Influence of Lipid-Soluble Gating Modifier Toxins on Sodium Influx in Neocortical Neurons

Abstract

The electrical signals of neurons are fundamentally dependent on voltage-gated sodium channels (VGSCs), which are responsible for the rising phase of the action potential. An array of naturally occurring and synthetic neurotoxins have been identified that modify the gating properties of VGSCs. Using murine neocortical neurons in primary culture, we have compared the ability of VGSC gating modifiers to evoke Na ϩ influx. gambierol. These data demonstrate that the ability of sodium channel gating modifiers to act as partial agonists is shared by compounds acting at both neurotoxin sites 2 and 5. The concentration-dependent increases in [Na ϩ ] i produced by PbTx-2, antillatoxin, veratridine, deltamethrin, aconitine, and gambierol were all abrogated by tetrodotoxin, indicating that VGSCs represent the sole pathway of Na ϩ entry after exposure to gating modifier neurotoxins.The electrical signals of neurons are fundamentally dependent on voltage-gated sodium channels (VGSCs), which are responsible for the rising phase of the action potential. These ion channels represent the molecular target for an array of naturally occurring and synthetic neurotoxins that specifically bind to at least six distinct receptor sites on the sodium channel ␣-subunit (Catterall et al., 2007). These toxins include hydrophilic toxins such as tetrodotoxin, saxitoxin, and -conotoxin (receptor site 1); lipid-soluble alkaloid toxins, including batrachotoxin, veratridine, acotinine, and grayanotoxin (receptor site 2); polypeptide toxins, such as ␣-scorpion toxins, sea anemone toxins, and some spider toxins (receptor site 3); ␤-scorpion toxins (receptor site 4); brevetoxins (PbTxs) and ciguatoxins originating from the marine dinoflagellates Karenia brevis and Gambierdiscus toxicus, respectively (receptor site 5); and ␦-conotoxins (receptor site 6). In addition, pyrethroid insecticides act at a site distinct from these better characterized neurotoxin receptor sites on the sodium channel ␣-subunit to enhance channel activity by shifting activation to more negative membrane potentials as well as by inhibiting inactivation (Ruigt et al., 1987). More recently, a structurally unique lipopeptide toxin, antillatoxin, produced by the marine cyanobacterium, Lyngbya majuscula, has been demonstrated to be a potent VGSC activator at yet another distinct receptor site (Berman et al., 1999;Li et al., 2001).The lipid-soluble toxins acting at neurotoxin receptor sites 2 and 5 have been characterized as allosteric modulators of sodium channel function (Catterall et al., 2007). These toxins bind at topologically distinct sites that favor the open state of the sodium channel and display complex allosteric interactions. Batrachotoxin is a neurotoxin that activates site 2 on

Antillatoxin is a sodium channel activator that displays unique efficacy in heterologously expressed rNav1.2, rNav1.4 and rNav1.5 alpha subunits

“…ATX binds to a site on the sodium channel α-subunit that remains to be defined and stimulates sodium influx in cerebellar granule and neocortical neurons [11,12]. Veratridine is a steroid-derived alkaloid purified from the plant Liliaceae family.…”

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

“…[Na ϩ ] i measurement and full in situ calibration of sodium-binding benzofuran isophthalate (SBFI) fluorescence ratio were performed as described previously (Cao et al, 2008). 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 , 0.1 mM glycine, pH 7.4) using an automated microplate washer (BioTek Instruments, Winooski, VT).…”

“…Primary cultures of cerebrocortical neurons were harvested from Swiss Webster mice on embryonic day 16 and cultured as described previously (Cao et al, 2008). Cells were plated onto poly-L-lysine-coated (Sigma-Aldrich, St. Louis, MO) 96-well (9 mm), clear-bottomed, black-well culture plates (Corning Life Sciences, Lowell, MA) at a density of 1.8 ϫ 10 6 cells/ml (150 l/well), 24-well (15.6 mm) culture plates at a density of 0.05 ϫ 10 6 cells/ml (0.5 ml/well), or 6-well (35 mm) culture dishes at a density of 2.25 ϫ 10 6 cells/ml (2 ml/well), respectively, and incubated at 37°C in a 5% CO 2 and 95% humid atmosphere.…”

“…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.…”

“…Previous work has demonstrated that ATX is a potent activator of voltagegated sodium channels (VGSCs) that elevates intracellular Na ϩ concentration ([Na ϩ ] i ) in intact neurons (Li et al, 2001;Cao et al, 2008). Moreover, ATX has been shown to be neurotoxic in cerebellar granule cells through an indirect activation of N-methyl-D-aspartate receptors (NMDARs) as a consequence of glutamate release (Li et al, 2001(Li et al, , 2004.…”