Antillatoxin, a Novel Lipopeptide, Enhances Neurite Outgrowth in Immature Cerebrocortical Neurons through Activation of Voltage-Gated Sodium Channels

Abstract

Antillatoxin (ATX) is a structurally novel lipopeptide that activates voltage-gated sodium channels (VGSC) leading to sodium influx in cerebellar granule neurons and cerebrocortical neurons 8 to 9 days in vitro (Li et al., 2001;Cao et al., 2008). However, the precise recognition site for ATX on the VGSC remains to be defined. Inasmuch as elevation of intracellular sodium ([Na ϩ ] i ) may increase N-methyl-Daspartate receptor (NMDAR)-mediated Ca 2ϩ influx, Na ϩ may function as a signaling molecule. We hypothesized that ATX may enhance neurite outgrowth in cerebrocortical neurons by elevating [Na ϩ ] i and augmenting NMDAR function. ATX (30 -100 nM) robustly stimulated neurite outgrowth, and this enhancement was sensitive to the VGSC antagonist, tetrodotoxin. To unambiguously demonstrate the enhancement of NMDA receptor function by ATX, we recorded single-channel currents from cell-attached patches. ATX was found to increase the open probability of NMDA receptors. Na ϩ -dependent up-regulation of NMDAR function has been shown to be regulated by Src family kinase (SFK) (Yu and Salter, 1998). The Src kinase inhibitor PP2 abrogated ATXenhanced neurite outgrowth, suggesting a SFK involvement in this response. ATX-enhanced neurite outgrowth was also inhibited by the NMDAR antagonist, (5R,10S)-(ϩ)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801), and the calmodulin-dependent kinase kinase (CaMKK) inhibitor, 1,8-naphthoylene benzimidazole-3-carboxylic acid (STO-609), demonstrating the requirement for NMDAR activation with subsequent downstream engagement of the Ca 2ϩ -dependent CaMKK pathway. These results with the structurally and mechanistically novel natural product, ATX, confirm and generalize our earlier results with a neurotoxin site 5 ligand. These data suggest that VGSC activators may represent a novel pharmacological strategy to regulate neuronal plasticity through NMDAR-dependent mechanisms.

Gambierol Inhibition of Voltage-Gated Potassium Channels Augments Spontaneous Ca2+Oscillations in Cerebrocortical Neurons

“…The Helios Gene Gun System (Bio-Rad, Hercules, CA) was used to deliver DiI-coated tungsten particles (1.3 mM) (Bio-Rad) into paraformaldehyde-fixed DIV 1 cerebrocortical neurons as previously described (Jabba et al, 2010).…”

Nanomolar Bifenthrin Alters Synchronous Ca2+Oscillations and Cortical Neuron Development Independent of Sodium Channel Activity

“…Nanomolar Bifenthrin Stimulates CREB Phosphorylation. Ca 21 influx mediated by mGluR5 glutamatergic neurotransmission is known to promote CREB phosphorylation, which in turn stimulates a number of genes involved in normal activity-dependent plasticity (Bengtson and Bading, 2012), as well as environmentally triggered alterations of dendritic architecture (Jabba et al, 2010;Wayman et al, 2012). We therefore examined whether bifenthrin-induced changes in the pattern of SCO were sufficient to increase CREB phosphorylation on Ser133.…”

Novel C-1 Substituted Cocaine Analogs Unlike Cocaine or Benztropine

“…The human SERT-expressing cells were those described previously by Eshleman et al (1995), and the human NET-expressing cells used were those described previously by Reith et al (2005). Voltage-gated sodium channel inhibition assays were performed with cultured neocortical neurons obtained from Swiss-Webster mice, as described briefly in the section below and in further detail elsewhere Jabba et al, 2010). All of the C-1 cocaine analogs were synthesized by us (Davis et al, 2010(Davis et al, , 2012.…”

“…Primary cultures of neocortical neurons were obtained from embryonic day 17 Swiss-Webster mice as described elsewhere Jabba et al, 2010). In brief, pregnant mice were euthanized by CO 2 asphyxiation, and embryos were removed under sterile conditions.…”

“…Intracellular sodium concentration measurement and full in situ calibration of sodium-binding benzofuran isophthalate (SBFI) fluorescence ratio were performed as described previously (Jabba et al, 2010). Cells grown in 96-well plates were washed four times with Locke's buffer (8.6 mM HEPES, 5.6 mM KCl, 154 mM NaCl, 5.6 mM glucose, 1.0 mM MgCl 2 , 2.3 mM CaCl 2 , and 0.1 mM glycine, pH 7.4) by using an automated microplate washer (BioTek Instruments, Winooski, VT).…”