Conantokins inhibit NMDAR-dependent calcium influx in developing rat hippocampal neurons in primary culture with resulting effects on CREB phosphorylation

Abstract

The effects of conantokin (con)-G, con-R [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17], and con-T on ion flow through N-methyl-Daspartate receptor (NMDAR) ion channels were determined in cultured primary rat hippocampal neurons. The potency of con-G diminished, whereas inhibition by con-R[1-17] and con-T did not change, as the neurons matured. Con-G, con-R[1-17], and con-T effectively diminished NMDAinduced Ca 2+ influx into the cells. A similar age-dependent decrease in con-G-mediated inhibition of the amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) was observed, compared to con-R[1-17] and con-T. The effects of the conantokins on NMDA-induced cAMP response element-binding protein (CREB) phosphorylation in immature (DIV 9) and mature (DIV 16) neurons showed that, at DIV 9, con-G, con-R[1-17], and con-T inhibited NMDA-mediated P-CREB levels, whereas in DIV 16 neurons the conantokins did not inhibit overall levels of NMDAinduced P-CREB. In contrast, P-CREB levels were enhanced through inhibition of the protein phosphatases, PP1 and PP2B (calcineurin). This ability of conantokins to sustain CREB phosphorylation can thus enhance neuronal survival and plasticity.

Hydroxyproline-induced Helical Disruption in Conantokin Rl-B Affects Subunit-selective Antagonistic Activities toward Ion Channels of N-Methyl-d-aspartate Receptors

“…Thus, the ␣-helical contents of the conantokin peptides were estimated based empirically on the following relationship: % conantokin helicity ϭ ( 222 peptide)/(( 222 Mg 2ϩ / conG) Ϫ ( 222 apo-conG)) ϫ 100. The data obtained showed that apo-conRl-B, in which 4-Hyp is present at sequence position 10 (Hyp 10 or O 10 ), contains Ͻ5% helical content (Fig. 1A), similar to other published data (54 1C) and exchange of the immediate upstream two amino acids to those that more closely resemble conG, also show similarly low ␣-helical values.…”

“…NMR Spectroscopy-The samples for NMR measurements were prepared by dissolving the purified peptide to a final concentration of 2 mM in a solution containing 10 15 N dimensions in two-dimensional heteronuclear spectra were referenced indirectly (34). The NMR data were processed and analyzed with the programs TopSpin (Bruker Instruments) and Sparky.…”

“…These data suggest that the Hyp 10 allows a large conformational rearrangement to a more helical structure in the presence of Mg 2ϩ . That Mg 2ϩ /conG [O 10 ] shows a full helical structure, at least as estimated by CD, suggests that Hyp 10 , in the context of the conG backbone, permits global folding of the peptide that resembles a full ␣-helix. In an overall sense, these data show that Mg 2ϩ binds to the Hyp 10 -containing peptides, thus allowing adoption of helix-like structures that are most likely composed of short local segments within the peptides.…”

“…5A) and conG [O 10 ] (Fig. 5B) revealed the introduction of a "kink" in the ␣-helical backbone due to the presence of Hyp 10 , which resulted in a root mean square deviation of Ͼ2 Å for each peptide. The average structure of conRl-B[⌬K At a lower temperature of 5°C, the Mg 2ϩ ⅐conRl-B complex exists as a less dynamic structure that resulted in an increased number (252) of NOE distance constraints.…”

“…The exact NMDAR subunit composition is variable and depends upon the stage of development and location of the heterotetramer in neuronal populations (7). Additionally, the variable compositions of functional NMDARs also determine the nature of their interactions with agonists/antagonists (8 functions as a secondary messenger, which further activates, by phosphorylation, the cAMP-response binding element, via a Ca 2ϩ /calmodulin-dependent pathway (9,10). Additionally, NMDARs also bi-directionally regulate activation of PI3K-Akt (11)(12)(13) and ERK1/2 (14,15).…”