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+]i) may increase N-methyl-d-aspartate receptor (NMDAR)-mediated Ca2+ influx, Na+ may function as a signaling molecule. We hypothesized that ATX may enhance neurite outgrowth in cerebrocortical neurons by elevating [Na+]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+-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 ATX-enhanced neurite outgrowth, suggesting a SFK involvement in this response. ATX-enhanced neurite outgrowth was also inhibited by the NMDAR antagonist, (5R,10S)-(+)-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 Ca2+-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.
Footnotes
This work was supported in part by the National Institutes of Health National Institute of Neurological Disorders and Stroke [Grant NS053398] (to W.H.G. and T.F.M.); and the National Institutes of Health National Center for Research Resources [Grant G20-RR024001].
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.109.161802.
↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
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ABBREVIATIONS:
- ATX
- antillatoxin
- PbTx
- brevetoxin
- VGSC
- voltage-gated sodium channel
- [Na+]i
- intracellular Na+ concentration
- NMDAR
- N-methyl-d-aspartate receptor
- SFK
- Src family kinase
- VGCC
- voltage-gated Ca2+ channel
- CaMK
- Ca2+/calmodulin-dependent protein kinase
- MAPK
- mitogen-activated protein kinase
- CaMKK
- CaMK kinase
- TTX
- tetrodotoxin
- MK-801
- (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate
- STO-609
- 1,8-naphthoylene benzimidazole-3-carboxylic acid
- PP2
- 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d] pyrimidine
- PP3
- 4-amino-7-phenylpyrazol [3,4-d] pyrimidine
- PBS
- phosphate-buffered saline
- PGP 9.5
- protein gene product 9.5
- DIV
- days in vitro
- PVP
- polyvinylpyrrolidone
- SBFI
- sodium-binding benzofuran isophthalate
- FMP
- FLIPR membrane potential
- CI
- confidence interval
- EM
- estimation of membrane potential
- Po
- open probability.
- Received September 21, 2009.
- Accepted December 18, 2009.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
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