Type B brevetoxins show tissue selectivity for voltage-gated sodium channels: comparison of brain, skeletal muscle and cardiac sodium channels

Toxicon 41 (2003) 919–927 www.elsevier.com/locate/toxicon

Type B brevetoxins show tissue selectivity for voltage-gated sodium channels: comparison of brain, skeletal muscle and cardiac sodium channels Marie-Yasmine Bottein Dechraoui, John S. Ramsdell* Marine Biotoxins Program, Center for Coastal Environmental Health and Biomolecular Research, NOAA-National Ocean Service, 219 Fort Johnson Road, Charleston, SC 29412, USA Received 19 November 2002; accepted 28 March 2003

Abstract Brevetoxins and ciguatoxins are two classes of phycotoxins which exert their toxic effect by binding to site-5 of voltagegated sodium channels. Sodium channels, a family of at least 10 structurally different proteins, are responsible for the rising phase of the action potential in membranes of neuronal, cardiac and muscular excitable cells. This work is a comparative study of the binding properties and the cytotoxic effects of ciguatoxins and brevetoxins on human embryonic cells (HEK) stably expressing either the skeletal muscle (Nav1.4), or the cardiac (Nav1.5) sodium channel a-subunit isoforms. We report that type A (PbTx-1) and type B (PbTx-3 and PbTx-2) brevetoxins as well as ciguatoxins target both cardiac and muscle channels; type B brevetoxins show isoform selectivity, presenting a lower affinity for the heart than the skeletal muscle channel. The lower selectivity of type B brevetoxins for heart sodium channels may result from a more rigid backbone structure than is found in type A brevetoxins and ciguatoxins. q 2003 Elsevier Science Ltd. All rights reserved. Keywords: Brevetoxins; Ciguatoxins; Ouabain; Veratridine; hH1a; m1; Nav1.4; Nav1.5; Sodium channel; Marine neurotoxins; HEK; Binding; Cytotoxicity

1. Introduction Excitable tissues such as nerve, skeletal muscle and heart contain voltage-gated sodium channels that mediate the rapidly activating and inactivating inward Naþ current of the action potential. At least 10 sodium channels have been documented through isolation of separate cDNA clones and subsequent amino acid sequencing. In the brain, sodium channel Nav1.1, 1.2 and 1.3 subtypes (or I, II, III and VI, respectively) (Noda et al., 1986) are highly expressed. In skeletal muscle the type Nav1.4 (or m1) (Trimmer et al., 1989) is expressed * Corresponding author. Tel.: þ1-843-762-8510; fax: þ 1-843762-8700. E-mail address: [email protected] (J.S. Ramsdell).

primarily, and the predominant form in the heart is the type Nav1.5 (or H1) (Rogart et al., 1989). These membrane proteins, products of different genes, are quite homologous in structure (at least 76%; for review see Catterall (2000)) but can be differentiated by their neurotoxin affinity (Bottein Dechraoui and Ramsdell, 2002), with the most widely recognized difference being sensitivity to tetrodotoxin (TTX) and saxitoxin (STX) (Barchi et al., 1981). Brevetoxins and ciguatoxins are two classes of lipidsoluble polyether neurotoxins known to activate sodium channels. Produced by marine dinoflagellates (Karenia brevis and Gambierdiscus toxicus, respectively), these toxins often accumulate in marine organisms and affect humans through seafood consumption. Intoxication signs in animals, as well as human symptoms associated

0041-0101/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0041-0101(03)00088-6

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with consumption of contaminated seafood have been well documented. Exposure to these toxins is mainly characterized by gastrointestinal and neurological disorders and, in most severe cases, by cardiovascular problems (Bagnis et al., 1979). By activating sodium channels at normal membrane resting potential and inhibiting normal inactivation (Westerfield et al., 1977; Huang et al., 1984; Benoit et al., 1986; Sheridan and Adler, 1989; Benoit et al., 1996) these toxins increase Naþ permeability of excitable cell membranes and thus affect various sodium-dependent signaling processes (Bidard et al., 1984; Molgo et al., 1992). Brevetoxin action on channel gating has been recorded on single channels (Purkerson et al., 1999) from rat neuroblastoma cells B50 (expressing Nav1.1 and 1.2 subtypes) and B104 (expressing type Nav1.3 and Nav1.x sodium channel mRNA). On neuromuscular preparations, ciguatoxins and brevetoxins have complex effects due to an indirect action (at low concentration) via the release of neurotransmitters, as well as a direct action (at high concentration) on skeletal or cardiac muscle sodium channels (Rodgers et al., 1984; Lewis and Endean, 1986; Lewis, 1988; Seino et al., 1988; Molgo et al., 1990; Sauviat et al., 2002). An electrophysiological study on rat cardiac ventricular cells showed that a high dose of PbTx-3 (20 mM) shifted the steady-state activation to negative potentials (Schreibmayer and Jeglitsch, 1992), as observed on axonal membranes (Huang et al., 1984). Radioligand binding studies on rat brain (Poli et al., 1986; Lewis et al., 1991; Dechraoui et al., 1999) have shown that brevetoxins and ciguatoxins specifically bind at Naþ channel receptor site-5, thought to exist at the interface of domains I and IV of the protein (Trainer et al., 1994). PbTx-3 binding properties have been explored on brain preparations of different species (marine mammals (Trainer and Baden, 1999); fish (Stuart and Baden, 1988; Trainer et al., 1990; Lewis, 1992); turtle (Trainer et al., 1990); insect (Cestele et al., 1996)) and on muscle preparation of fish (Yotsu-Yamashita et al., 2000). To our knowledge, brevetoxin and ciguatoxin binding characteristics have not been reported on mammalian muscle and heart. We have previously reported the potent action of PbTx-1 on human embryonic kidney cells (HEK-293) expressing human heart voltage-gated sodium channels (HEK-hH1a) and used these cells to modify the cell-based assay for the detection of brevetoxins (Fairey et al., 2001). In this paper we examine the binding properties and cytotoxic activity of brevetoxins and ciguatoxins on a HEK cell line stably expressing either the skeletal muscle or the heart sodium channel isoforms. 2. Materials and methods 2.1. Chemicals All tissue culture reagents were from Gibco BRL/Life Technology. Brevetoxins PbTx-1 and PbTx-3 were

purchased from Calbiochem and PbTx-2 was purified from cultures of K. brevis by Drs Steve Morton and Peter Moeller, NOAA Marine Biotoxins Program, Charleston, SC, USA. STX was purchased from NRC Canada and the Pacific ciguatoxin P-CTX3C (Satake et al., 1993) was provided by Professor T. Yasumoto. The 42-[3H]PbTx-3 (666 GBq/mmol) and 11-[3H] STX (777 GBq/mmol) were products of Amersham. 2.2. Cells HEK-293 cells (ATCC CRL 1573) were purchased from the American Type Culture Collection. Stably transfected cells HEK-m1 and HEK-hH1a were provided by Dr D.A. Hanck (University of Chicago) (Sheets and Hanck, 1999). HEK cells were cultured in DMEM enriched with 10% (v/v) fetal bovine serum. Cells were grown until confluence in a monolayer at 37 8C in a humidified atmosphere of 5% CO2. The medium for transfected cell lines was supplemented with 250 mg/ml of Geneticin (G-418). 2.3. HEK-293 cells membrane preparation For cell membrane preparation, HEK-m1, HEK-hH1a and wild type HEK-293 cells were grown until confluence in 150 £ 25 mm2 cell culture dish (Corning). They were washed once with ice-cold phosphate-buffered saline (PBS) and scraped into a hypotonic 5 mM Tris – HCl buffer (pH 7.4) containing 5 mM EDTA and 0.1 mM PMSF. Cells were lysed using a Polytron for 30 s or until .90% of the cells were lysed as observed under a microscope. The nuclei and unlysed cells were removed by centrifugation at 1000g for 5 min at 4 8C. The supernatants were reserved and membrane homogenates were collected by centrifugation at 48 000g for 40 min at 4 8C. The pellets were resuspended in a small volume of binding buffer without BSA, probe sonicated 3 £ for 20 s. Aliquots were dispensed and stored at 280 8C until use. Total protein concentration was determined on each preparation using the microassay procedure of the BioRad protein assay with BSA as standard (Bio-Rad, Richmond, CA) according to the manufacturer’s instructions. 2.4. Rat brain membrane preparation Rat brain membrane homogenates were prepared according to the procedure described by Doucette et al. (1997) and Van Dolah et al. (1994) with some minor modification. Briefly, frozen brains (2 80 8C) were ground in an ice cold homogenization buffer (20 mM Tris; 140 mM NaCl, pH 7.1) containing 1 mM PMSF using a Teflon/glass tissue homogenizer (Brinkman Polytron). The homogenate was centrifuged at 1000g for 5 min at 4 8C to eliminate tissue debris. The supernatant was further

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centrifuged at 54 000g for 40 min at 4 8C. The final pellet was resuspended in ice-cold sample buffer (75 mM HEPES and 140 mM NaCl, pH 7.5). The crude membrane preparations were stored at 2 80 8C until use.

2.5. General procedure for receptor binding experiments The binding of [3H]PbTx-3 and its specific inhibitors to selected membrane preparations are compared in this work. Total binding was measured using the test tube format receptor binding assay previously described (Brown, 1986; Dechraoui et al., 1999). For saturation experiments, membranes were incubated with increasing concentrations of [3H]STX (0.2– 10 nM) in 500 ml of incubation medium (containing in mM, 50 Hepes pH 7.4, 130 choline chloride, 5 glucose, 0.5 MgSO4, 5.4 KCl, 0.01% emulphore EL620 and 1 mg/ml BSA). For competition experiments, membranes were incubated with [3H]PbTx-3 (3 nM) and increasing concentrations of competitors in 500 ml of the same binding medium. After 1 h incubation at 4 8C, the reaction was stopped by adding 3 ml ice-cold binding buffer to each tube. The membranes were then collected immediately on GF/C (Whatman) glass fiber filters and washed two more times with 3 ml of buffer. To measure the non-specific binding, excess unlabeled PbTx-3 (1 mM) or STX (1 mM) was added to the incubation medium. Bound radioactivity was determined using 3 ml of BD ScintiVerse (Fisher) scintillation cocktail and counted for 1 min (RackBeta 1212, LKB WALLAC).

2.6. MTS-cytotoxicity assay Site-5 neurotoxins were assessed for their effects on HEK-m1, HEK-hH1a and wild type HEK-293 cells using the simple and accurate Cell Titer 96 Aqueous colorimetric assay (Promega). Briefly, cells were harvested by the application of trypsin-EDTA solution and plated 24 h prior treatment in a 96-well plate (Costar, Cambridge, MA) at a density of 30 000 cells in 100 ml of complete growth medium per well. Neurotoxin activity was assessed with a 22 h treatment period in the presence of ouabain and veratridine at concentrations adapted to each cell line. Cell viability was measured by adding in each well (for 2 – 3 h at 37 8C) 20 ml of a solution including a novel tetrazolium compound MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium, inner salt) and an electron coupling reagent (phenazine methosulfate or PMS). The absorbance of the aqueous soluble formazan produced by the metabolically active cells was measured directly at 492 nm using an automated microplate-based reader FLUOstar (BMG Labtechnologies).

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2.7. Calculation and statistical methods Radioactivity collected on filters was corrected for non-specific binding. Effects of inhibitors are presented as percent inhibition. IC50 (concentration giving 50% of the maximum response), Ki (inhibition constant), Kd (dissociation constant) and Bmax (maximum binding) values were calculated by non-linear regression using Prism software (version 3.0; GraphPad Software, Inc., San Diego, CA, USA). The assay was performed in duplicate and repeated three times for each membrane preparation. Cytotoxicity results (samples were tested in triplicate at various dilutions) were normalized to the percentage of cells counted for the control condition (as defined in Section 3, on a minimum of six wells). Each experimental condition was repeated in triplicate.

3. Results 3.1. Binding parameters of the expressed sodium channels In order to determine binding parameters of receptors expressed at the cell surface, binding experiments using [3H]PbTx-3 were performed on membranes from HEK 293 cells stably transfected with sodium channels Nav 1.4 (m1) or Nav 1.5 (hH1a) a-subunit DNA. [3H]PbTx-3 specifically bound to both HEK-m1 and HEK-hH1a cells membrane preparations. Total binding was linear with increasing concentration up to 500 mg of total protein (Fig. 1). Therefore, all subsequent binding experiments were performed with 400 mg of membrane protein. Under these conditions, the specific binding represented 70% of the total binding. Membranes prepared from untransfected cells lacked specific [3H]PbTx-3 binding (Fig. 1).

Fig. 1. HEK-m1 and HEK-hH1a tissue linearity curve for [3H]PbTx3 binding. Tritiated brevetoxin (3 nM) was incubated 1 h with increasing amount of membrane preparation in standard binding medium. Straight lines represent the total binding of brevetoxin on HEK-m1, HEK-hH1a and wild type HEK-293 membrane preparation. Error bars represent standard deviation ðn ¼ 3Þ:

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the skeletal muscle and cardiac sodium channels were determined by competition experiment using [3H]PbTx-3 (3 nM final concentration). Competition curves for tritiated PbTx-3 are presented in Fig. 3 and the calculated inhibitory concentrations at 50% (IC50) are provided in Table 1. For comparison, competition data obtained with rat brain membrane in the presence of the same radioligand concentration (3 nM [3H]PbTx-3) are also reported in Fig. 3 and Table 1. PbTx-1, PbTx-2 and P-CTX3C each competed for [3H]PbTx-3, on both muscle (Nav1.4) and cardiac (Nav1.5) sodium channels. Using HEK-hH1a membrane preparations, the brevetoxin PbTx-1 receptor affinity was not statistically different than when using brain preparations (t-test, P , 0:05); in contrast a significant difference (t-test, P , 0:05) in receptor affinity of PbTx3 and PbTx-2 between HEK-hH1a and brain membranes was recorded. The IC50 values were 7- and 8-fold lower with rat brain than with HEK-hH1a membrane, respectively. Using HEK-m1 membrane preparations, all brevetoxins presented a similar receptor affinity (within 95% confidence limits) with an IC50 averaging 8 nM. Furthermore, no significant differences between the binding affinity of P-CTX3C to HEK-m1 or HEK-hH1a membrane preparations were observed. 3.3. PbTx-1 and PbTx-3 cytotoxic effect on HEK-m1 and HEK-hH1a cells

Fig. 2. Binding of [3H]STX to HEK-m1 and HEK-hH1a transfected cells membranes. HEK-m1 and HEK-hH1a (400 and 500 mg of total protein, respectively) were incubated at 4 8C with increasing concentration of radioligand. Bound toxins were measured using the rapid filtration assay as described in Section 2. Non-specific binding (NSB) was measured in the presence of excess unlabeled STX (1 mM) and was subtracted from total binding (TB) to yield specific binding (SB). The Scatchard transformation of these data is shown in insets.

Estimation of the total amount of sodium channel (Bmax) on the different cell lines expressing Nav1.4 and Nav1.5 subtypes was evaluated by a saturation experiment using tritiated STX. The [3H]STX specific binding was concentration dependent and saturable (Fig. 2), while saturation curves yielded a linear Scatchard transformation (insets Fig. 2). The calculated Bmax values were 111.0 ^ 7 and 185.1 ^ 9 fmol/mg of total proteins with a Kd for [3H]STX of 1.13 ^ 0.24 and 10.7 ^ 0.92 nM for HEK-m1 and HEKhH1a, respectively. 3.2. Comparison of brevetoxin and ciguatoxin derivative affinities for Nav1.4 and Nav1.5 Na channels The binding affinities of three brevetoxins, PbTx-1, PbTx-2, PbTx-3 and one ciguatoxin P-CTX3C, for

Skeletal muscle and heart sodium channels expressed on human embryonic kidney cells were assessed for their ability to be activated by brevetoxins PbTx-1 and PbTx-3. For this purpose, we have adapted the neurotoxin cytotoxicity assay developed for neuroblastoma cells (Neuro2a) to HEK-m1 and HEK-hH1a cells. The MTS tetrazolium dye used as a replacement for MTT (Mosmann, 1983; Manger et al., 1993) as an endpoint for the cell assay, gave a linear response between cell number and absorbance at 492 nm of the formazan produced by living HEK cells (result not shown). The background absorbance found for 100% cell mortality was around 1 (result not shown). The sodium channel marine neurotoxin cytotoxicity assay previously developed using Neuro-2a cells (Kogure et al., 1988; Manger et al., 1993) requires the presence of both veratridine and ouabain in the incubation medium. As previously reported by Fairey et al. (2001), the concentrations of ouabain and veratridine need to be modified when using HEK cells. In Fig. 4, we have reported dose – response curves of ouabain and veratridine for HEK-m1, HEK-hH1a and wild type HEK-293 cells. All HEK cells presented the same sensitivity to ouabain, with an EC50 averaging 33.2 nM (Fig. 4A). Veratridine (up to 100 mM) was not cytotoxic to HEK-293 (result not shown) or HEK-m1 cells (Fig. 4B). Only HEK-hH1a cells were sensitive to veratridine, which showed an EC50 of

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Fig. 3. Inhibition of [3H]PbTx-3 specific binding by type A and type B brevetoxins and ciguatoxin CTX-3C (chemical structures of the polyether toxins are presented in the bottom of the figure) on HEK-m1 and HEK-hH1a membrane preparation. Membranes of rat brain, HEK-hH1a or HEK-m1 cells were incubated with [3H]PbTx-3 (3 nM) and increasing concentrations of PbTx-1, PbTx-2, PbTx-3 or P-CTX3C in the standard conditions developed in Section 2. IC50 values are reported in Table 1.

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Table 1 Brevetoxin and ciguatoxin affinity for heart, brain and skeletal muscle sodium channels given as the inhibitory concentration (IC50) in nM ^ SE calculated from competitive binding experiments IC50 (nM)

Brain

HEK-m1

HEK-hH1a

PbTx-3 PbTx-2 PbTx-1 P-CTX3C

3.51 ^ 0.81 4.29 ^ 0.95 3.02 ^ 0.84 0.48 ^ 0.09

7.70 ^ 0.89 8.44 ^ 0.81 8.48 ^ 0.83 0.80 ^ 0.08

23.5 ^ 0.84 34.1 ^ 0.85 2.90 ^ 0.83 1.10 ^ 0.09

8.6 mM. Neither PbTx-1 nor PbTx-3, added alone, reduced transfected HEK cells viability. However, as shown in Fig. 5, brevetoxin dose-dependent cytotoxicity was measured in the presence of both ouabain (5 nM) and veratridine (50 and 5 mM for HEK-m1 and HEK-hH1a cells, respectively). Under these conditions, HEK-hH1a cells were less sensitive to brevetoxins than HEK-m1

Fig. 5. Brevetoxin induced HEK-m1 and HEK-hH1a cytotoxicity. HEK-m1 and HEK-hH1a cells were treated with ouabain (0.05 mM) and veratridine (5 and 50 mM, respectively) in the presence of various concentrations of PbTx-1 and PbTx-3. Viability is expressed as percent of viable cells relative to a ouabain/veratridine control ðn ¼ 6Þ: EC50 calculated values of the sigmoidal curves are reported in Fig. 6.

cells. PbTx-1 was 5- and 3-fold more potent than PbTx-3 on HEK-hH1a and HEK-m1, respectively. Brevetoxin EC50 values for HEK cells expressing Nav1.4 and Nav1.5 sodium channels are reported in Fig. 6.

Fig. 4. Comparison of the cytotoxic effect of ouabain and veratridine on HEK-m1 and HEK-hH1a cells. Cells were treated for 22 h with various concentrations of ouabain (top) or veratridine (bottom) and cell viability was measured using the MTS assay as described in Section 2. Results are expressed in absorbance units with a control at 2.1 units (no treatment, n ¼ 6) and the background level (no cells, n ¼ 6) at 1.31 units.

Fig. 6. Sodium dependent cytotoxicity of PbTx-1 and PbTx-3. Effective concentration at 50% (EC50) were determined using Prisme software. Bars represent the mean of three experiments performed in triplicate.

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4. Discussion The differential pattern of expression of voltage-gated sodium channels between species, tissues and during development suggests that sodium channel isoforms have unique characteristics. In this study we have characterized sodium channels from human heart (Nav1.5 type) and rat skeletal muscle (Nav1.4 type) for their site-5 neurotoxin activity and found that type B brevetoxins, the predominant brevetoxins occurring during red tides, exhibit a substantial isoform-selectivity. Nav1.4 or Nav1.5 a-subunit sodium channels were compared for their sensitivity to site-5 neurotoxins by receptor binding and cytotoxicity assays. Membrane preparations of HEK cells expressing either Nav1.4 or Nav1.5 specifically bound [3H]PbTx-3 and [3H]STX. The calculated Kd for STX were 1.13 and 10.7 nM for the skeletal muscle and the cardiac sodium channels, respectively, values similar to previous determination from radiolabeled toxin binding experiment on native tissue (Rogart, 1986). The membrane preparation exhibited a low number of total binding sites, about 30 times lower than in rat brain membrane preparations, which led to the use of a high total protein level (400 mg) in the assay and yielded a non-specific binding around 30% of the total binding. The homologous and heterologous [3H]PbTx-3 inhibition experiments showed that brevetoxins and ciguatoxins targeted both cardiac and skeletal muscle sodium channels. HEK-m1 and HEK-hH1a membrane preparations bound ciguatoxins with a higher affinity than they did for brevetoxins, as previously described using rat brain membrane preparations. However, detailed analysis of the calculated IC50 pointed out some toxin isoform selectivity. The inhibitory constants Ki for PbTx-3 calculated from the IC50 of homologous competition experiment using the Cheng Prussof equation were 4.7 and 20.5 nM for the Nav1.4 and Nav1.5 sodium channels, respectively. Previous studies showed that PbTx-3 bound to brain type II sodium channel (Nav1.2) transiently expressed in human embryonic kidney (TSA 201) cells with a Kd of 8 nM (Trainer et al., 1994). Otherwise, the Nav1.5 a-subunit bound type B brevetoxins (including PbTx-3 and PbTx-2) with a much lower affinity (8 fold) than type A brevetoxins (PbTx-1). This toxin selectivity was not observed for the Nav1.4 sodium channel isoform which did not distinguish type A and B brevetoxins with both exhibiting IC50 values averaging 8 nM. Very similar affinities between both types A and B native brevetoxin (except PbTx-6) have also been recently demonstrated using a rat brain membrane preparation (IC50 of 3.1 and 4.0 nM (Rein et al., 1994); 2.0 and 2.2 nM (Dechraoui et al., 1999) for PbTx-1 and PbTx-3, respectively). We have observed the activity of a type A (PbTx-1) and a type B (PbTx-3) brevetoxin on Nav1.4 and Nav1.5 sodium channels by measuring their induced cytotoxicity in the presence of ouabain and veratridine. As previously reported for HEK-hH1a cells (Fairey et al., 2001), HEK-m1 as well as

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HEK-293 cell lines presented a high sensitivity to ouabain, leading to the use of a much lower concentration of ouabain than conventionally used for the Neuro-2a cytotoxicity assay (0.05 vs 500 mM). Interestingly, we found that veratridine showed a sodium channel isoform specificity, cytotoxic by itself on HEK-hH1a cells but not on HEK-m1 and Neuro2a cells at concentrations up to 100 mM. Brevetoxin induced cytotoxicity was therefore observed in the presence of ouabain and 5 and 50 mM of veratridine for HEK-hH1a and HEK-m1 cells, respectively. Under these experimental conditions, Nav1.5 sodium channels appeared globally less sensitive to brevetoxins than the Nav1.4 isoform. This isoform specificity was greater for the type B PbTx-3 brevetoxin, confirming the toxin selectivity of the heart sodium channel determined by receptor binding experiment. Type B brevetoxins, composed of 11-membered rings, are characterized by a more rigid backbone than their Type A brevetoxin analogues with 10-membered rings. Toxin structure-activity dependency for brain sodium channel binding has been well studied using native or derivative toxins, and specific regions of the toxin molecule have been correlated with toxin activities on what is called a pharmacophore site (Gawley et al., 1992; Rein et al., 1994; Gawley et al., 1995; Purkerson-Parker et al., 2000). Our results reveal that specific voltagegated sodium channel subtypes also provide insight into the site-5 neurotoxins’ interaction with the sodium channels. Thus, Nav1.5 has a lower affinity to the type B brevetoxins than the Nav1.4 or Nav1.2 isoforms. The amino sequence of Nav1.2 show 69 and 61% identities against Nav1.4 and Nav1.5 a subunits, respectively (Clare et al., 2000), and 81 and 76% identities (NCBI Blast) within the putative brevetoxin receptor site5 amino acid sequences identified by Trainer et al. (1994). A single amino acid difference on receptor site-1 led to marked pharmacological difference of the cardiac Naþ channel, compared to brain or skeletal muscle isoforms, which is manifest in its resistance to TTX (Barchi et al., 1981; Cohen and Barchi, 1981; Frelin et al., 1986; Noda et al., 1989; Satin et al., 1992) and all site-1 neurotoxins. Heart sodium channels are otherwise characterized by their lower sensitivity (compared to skeletal muscle or brain sodium channel) to many other toxins such as the LqIT and LqII a-scorpion toxins, the wasp pompilidotoxins (Kinoshita et al., 2001) and some m-conotoxins. Concerning the site-5 neurotoxins, the primary sequence differences of the Nav1.5 receptor site-5 is the most likely basis to differences in toxin binding and cytotoxic activity. In conclusion, the present work compares and characterizes for the first time the specific binding and activity of the marine toxins, ciguatoxins and brevetoxins, on heart and skeletal muscle sodium channels. The Nav1.5 sodium channel was substantially more selective to site-5 neurotoxins. The affinity of the type B brevetoxins differed between

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type Nav1.5 and Nav1.2 sodium channels, indicating that those toxins are potential tools to discriminate sodium channels. Our findings demonstrate that the brevetoxin activation of sodium channels not only depend on toxin structures for a given channel isoform but also on the diversity of the different channels. Application of the newly identified selectivity of brevetoxins for the cardiac channel, together with site-mediated mutagenesis, should better define the molecular basis for brevetoxin selectivity for the heart channel. This may provide important insight into the sensitivity of the cardiovascular system to the most common brevetoxin molecules associated with harmful algal blooms.

Acknowledgements This work was performed while the author held a National Research Council Associateship Award at Marine Biotoxins Program, NOAA/NOS/CCEHBR. We thank Dr M. Barbier for her help in the determination of sequence homologies. This work was funded by the National Oceanic and Atmospheric Administration (NOAA-NOS). The National Ocean Service (NOS) does not approve, recommend, or endorse any proprietary product or material mentioned in this publication. No reference shall be made to NOS, or to this publication furnished by NOS, in any advertising or sales promotion which would indicate or imply that NOS approves, recommends, or endorses any proprietary product or proprietary material mentioned herein or which has as its purpose any intent to cause directly or indirectly the advertised product to be used or purchased because of NOS publication.

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