con-G), conantokin-T (con-T), and a 17-residue truncated form of conantokin-R (con-R[1-17]) to inhibit N-methyl-d-aspartate (NMDA)-evoked currents in human embryonic kidney 293 cells transiently expressing various combinations of NR1a, NR1b, NR2A, and NR2B receptor subunits. Con-T and con-R[1-17] attenuated ion currents in cells expressing NR1a/NR2A or NR1a/NR2B. Con-G did not affect NMDA-evoked ionic currents in cells expressing NR1a/NR2A, but it showed inhibitory activity in cells expressing NR1a/NR2B receptors and the triheteromeric combination of NR1a/NR2A/NR2B. An Ala-rich con-G analog, con-G[Q6G/gamma7K/N8A/gamma10A/gamma14A/K15A/S16A/N17A] (Ala/con-G, where gamma is Gla), in which all nonessential amino acids were altered to Ala residues, manifested subunit specificity similar to that of con-G, suggesting that the replaced residues are not responsible for selectivity in the con-G framework. A sarcosine-containing con-T truncation analog, con-T[1-9/G1Src/Q6G], inhibited currents in NR1a/NR2A and NR1a/NR2B receptors, eliminating residues 10-21 as mediators of the broad subunit selectivity of con-T. In contrast to the null effects of con-G and Ala/con-G at a NR1a/NR2A-containing receptor, some inhibition ( approximately 40%) of NMDA-evoked currents was effected by these peptides in cells expressing NR1b/NR2A. This finding suggests that the presence of exon 5 in NR1b plays a role in the activity of the conantokins. Analysis of various conantokin analogs demonstrated that Leu(5) of con-G is an important determinant of conantokin selectivity. Taken as a whole, these results suggest that the important molecular determinants on conantokins responsible for NMDA receptor activity and specificity are discretely housed in specific residues of these peptides, thus allowing molecular manipulation of the NMDA receptor inhibitory properties of the conantokins.

Subtype-selective antagonism of N-methyl-d-aspartate receptor ion channels by synthetic conantokin peptides

“…It has previously been shown in several laboratories that conantokins function as subunitselective inhibitors of current flow through the NMDAR channel, in major part through interactions with the glutamate site on the NR2 subunit with NR1/NR2B Klein et al, 2001) and NR1/2A,2B (Barton et al, 2004;Klein et al, 2001) subunit combinations providing receptors that are inhibited by conantokins. Conantokins, at least as exemplified by con-G, do not appear to inhibit receptor function through the NR1-specific glycine site, although the potency of inhibition by conantokins of ion flow through NMDAR channels does vary somewhat depending upon the exact NR1 splice variant present Ragnarsson et al, 2006).…”

N-methyl-d-aspartate receptor inhibition by an apolipoprotein E-derived peptide relies on low-density lipoprotein receptor-associated protein

“…HEK293 cells (CRL-1573 from ATCC, Manassas, VA) were transiently transfected with NMDAR subunits using calcium phosphate precipitation (Klein et al, 2001) The cells were grown to ~50% confluency in 60 mm poly-D-lysine-coated dishes and transfected (10 µg of DNA/dish), with combinations of NR1a, NR1b, NR2A, and NR2B-containing plasmids, along with pEGFP-N1 for positive selection of transfected cells, at ratios of 1:3:6 for GFP:NR1a/ b:NR2A/B (Klein et al, 2001). …”

“…Whole cell recordings were obtained at room temperature (Klein et al, 2001;Sheng et al, 2007) Ketamine was removed from cells immediately prior to recordings by 3 × 3 mL washes (2 min each) with Mg 2+ -free extracellular solution, which consisted of 140 mM NaCl/3 mM KCl/2 mM CaCl 2 /10 mM Na-Hepes/20 mM dextrose, pH 7.35 (in some experiments, the extracellular level of NaCl was varied). Borosilicate glass recording pipettes (Drummond Scientific, Broomall, PA), with a resistance of 2-4 MΩ, were back-filled with a solution containing 140 mM CsF/1 mM CaCl 2 /2 mM MgCl 2 /10 mM EGTA/10 mM Cs-Hepes/2 mM tetraethylammonium chloride/4 mM Na 2 ATP, pH 7.35.…”

Developmental Biology of Neoplastic Growth

“…The molecular binding site of con-G could be heterotopic, because the inhibitory activity is enhanced by polyamines such as spermine , which binds to an extracellular allosteric modulatory site. Con-G also exhibits a high degree of subunit selectivity; inhibition of NR1/NR2B NMDA receptors is potent, whereas NR1/NR2A receptors (or those containing NR2C or NR2D) are relatively unaffected by the peptide Klein et al 2001). This degree of selectivity does not extend to all conantokins, however, as con-R and con-T inhibit both NR2A-and NR2B-containing receptors (White et al 2000;Klein et al 2001).…”

“…Con-G also exhibits a high degree of subunit selectivity; inhibition of NR1/NR2B NMDA receptors is potent, whereas NR1/NR2A receptors (or those containing NR2C or NR2D) are relatively unaffected by the peptide Klein et al 2001). This degree of selectivity does not extend to all conantokins, however, as con-R and con-T inhibit both NR2A-and NR2B-containing receptors (White et al 2000;Klein et al 2001). Conantokins, and in particular con-G, have attracted significant attention for their potential as therapeutics in a variety of neuropathologies ).…”