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.2007 Jul;53(1):145-56.
doi: 10.1016/j.neuropharm.2007.04.016. Epub 2007 May 10.

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

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

Zhenyu Sheng et al. Neuropharmacology.2007 Jul.

Abstract

Conantokin-G (con-G), conantokin-T (con-T), a truncated conantokin-R (con-R[1-17]), that functions the same as wild-type con-R, and variant sequences of con-T, were chemically synthesized and employed to investigate their selectivities as antagonists of glutamate/glycine-evoked ion currents in human embryonic kidney-293 cells expressing various combinations of NMDA receptor (NMDAR) subunits (NR), viz., NR1a/2A, NR1a/2B, NR1b/2A and NR1b/2B. Con-G did not substantially affect ion flow into NR1a,b/NR2A-transfected cells, but potently inhibited cells expressing NR1a,b/NR2B, showing high NR2B selectivity. Con-T and con-R served as non-selective antagonists of all of four NMDAR subunit combinations. C-terminal truncation variants of the 21-residue con-T were synthesized and examined in this regard. While NMDAR ion channel antagonist activity, and the ability to adopt the Ca(2+)-induced alpha-helical conformation, diminished as a function of shortening the COOH-terminus of con-T, NMDAR subtype selectivity was enhanced in the con-T[1-11], con-T[1-9], and con-T[1-8] variants toward NR2A, NR1b, and NR1b/2A, respectively. Receptor subtype selectivity was also obtained with Met-8 sequence variants of con-T. Con-T[M8A] and con-T[M8Q] displayed selectivity with NR2B-containing subunits, while con-T[M8E] showed enhanced activity toward NR1b-containing NMDAR subtypes. Of those studied, the most highly selective variant was con-T[M8I], which showed maximal NMDAR ion channel antagonism activity toward the NR1a/2A subtype. These studies demonstrate that it is possible to engineer NMDAR subtype antagonist specificity into con-T. Since the subunit composition of the NMDAR varies temporally and spatially in developing brain and in various disease states, conantokins with high subtype selectivities are potentially valuable drugs that may be used at specific stages of disease and in selected regions of the brain.

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Figures

Fig. 1
Fig. 1
Time course of current flow through a recombinant NMDARs in transfected HEK293 cells. (A) Representative trace illustrating the electrophysiological response to NR1b/2B to 100 μM NMDA/10 μM glycine, showing the rapid onset and offset responses to NMDA at the beginning and at the conclusion of the agonist applications. (B) Whole-cell currents from cells with different subunit combinations at various concentrations of NMDA, were normalized to the response produced by 100 μM NMDA in the presence of 10 μM glycine. The response at 500 μM NMDA was set to 100%. In this example, the EC50 value for NMDA obtained with NR1b/2B was 19.0 ± 1.5 μM. (C) The EC50 values for NMDA obtained with all four different NMDAR subunit combinations. (D) Representative trace illustrating the electrophysiology data obtained to evaluate the τobs and τoff at 50 μM con-R[1-17] with 100 μM NMDA/10 μM glycine present in all test solutions. The times and duration of application of test solutions are indicated by the solid bars. The onset and offset curves of conantokin inhibition were fit using a single exponential. (E) Plots of kobs values against the concentration of con-R[1-17] used as an inhibitor of current flow through NMDARs composed of NR1b/NR2A transfected into HEK293 cells. The holding potential was maintained at -70 mV.
Fig. 2
Fig. 2
Time course for inhibition of current flow through recombinant NMDAR subunit combinations by con-T[1-13], con-T[1-11], con-T[1-9], and con-T[1-8]. Receptor subunits NR1a/NR2A, NR1b/NR2A, NR1a/NR2B, and NR1b/NR2B were cotransfected into HEK293 cells. Data are shown with 50 μM of each conantokin applied at a time and for the duration indicated by horizontal line above the traces, with 100 μM NMDA/10 μM glycine present in all test solutions. The onset and offset curves of conantokin inhibition were fit using a single exponential. The holding potential was maintained at - 70 mV. The solid line above each panel represents the duration of application of the control solution, 100 μM NMDA + 10 μM glycine, and the dashed line indicates the duration of application of the relevant conantokin.
Figure 3
Figure 3
Comparison between Kd values for NR1a/2A, NR1a/2B, NR1b/2A, and NR1b/2B with: (A) con-T[1-11] (p = 0.006 for comparison between NR1a/2A and NR1a/2B, p = 0.004 for comparison between NR1b/2A and NR1b/2B); (B) con-T[1-9] (p = 0.006 for comparison between NR1a/2A and NR1b/2B; p = 0.003 for comparison between NR1a/2A and NR1b/2B; p = 0.006 between NR1a/2B and NR1b/2B); and (C) con-T[M8N] (p = 0.02 for comparison between NR1a/2A and NR1a/2B; p = 0.02 for comparison between NR1b/2A and NR1b/2B).
Fig. 4
Fig. 4
Time course for inhibition of current flow through recombinant NMDAR subunit combinations by Met-8 based variants of con-T. Receptor subunits NR1a/NR2A, NR1b/NR2A, NR1a/NR2B, and NR1b/NR2B were cotransfected into HEK293 cells. Data are shown with 40 μM conantokin under conditions described in Figure 2. A. Current flow data for con-T[M8E], con-T[M8I], con-T[M8Q]. B. Data for con-T[M8F], con-T[M8A], con-T[M8N]. The solid line above each trace represents the duration of application of the control solution, 100 μM NMDA + 10 μM glycine, and the dashed line indicates the duration of application of the relevant conantokin.
Fig. 4
Fig. 4
Time course for inhibition of current flow through recombinant NMDAR subunit combinations by Met-8 based variants of con-T. Receptor subunits NR1a/NR2A, NR1b/NR2A, NR1a/NR2B, and NR1b/NR2B were cotransfected into HEK293 cells. Data are shown with 40 μM conantokin under conditions described in Figure 2. A. Current flow data for con-T[M8E], con-T[M8I], con-T[M8Q]. B. Data for con-T[M8F], con-T[M8A], con-T[M8N]. The solid line above each trace represents the duration of application of the control solution, 100 μM NMDA + 10 μM glycine, and the dashed line indicates the duration of application of the relevant conantokin.
Fig. 5
Fig. 5
CD analysis of the secondary structure of con-T variants in the presence of Ca2+. (A) CD spectra of truncation variants (100 μM) of con-T. (❍) con-T[1-13]; (●) con-T[1-11]; (△) con-T[1-9]; and (◆) con-T[1-8]. (B) CD spectra of sequence position-8 variants of con-T. (formula image) extracellular buffer; (●) con-T; (❍) con-T[M8A]; (◆) con-T[M8N]; (□) con-T[M8F]; (■) con-T[M8E]; (△) con-T[M8Q]; (◆) con-T[M8I]. The buffer for all spectra was 140 mM NaCl, 3 mM KCl, 2 mM CaCl2, 10 mM Na-Hepes, and 20 mM dextrose, pH 7.35, at room temperature (extracellular buffer).
Fig. 5
Fig. 5
CD analysis of the secondary structure of con-T variants in the presence of Ca2+. (A) CD spectra of truncation variants (100 μM) of con-T. (❍) con-T[1-13]; (●) con-T[1-11]; (△) con-T[1-9]; and (◆) con-T[1-8]. (B) CD spectra of sequence position-8 variants of con-T. (formula image) extracellular buffer; (●) con-T; (❍) con-T[M8A]; (◆) con-T[M8N]; (□) con-T[M8F]; (■) con-T[M8E]; (△) con-T[M8Q]; (◆) con-T[M8I]. The buffer for all spectra was 140 mM NaCl, 3 mM KCl, 2 mM CaCl2, 10 mM Na-Hepes, and 20 mM dextrose, pH 7.35, at room temperature (extracellular buffer).
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