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TOXICOLOGY

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

Creighton University, School of Medicine, Department of Pharmacology, Omaha, Nebraska (Z.C., J.G., T.F.M.); Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California at San Diego, La Jolla, California (W.H.G.); University of North Carolina, Center for Marine Science Research, Wilmington, North Carolina (D.G.B.); and Department of Chemistry, University of Utah, Salt Lake City, Utah (J.D.R.)

Received for publication February 20, 2008
Accepted April 25, 2008.

	Abstract

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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⁺ influx. Intracellular sodium concentration ([Na⁺]_i) was monitored using the Na⁺-sensitive fluorescent dye, sodium-binding benzofuran isophthalate. All sodium channel gating modifier compounds tested produced a rapid and concentration-dependent elevation in neuronal [Na⁺]_i. The increment in [Na⁺]_i exceeded 40 mM at high concentrations of brevetoxins, batrachotoxin, and the novel lipopeptide, antillatoxin. The maximal increments in neuronal [Na⁺]_i produced by neurotoxin site 2 alkaloids, veratridine and aconitine, and the pyrethroid deltamethrin were somewhat lower with maximal [Na⁺]_i increments of less than 40 mM. The rank order of efficacy of sodium channel gating modifiers was brevetoxin (PbTx)-1 > PbTx-desoxydioxolane > batrachotoxin > antillatoxin > PbTx-2 = PbTx-3 > PbTx-3-naphthoate > veratridine > deltamethrin > aconitine > 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⁺]_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⁺ entry after exposure to gating modifier neurotoxins.

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 the -subunit of VGSC. Batrachotoxin produces a shift in the threshold for VGSC activation to more hyperpolarized membrane potentials, resulting in persistent channel opening at resting potential (Wang and Wang, 2003). Batrachotoxin and other site 2 ligands also inhibit VGSC inactivation, further contributing to persistent channel opening (Wang and Wang, 2003).

The specific binding of [³H]batrachotoxin to neurotoxin site 2 is sensitive to conformational changes induced by the binding of toxins to other sites on the -subunit (Catterall et al., 1981). The binding of [³H]batrachotoxin to neurotoxin site 2 is enhanced by the interaction of brevetoxins with site 5 (Sharkey et al., 1987). Using reconstituted VGSC preparations, the most potent brevetoxin analog, PbTx-1, enhanced [³H]batrachotoxin binding greater than 5-fold (Trainer et al., 1993). It is noteworthy that the less toxic brevetoxin, PbTx-9, caused only a small increase in [³H]batrachotoxin binding. These results are consistent with brevetoxin analogs possessing distinct efficacies as activators of neurotoxin site 5 (LePage et al., 2003). Lipophilic toxins, in addition to batrachotoxin, that act at neurotoxin site 2 include the alkaloids veratridine and aconitine (Cestèle and Catterall, 2000). These ligands interact with neurotoxin site 2 in a mutually exclusive manner and increase Na⁺ permeability of neuroblastoma cells to different extents at saturation (Catterall, 1975). Sodium influx studies using rat brain synaptosomes have confirmed that batrachotoxin, veratridine, and aconitine activate VGSCs by an interaction with a common receptor site 2, at which batrachotoxin is a full agonist, and aconitine and veratridine are partial agonists (Tamkun and Catterall, 1981). These actions of sodium channel activators have been quantitatively described by an allosteric model that assumes toxin high-affinity binding to activated sodium channels with a shift in the conformational equilibrium toward the activated or open state (Catterall, 1977a).

VGSCs are vital for normal central nervous system functioning, and recent studies have additionally shown that intracellular sodium concentration ([Na⁺]_i) may act as a signaling molecule. Yu and Salter (1998, 1999) reported that increases in intracellular Na⁺ increase NMDA receptor mediated whole-cell currents and NMDA receptor single-channel activity by increasing both open probability and mean open time of the channel. Using veratridine, these investigators demonstrated that influx of Na⁺ through a tetrodotoxin (TTX)-sensitive VGSC was sufficient to produce potentiation of NMDA channel activity. Moreover, previous studies have found that PbTx-2 augments NMDA receptor-mediated Ca²⁺ influx in both spontaneously oscillating mature and nonoscillatory immature cerebrocortical neurons (Dravid at al., 2005). PbTx-2 also enhanced the effect of bath-applied NMDA on extracellular signal-regulated kinase 2 activation.

Therefore, we have quantified the potencies and efficacies of an array of lipophilic VGSC gating modifiers by measuring Na⁺ influx in murine neocortical neurons. This was accomplished using the Na⁺-sensitive fluorescent dye, sodium-binding benzofuran isophthalate (SBFI). Application of this assay to primary cultures of neurons afforded a highly quantitative assessment of the increments in neuronal sodium level produced by gating modifier toxins.

	Materials and Methods

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Materials. Trypsin, penicillin, streptomycin, heat-inactivated fetal bovine serum, horse serum, and soybean trypsin inhibitor were obtained from Atlanta Biologicals (Norcross, GA). Minimum essential medium, DNase, poly-L-lysine, cytosine arabinoside, veratridine, aconitine, and deltamethrin were from Sigma-Aldrich (St. Louis, MO), and batrachotoxin was from BIOMOL Research Laboratories (Plymouth Meeting, PA). The fluorescent dye SBFI-acetoxymethyl ester and Pluronic acid F-127 were obtained from Invitrogen (Carlsbad, CA). Brevetoxins-1, -2, and -3 were isolated and purified from K. breve cultures at the Center for Marine Sciences at the University of North Carolina (Wilmington, NC). The semisynthetic brevetoxin analogs were synthesized as described elsewhere by Purkerson-Parker et al. (2000). Gambierol was synthesized as described by Johnson et al. (2006). Antillatoxin was authentic natural (–)-antillatoxin, isolated as described by Orjala et al. (1995). The structures of all gating modifier toxins evaluated are depicted in Fig. 1.

View larger version (21K): [in this window] [in a new window]   Fig. 1. Structures of sodium channel gating modifiers.

[Na⁺]_i Measurement. The cells 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 MgCl₂, 2.3 mM CaCl₂, 0.0001 mM glycine, pH 7.4) using an automated cell washer (Bio-Tek Instruments, Winooski, VT). The background fluorescence of each well in the plate was measured and averaged before dye loading. Cells were then incubated for 1 h at 37°C with dye loading buffer (100 µl/well) containing 10 µM SBFI-acetoxymethyl ester and 0.02% Pluronic F-127. After 1 h of incubation in dye-loading medium, cells were washed five times with Locke's buffer, leaving a final volume of 150 µl in each well. The plate was then transferred to the plate chamber of a FLEXstation II (Molecular Devices, Sunnyvale, CA). Cells were excited at 340 and 380 nm, and Na⁺-bound SBFI emission was detected at 505 nm. Fluorescence readings were taken once every 5 s for 60 s to establish the baseline, and then 50 µl of neurotoxin containing solution (4x) was added to each well from the compound plate at the rate of 26 µl/s, yielding a final volume of 200 µl/well. The cells were exposed to the VGSC gating modifiers for another 240 s. Full in situ calibration of the SBFI fluorescence ratio was performed as described previously (Diarra et al., 2001) using calibration media containing 0.6 mM MgCl₂, 0.5 mM CaCl₂, 10 mM HEPES, Na⁺ and K⁺ such that [Na⁺] + [K⁺] = 130 mM, 100 mM gluconate, and 30 mM Cl^– (titrated with 10 mol/l KOH to pH 7.4). Gramicidin D (5 µM) (Na⁺ ionophore), monensin (10 µM) (Na⁺/H⁺ carrier), and ouabain (100 µM) (Na⁺/K⁺-ATPase inhibitor) were added to equilibrate the intracellular and extracellular sodium concentration. After five washes, the Locke's buffer was replaced by 150 µl of sodium containing calibration solution (0–130 mM). The plate was then loaded onto the FLEXstation chamber for recording of emitted fluorescence during excitation at 340 and 380 nm.

Data Analysis. The raw emission data at each excitation wavelength were exported to an Excel work sheet and corrected for background fluorescence. The SBFI fluorescence ratios (340:380) versus time were then analyzed, and time-response and concentration-response graphs were generated using GraphPad Prism software (GraphPad Software Inc., San Diego, CA). The EC₅₀ and maximal response values for VGSC gating modifiers were determined by nonlinear regression analysis using a logistic equation. In select experiments, SBFI fluorescence ratios were analyzed by one-way analysis of variance. A Dunnett's post hoc analysis was performed to compare these values for different treatments.

	Results

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Calibration of [Na⁺]_i in Neocortical Neurons. To quantify VGSC gating modifier-induced [Na⁺]_i elevation in neocortical neurons, the relationship between the radiometric SBFI signal and [Na⁺]_i was established in situ. This in situ calibration is required inasmuch as SBFI, similar to the Ca²⁺ indicator fura-2, displays different spectral properties in an intracellular environment than in a cell-free system. The emitted fluorescence intensities were recorded during excitation at 340 and 380 nm and were converted to a ratio (340:380) after background correction (Fig. 2a). To convert the ratio of emitted SBFI signals into a [Na⁺]_i value, the following equation (eq. 1) was used:

(1)

where β is the ratio of the fluorescence of the free (unbound) dye to bound dye at the second excitation wavelength (380 nm), K_d is the apparent dissociation constant of SBFI for Na⁺, R is the background-subtracted SBFI fluorescence ratio, and R_min and R_max are the minimum and maximal fluorescence values, respectively. The data points relating [Na⁺]_i to R were fitted by a three-parameter hyperbolic equation having the form:

(2)

where a and b are constants and equal to R_max – R_min and βK_d, respectively (Diarra et al., 2001). These data relating [Na⁺]_i to R (Fig. 2b) were well described (r² = 0.987) by eq. 2. The derived parameters were R_min = 1.460 ± 0.012, a = 2.950 ± 0.029, and b = 39.14 ± 1.26. The value for R_min obtained by this method was identical to the value of R_min derived experimentally at [Na⁺] = 0 mM. Therefore, the corresponding values for R_max and βK_d were R_max = 4.41 ± 0.04 and βK_d = 39.14 ± 1.26 mM.

View larger version (17K): [in this window] [in a new window]   Fig. 2. Full calibration of SBFI at 37°C. Neocortical neurons cultured in a 96-well plate were exposed to the calibration solution containing eight sodium concentrations from 0 to 130 mM (see Materials and Methods). The emitted fluorescences during excitation at 340 and 380 nm were recorded and converted to the ratio of 340:380 after background substraction. a, relationship between eight different [Na+]i and SBFI ratio values. b, three-parameter hyperbolic fit (eq. 2) of [Na+] versus mean ratio values (R) obtained from three calibration experiments (r2 = 0.987). c, Hanes plot (eq. 3) of calibration data show a linear regression fit of the data (r2 = 0.997).

(3)

As shown in Fig. 2c, a plot of [Na⁺]/(R – R_min) versus [Na⁺]_i yields a straight line (r² = 0.9965). The slope [1/(R_max – R_min)] provides a means to estimate of R_max, whereas the intercept on the abscissa is equal to –βK_d. The value for R_min was obtained from the experimental data. The values of R_max and βK_d calculated from Hanes plot were 4.35 ± 0.04 and 39.37 ± 1.42 mM, respectively, and were not significantly different from the values derived from the three-parameter hyperbolic fit that were 4.41 ± 0.04 (R_max) and 39.14 ± 1.26 (βK_d) mM.

VGSC Gating Modifiers Elevate Intracellular Sodium Concentration. Given the role of intracellular sodium as a putative regulator of NMDA receptor-mediated signaling, it was important to quantify VGSC gating modifier-induced elevation of [Na⁺]_i (Yu, 2006). Therefore, we assessed gating modifier-induced elevation of [Na⁺]_i in neocortical neurons loaded with SBFI. Fluorescence emitted during excitation at 340 nm was unaffected by changes in [Na⁺]_i. As depicted in Fig. 3, exposure to PbTx-2 (300 nM) produced a rapid decrease in SBFI fluorescence emitted by excitation at 380 nm, whereas emitted fluorescence during excitation at 340 nm was unaffected. The lack of effect on fluorescence emission after excitation at 340 nm indicates that PbTx-2 did not produce significant cell swelling in neocortical neurons. The in situ SBFI calibrations revealed that the basal [Na⁺]_i concentration in DIV 9 cerebrocortical neurons was 10.3 ± 0.22 mM. This value is in agreement with the 8.9 mM concentration determined in cultured hippocampal neurons (DIV 14–21) (Rose and Ransom, 1997). This basal level of [Na⁺] was decreased slightly, although significantly (P < 0.05), by exposure to 1 µM tetrodotoxin, to a value of 9.37 ± 0.17 mM.

View larger version (21K): [in this window] [in a new window]   Fig. 3. Representative emitted fluorescence intensities before and after application of 300 nM PbTx-2. Emitted fluorescence during excitation at 340 nm (diamond) was insensitive to 300 nM PbTx-2 exposure, whereas emitted fluorescence during excitation at 380 nm (circle) decreased after application of 300 nM PbTx-2. Arrow, time of addition of PbTx-2.

View larger version (48K): [in this window] [in a new window]   Fig. 4. Time-response relationships for VGSC gating modifier-induced [Na+]i changes in neocortical neurons. [Na+]i responses after exposure to different concentrations of PbTx-1 (a), PbTx-2 (b), PbTx-3 (c), PbTx-desoxydioxolane (d), PbTx-3--naphthoate (e) antillatoxin (f), deltamethrin (g), batrachotoxin (h), veratridine (i), and aconitine (j). These data were obtained from at least four different cultures performed in duplicate.

View larger version (15K): [in this window] [in a new window]   Fig. 8. a, time-response relationships for gambierol-induced [Na+]i changes in neocortical neurons. These data were obtained from three different cultures performed in duplicate. b, nonlinear regression analysis of gambierol inhibition of the integrated SBFI response [area under the curve (AUC)] to PbTx-1 (30 nM) in neocortical neurons. Histogram represents the responses to PbTx-1 (30 nM) and gambierol (10 µM) alone. Each data point is the mean (±S.E.M.) of six determinations.

View larger version (31K): [in this window] [in a new window]   Fig. 5. Nonlinear regression analysis of the peak SBFI (340/380) response versus the concentration of brevetoxins (PbTx-1, PbTx-2, PbTx-3, PbTx-desoxydioxolane, PbTx-3--naphthoate), batrachotoxin, antillatoxin, veratridine, deltamethrin, aconitine, and gambierol.

Although all of the [Na⁺]_i responses to gating modifier compounds reported herein were derived from DIV 7 to 9 cerebrocortical neurons, we have found robust responses to these compounds as early as DIV 2. Both PbTx-2 and antillatoxin evoke maximal [Na⁺]_i responses in DIV 2 cerebrocortical neurons that are comparable with those found in DIV 7 to 9 neurons (data not shown).

TTX Antagonism of Sodium Channel Gating Modifier-Induced Elevation of [Na⁺]_i. To confirm the role of VGSCs in gating modifier-induced elevation of [Na⁺]_i in neocortical neurons, we examined the influence of TTX (1 µM) on this response. As shown in Fig. 6, the concentration-dependent increases in [Na⁺]_i produced by antillatoxin, PbTx-2, veratridine, deltamethrin, aconitine, and gambierol were all abrogated by TTX. These data suggest that the observed gating modifier-induced elevation of [Na⁺]_i is dependent on the activation of VGSCs. Due to the limited availability of the brevetoxin analogs, we only tested TTX with PbTx-2 inasmuch as this is the most abundant naturally occurring brevetoxin, and all brevetoxin analogs tested have been shown to inhibit the binding of [³H]PbTx-3 to neurotoxin site 5 (Gawley et al., 1995; Michelliza S et al., 2007).

View larger version (40K): [in this window] [in a new window]   Fig. 6. Effects of TTX on PbTx-2 (a)-, antillatoxin (b)-, veratridine (c)-, aconitine (d)-, deltamethrin (e)-, and gambierol (f)-induced elevation of [Na+]i.

View larger version (25K): [in this window] [in a new window]   Fig. 7. PbTx-2 (10 nM) enhances deltamethrin (1 µM)-induced elevation of neocortical [Na+]i. Each data point represents the mean of seven determinations.

	Discussion

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Based on the original work of Hodgkin and Huxley (1952) with squid axons, a single action potential was calculated to change minimally the Na⁺ electrochemical gradient (Hille, 1991). However, the situation in mammalian neurons with fine axons, dendrites, and spines is much different due to greater surface/volume ratios. Thus, a single action potential may elevate [Na⁺]_i substantially (Hille, 1991). Using two-photon imaging to measure Na⁺ transients in spines and dendrites of CA 1 pyramidal neurons in hippocampal slices, Rose et al. (1999) demonstrated action potential-induced [Na⁺]_i increments reached values of 3 to 4 mM after a train of just 20 action potentials. The influence of synaptic activity on [Na⁺]_i in apical dendrites and spines was subsequently demonstrated in rat hippocampal slice preparations by Rose and Konnerth (2001). These authors demonstrated that [Na⁺]_i increased by 30 to 40 mM during short bursts of synaptic stimulation and reached 100 mM during a longterm potentiation protocol. The influence of [Na⁺]_i dynamics on NMDA receptor function has been demonstrated in hippocampal neurons, where elevation of [Na⁺]_i increased the open probability of NMDA receptors (Yu and Salter, 1998; Yu, 2006). An increment of [Na⁺]_i of 10 mM was sufficient to produce significant increases in NMDA receptor single-channel activity. This Na⁺-dependent regulation of NMDA receptor function was moreover shown to be controlled by Src family kinase-induced phosphorylation of the receptor (Yu and Salter, 1998; Yu, 2006).

Lipid-soluble toxins acting at neurotoxin sites 2 and 5 have been shown to affect gating and to be allosterically coupled. Catterall has provided evidence that these neurotoxins induce conformational changes that alter the equilibrium between open and closed/inactivated states of the sodium channel (Catterall, 1977a,b, 1980). This work further indicated that the site 2 toxins aconitine, veratridine, grayanotoxin, and batrachotoxin act as full or partial agonists and cause persistent activation of sodium channels that can be quantitatively fit by an allosteric model (Catterall, 1977a, 1980).

We have previously demonstrated that a group of site 5 ligands, including both naturally occurring and semisynthetic brevetoxin analogs, produce different maximal responses at saturating concentrations (LePage et al., 2003). These previous studies used alterations in [Ca²⁺]_i in intact neurons to demonstrate that the brevetoxin PbTx-1 was a full agonist, whereas PbTx-3 and its analogs were partial agonists. In the present study, these data were extended by directly comparing the efficacies of an array of gating modifier toxins as stimulators of Na⁺ influx in cerebrocortical neurons. Measurement of [Na⁺]_i transients in neocortical neurons represents a more direct measure of sodium channel activation than the [Ca²⁺]_i transients used previously (LePage et al., 2003).

To explore the relative efficacies of gating modifier toxins, we used SBFI-loaded neocortical neurons to evaluate their respective concentration-response relationships. All neurotoxins tested evoked a rapid and concentration-dependent increase in neuronal [Na⁺]_i. In agreement with the original observations of Catterall (1975) and Catterall et al. (1978), our results with the site 2 activators batrachotoxin, veratridine, and aconitine indicate that these compounds produce concentration-dependent elevations of [Na⁺]_i in neocortical neurons, with markedly distinct efficacies. Veratridine and aconitine were partial agonists, whereas batrachotoxin acted as a full agonist at neurotoxin site 2 in neocortical neurons. The naturally occurring brevetoxins PbTx-1, PbTx-2, and PbTx-3 also produced rapid and concentration-dependent increments in [Na⁺]_i, with PbTx-1 proving to be the most potent and efficacious toxin. The primacy of PbTx-1 as an activator of Na⁺ influx in neocortical neurons suggests that this compound is a full agonist at neurotoxin site 5. The lower maximal responses to PbTx-2 and PbTx-3, accordingly, suggest that these compounds are partial agonists at neurotoxin site 5, inasmuch as a previous study has shown that all three toxins interact with the VGSC in a mutually exclusive manner (Gawley et al., 1995). The greater efficacy of PbTx-1 at neurotoxin site 5 relative to other naturally occurring brevetoxins accords with the results of Trainer et al. (1993), who showed PbTx-1 to be the most effective brevetoxin in enhancing the binding of [³H]batrachotoxin to rat brain VGSCs. It has been suggested that the greater conformational flexibility in the backbone structure of PbTx-1compared with that of PbTx-2 may underlie its efficacy as a modulator of [³H]batrachotoxin binding to neurotoxin site 2 (Cestèle et al., 1995). The present results with the brevetoxin analog PbTx-3-naphthoate are also in agreement with our earlier studies monitoring brevetoxin-induced Ca²⁺ influx in cerebellar granule neurons in that this compound acts as a partial agonist in stimulating Na⁺ influx in neocortical neurons (LePage et al., 2003). The brevetoxin analog PbTx-desoxydioxolane displayed an efficacy comparable with that of PbTx-1 but was more than 15-fold less potent.

In addition to the demonstration of partial agonism of site 2 and site 5 gating modifiers, we now report that antillatoxin has an efficacy at VGSCs that is comparable with PbTx-2 and PbTx-3. Antillatoxin-induced neurotoxicity and Ca²⁺ influx are abrogated by tetrodotoxin, indicating that this compound is an activator of VGSCs (Li et al., 2001). The ability of antillatoxin to activate VGSCs was directly demonstrated by the TTX-sensitive stimulation of ²²Na⁺ influx in cerebellar granule neurons (Li et al., 2001). These results were confirmed and extended in the present report, showing that antillatoxin is a potent and efficacious stimulator of [Na⁺]_i transients in neocortical neurons. Its efficacy was comparable with that of the site 5 ligands PbTx-2 and PbTx-3. Moreover, the EC₅₀ value of antillatoxin of 78.9 nM was similar to the previously reported value of 98.2 nM as a stimulator of ²²Na⁺ influx. Therefore, antillatoxin seems to represent a novel activator of VGSCs whose site of action remains to be established (Li et al., 2001).

Pyrethroids are synthetic insecticide compounds that resemble the natural pyrethrin toxins. Their primary molecular target as insecticides and neurotoxins is the VGSC (Trainer et al., 1997). Pyrethroids such as deltamethrin shift the VGSC activation voltage to more negative values and inhibit inactivation. These activities result in a persistent activation of VGSCs as reflected in an enhancement of ²²Na⁺ influx similar to other gating modifiers (Lombet et al., 1988). We demonstrate here that deltamethrin produces a concentration-dependent stimulation of Na⁺ influx in neocortical neurons, with an efficacy of 0.53.

The lowest efficacy neurotoxin evaluated was gambierol (efficacy = 0.11). Gambierol is a polyether ladder toxin derived from the marine dinoflagellate G. toxicus, which also produces the potent site 5 activator ciguatoxin (LePage et al., 2005, 2007). Gambierol has been shown to block voltagegated K⁺ channels at depolarized membrane potentials under voltage-clamp conditions in Xenopus oocytes (Cuypers et al., 2008). However, whether the development of K⁺ channel block by gambierol requires depolarizing steps remains to be determined. At the resting membrane potential of cerebellar granule cells, we have shown previously that, unlike ciguatoxin, gambierol has no effect on intracellular Ca²⁺ concentration and is without neurotoxic action in these cells (LePage et al., 2007). By directly monitoring [Na⁺]_i transients in the present studies, we have found gambierol to be a low-efficacy activator of VGSCs. These data are in agreement with those of Louzao et al. (2006), who showed that gambierol acts as a partial agonist at site 5 of VGSCs in a neuroblastoma cell line. The involvement of neurotoxin site 5 in the Na⁺ influx produced by gambierol reported herein was demonstrated by the ability of gambierol to produce a concentration-dependent inhibition of PbTx-1-induced elevation of [Na⁺]_i in neocortical neurons. These data parallel the original observations of Catterall (1977a) demonstrating partial agonism at neurotoxin site 2 in that treatment of cells with a combination of a good activator (batrachotoxin) and a poor activator (aconitine) resulted in an inhibition of the response to the good activator by the poor activator. Thus, a low-efficacy agonist can be treated as an inhibitor of a high-efficacy agonist. Therefore, our demonstration of gambierol inhibition of PbTx-1-induced Na⁺ influx parallels the earlier findings of Catterall and further demonstrates that the ability of neurotoxins to act as partial agonists is shared by compounds acting at neurotoxin sites 2 and 5. An additional parallel for sites 2 and 5 gating modifier compounds documented herein is in regards to the maximal increment in [Na⁺]_i produced by the respective full agonists, batrachotoxin and PbTx-1. The similarity in the maximal responses to batrachotoxin and PbTx-1 may reflect the partial overlap of neurotoxin sites 2 and 5 on S6 segments of the VGSC -subunit (Wang and Wang, 2003). Receptor mapping studies have found that both site 2 and site 5 ligands interact with residues in the S6 segments of domain I on the VGSC -subunit (Wang and Wang, 2003).

Thus, when considered together, the lipophilic gating modifier toxins (brevetoxins, batrachotoxin, veratridine, aconitine, gambierol, and deltamethrin) as well as the novel lipopeptide antillatoxin are all capable of exerting a TTX-sensitive elevation of [Na⁺]_i in neocortical neurons. Although these sodium channel activators display differing efficacies, they are all able to produce [Na⁺]_i increments of 4 to 60 mM. Therefore, these [Na⁺]_i increments are sufficient to increase NMDA receptor channel activity. Sodium channel gating modifier toxins are all potentially capable of up-regulating NMDA receptor signaling (Dravid et al., 2005).

	Footnotes

 
This study was supported in part by National Institutes of Health Grants ES10594 (to D.G.B.), GM56677 (to J.D.R.), and NS053398 (to W.H.G. and T.F.M.).

doi:10.1124/jpet.108.138230.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

ABBREVIATIONS: VGSC, voltage-gated sodium channel; PbTx, brevetoxin; NMDA, N-methyl-D-aspartate; TTX, tetrodotoxin; SBFI, sodium-binding benzofuran isophthalate; DIV, day(s) in vitro.

Address correspondence to: Dr. Thomas F. Murray, Creighton University School of Medicine, Department of Pharmacology, Omaha, NE 68178. E-mail: tfmurray{at}creighton.edu

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