Influence of Lipid-Soluble Gating Modifier Toxins on Sodium Influx in Neocortical Neurons
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0022-3565/08/3262-604–613$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 326, No. 2 Copyright © 2008 by The American Society for Pharmacology and Experimental Therapeutics 138230/3358916 JPET 326:604–613, 2008 Printed in U.S.A. Influence of Lipid-Soluble Gating Modifier Toxins on Sodium Influx in Neocortical Neurons Zhengyu Cao, Joju George, William H. Gerwick, Daniel G. Baden, Jon D. Rainier, and Thomas F. Murray 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 February 20, 2008; accepted April 25, 2008 Downloaded from ABSTRACT The electrical signals of neurons are fundamentally dependent tridine and aconitine, and the pyrethroid deltamethrin were ϩ on voltage-gated sodium channels (VGSCs), which are respon- somewhat lower with maximal [Na ]i increments of less than 40 sible for the rising phase of the action potential. An array of mM. The rank order of efficacy of sodium channel gating mod- jpet.aspetjournals.org naturally occurring and synthetic neurotoxins have been iden- ifiers was brevetoxin (PbTx)-1 Ͼ PbTx-desoxydioxolane Ͼ tified that modify the gating properties of VGSCs. Using murine batrachotoxin Ͼ antillatoxin Ͼ PbTx-2 ϭ PbTx-3 Ͼ PbTx- neocortical neurons in primary culture, we have compared the 3␣-naphthoate Ͼ veratridine Ͼ deltamethrin Ͼ aconitine Ͼ ability of VGSC gating modifiers to evoke Naϩ influx. Intracel- gambierol. These data demonstrate that the ability of sodium ϩ lular sodium concentration ([Na ]i) was monitored using the channel gating modifiers to act as partial agonists is shared Naϩ-sensitive fluorescent dye, sodium-binding benzofuran by compounds acting at both neurotoxin sites 2 and 5. The isophthalate. All sodium channel gating modifier compounds concentration-dependent increases in [Naϩ] produced by i at ASPET Journals on January 19, 2020 tested produced a rapid and concentration-dependent eleva- PbTx-2, antillatoxin, veratridine, deltamethrin, aconitine, and ϩ ϩ tion in neuronal [Na ]i. The increment in [Na ]i exceeded 40 gambierol were all abrogated by tetrodotoxin, indicating that mM at high concentrations of brevetoxins, batrachotoxin, and VGSCs represent the sole pathway of Naϩ entry after exposure the novel lipopeptide, antillatoxin. The maximal increments in to gating modifier neurotoxins. ϩ neuronal [Na ]i produced by neurotoxin site 2 alkaloids, vera- The electrical signals of neurons are fundamentally depen- and ciguatoxins originating from the marine dinoflagellates dent on voltage-gated sodium channels (VGSCs), which are Karenia brevis and Gambierdiscus toxicus, respectively (recep- responsible for the rising phase of the action potential. These tor site 5); and ␦-conotoxins (receptor site 6). In addition, pyre- ion channels represent the molecular target for an array of throid insecticides act at a site distinct from these better char- naturally occurring and synthetic neurotoxins that specifi- acterized neurotoxin receptor sites on the sodium channel cally bind to at least six distinct receptor sites on the sodium ␣-subunit to enhance channel activity by shifting activation to ␣ channel -subunit (Catterall et al., 2007). These toxins in- more negative membrane potentials as well as by inhibiting clude hydrophilic toxins such as tetrodotoxin, saxitoxin, and inactivation (Ruigt et al., 1987). More recently, a structurally -conotoxin (receptor site 1); lipid-soluble alkaloid toxins, unique lipopeptide toxin, antillatoxin, produced by the marine including batrachotoxin, veratridine, acotinine, and grayano- cyanobacterium, Lyngbya majuscula, has been demonstrated to toxin (receptor site 2); polypeptide toxins, such as ␣-scorpion be a potent VGSC activator at yet another distinct receptor site toxins, sea anemone toxins, and some spider toxins (receptor (Berman et al., 1999; Li et al., 2001). site 3); -scorpion toxins (receptor site 4); brevetoxins (PbTxs) The lipid-soluble toxins acting at neurotoxin receptor sites 2 and 5 have been characterized as allosteric modulators of 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 sodium channel function (Catterall et al., 2007). These toxins T.F.M.). bind at topologically distinct sites that favor the open state of Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. the sodium channel and display complex allosteric interac- doi:10.1124/jpet.108.138230. tions. Batrachotoxin is a neurotoxin that activates site 2 on 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. 604 Gating Modifier-Induced Sodium Influx 605 the ␣-subunit of VGSC. Batrachotoxin produces a shift in the Materials and Methods threshold for VGSC activation to more hyperpolarized mem- brane potentials, resulting in persistent channel opening at Materials. Trypsin, penicillin, streptomycin, heat-inactivated fe- tal bovine serum, horse serum, and soybean trypsin inhibitor were resting potential (Wang and Wang, 2003). Batrachotoxin and obtained from Atlanta Biologicals (Norcross, GA). Minimum essen- other site 2 ligands also inhibit VGSC inactivation, further tial medium, DNase, poly-L-lysine, cytosine arabinoside, veratridine, contributing to persistent channel opening (Wang and Wang, aconitine, and deltamethrin were from Sigma-Aldrich (St. Louis, 2003). MO), and batrachotoxin was from BIOMOL Research Laboratories The specific binding of [3H]batrachotoxin to neurotoxin site (Plymouth Meeting, PA). The fluorescent dye SBFI-acetoxymethyl 2 is sensitive to conformational changes induced by the bind- ester and Pluronic acid F-127 were obtained from Invitrogen (Carls- ing of toxins to other sites on the ␣-subunit (Catterall et al., bad, CA). Brevetoxins-1, -2, and -3 were isolated and purified from K. breve cultures at the Center for Marine Sciences at the University of 1981). The binding of [3H]batrachotoxin to neurotoxin site 2 North Carolina (Wilmington, NC). The semisynthetic brevetoxin an- is enhanced by the interaction of brevetoxins with site 5 alogs were synthesized as described elsewhere by Purkerson-Parker (Sharkey et al., 1987). Using reconstituted VGSC prepara- et al. (2000). Gambierol was synthesized as described by Johnson et tions, the most potent brevetoxin analog, PbTx-1, enhanced al. (2006). Antillatoxin was authentic natural (Ϫ)-antillatoxin, iso- [3H]batrachotoxin binding greater than 5-fold (Trainer et al., lated as described by Orjala et al. (1995). The structures of all gating 1993). It is noteworthy that the less toxic brevetoxin, PbTx-9, modifier toxins evaluated are depicted in Fig. 1. caused only a small increase in [3H]batrachotoxin binding. Neocortical Neuron Culture. Primary cultures of neocortical Downloaded from These results are consistent with brevetoxin analogs possess- neurons were obtained from embryonic day 16 Swiss-Webster mice (LePage et al., 2005). In brief, pregnant mice were euthanized by ing distinct efficacies as activators of neurotoxin site 5 CO2 asphyxiation, and embryos were removed under sterile condi- (LePage et al., 2003). Lipophilic toxins, in addition to batra- tions. Neocortices were collected, stripped of meninges, minced by chotoxin, that act at neurotoxin site 2 include the alkaloids trituration with a Pasteur pipette, and treated with trypsin for 20 veratridine and aconitine (Ceste`le and Catterall, 2000). min at 37°C. The cells were then dissociated by two successive These ligands interact with neurotoxin site 2 in a mutually trituration and sedimentation steps in soybean trypsin inhibitor and jpet.aspetjournals.org exclusive manner and increase Naϩ permeability of neuro- DNase containing isolation buffer, centrifuged, and resuspended in blastoma cells to different extents at saturation (Catterall, Eagle’s minimal essential medium with Earle’s salt and supple- mented with 2 mM L-glutamine, 10% fetal bovine serum, 10% horse 1975). Sodium influx studies using rat brain synaptosomes serum, 100 IU/ml penicillin, and 0.10 mg/ml streptomycin, pH 7.4. have confirmed that batrachotoxin, veratridine, and aconi- Cells were plated onto poly-L-lysine-coated 96-well (9-mm) clear- tine activate VGSCs by an interaction with a common recep- bottomed, black-well culture plates (Corning Life Sciences, Acton, tor site 2, at which batrachotoxin is a full agonist, and aconi- MA) at a density of 1.5 ϫ 105 cells/cm2 and incubated at 37°C in a 5% CO and 95% humidity atmosphere. Cytosine arabinoside (10 M) tine and veratridine are partial agonists (Tamkun and 2 at ASPET Journals on January 19, 2020 Catterall, 1981). These actions of sodium channel activators was added to the culture medium on day 2 after plating to prevent have been quantitatively described by an allosteric model proliferation of non-neuronal cells. The culture media was changed that assumes toxin high-affinity binding to activated sodium both on days 5 and 7 using a serum-free growth medium containing neurobasal medium supplemented with B-27, 100 IU/ml penicillin, channels with a shift