Brevetoxins: Unique Activators of Voltage-Sensitive Sodium Channels

Chapter 11 Brevetoxins: Unique Activators of Voltage-Sensitive Sodium Channels 1

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Vera L. Trainer , Richard A. Edwards , Alina M. Szmant , Adam M. Stuart , Thomas J. Mende , and Daniel G. Baden1,2 1

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Baden

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Department of Biochemistry and Molecular Biology, School of Medicine, University of Miami, P.O. Box 016129, Miami, FL 33101 Rosenstiel School of Marine and Atmospheric Science, Division of Biology and Living Resources, University of Miami, 4600 Rickenbacker Causeway, Miami, FL 33149

Downloaded by CORNELL UNIV on April 25, 2015 | http://pubs.acs.org Publication Date: January 29, 1990 | doi: 10.1021/bk-1990-0418.ch011

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E i g h t polyether toxins isolated from Florida's red tide dinoflagellate Ptychodiscus brevis are based structurally o n two different carbon backbones. All toxins examined exert their toxic effects by specific binding to Site 5 associated with voltage-sensitive sodium channels. Exposure to brevetoxins leads to activation o f sodium channels at n o r m a l resting potential. Specific binding o f tritiated brevetoxin PbTx-3 to synaptosomes has been undertaken i n rats, fish, and turtles. Dissociation constants are comparable i n the nanomolar concentration range i n each system, and binding maxima are i n the p m o l / m g protein range i n all cases. Derivative brevetoxins specifically displace tritiated PbTx-3 from its site o f action and IC data for derivative brevetoxins are comparable i n each o f the three species. Derived i n h i b i t i o n constants are i n the nanomolar concentration range, with the most toxic brevetoxins being most most potent at displacing labeled toxin from its site. 50

T h e marine dinoflagellate Ptychodiscus brevis (formerly Gymnodinium breve) is responsible for toxic red tides along the G u l f o f M e x i c o coast o f F l o r i d a and Texas ( i ) , and entrained blooms have been transvected around the F l o r i d a peninsula and up the Eastern coast o f the U n i t e d States as far north as N o r t h C a r o l i n a . Toxicity o f this red tide organism is due to synthesis and intracellular maintenance o f a multiplicity o f potent polyether neurotoxins (2), k n o w n as the brevetoxins (Figure 1) (3). Since this organism is toxic i n laboratory culture, most o f the recent brevetoxin w o r k has been undertaken employing laboratory cultures o f P. brevis and the toxins derived from these cultures (2—5). T h e toxicological consequences o f P. brevis red tides are mass mortality o f fishes exposed to the red tide; toxic shellfish which, i f consumed, result i n human neurotoxic shellfish poisoning; and an irritating aerosol which results from contact w i t h P. brevis cell particles entrapped i n seaspray. I n all cases, the threshhold levels for intoxication are i n the picomolar to nanomolar concentration ranges, implying a specific locus or loci o f action for brevetoxins (reviewed i n 6). that

Electrophysiological protocols utilizing crayfish and squid giant axons revealed external application of brevetoxin caused a concentration-dependent

0097-6156/90/0418-0166$06.00/0 o 1990 American Chemical Society

In Marine Toxins; Hall, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1990.


TRAINER ET AL.

Brevetoxins and Voltage-Sensitive Sodium Channels

Figure 1. T h e brevetoxins are based o n two different backbone tures, as indicated (4). Type 1 toxins (top) include: PbTx-2 PbTx-3 PbTx-5 PbTx-6 PbTx-8

[R^H, [R^H, [R^Ac, [R^H, [R^H,

struc-

R =CH C(=CH )CHO)]; R2=CH C(=CH )CH OH)]; R =CH C(=CH )CHO]; R = C H C ( = C H ) C H O , 27,28 epoxide]; R =CH COCH Cl]. 2

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Type 2 toxins (bottom) include: PbTx-1 [ R = C H O ] ; PbTx-7 [ R = C H O H ] . 2

N o structual information is available o n PbTx-4.


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MARINE TOXINS: ORIGIN, STRUCTURE, AND MOLECULAR PHARMACOLOGY

depolarization, repetitive discharges, and a depression o f the action potential leading to a block o f excitability (Figure 2). V o l t a g e clamp experiments illustrated that only sodium currents were affected (7). E a r l y experiments utilizing neuroblastoma cells illustrated that application o f brevetoxin-A (PbTx-1) (8) induced an influx o f Na i n a dose-dependent manner. This work was followed by experiments using brevetoxin PbTx-3 and rat brain synaptosomes, again illustrating a dose-dependent uptake o f N a following toxin application (Figure 3) (4). Catterall and R i s k (8) demonstrated that brevetoxins did not interfere w i t h binding o f sodium channel-specific neurotoxins which bind at sites 1-3, and Catterall and G a i n e r illustrated the lack o f brevetoxin interaction at site 4 (9). That brevetoxins b i n d at a unique site associated w i t h voltage-sensitive sodium channels ( V S S C ) was suggested by this data (8, 9). Specific binding o f brevetoxins to synaptosomes was first demonstrated by P o l i et al. (Figure 4) (4) by utilizing brevetoxin PbTx-2 synthetically reduced with sodium borotritiide to yield tritiated PbTx-3 w i t h specific activities approaching 20 C i / m m o l (10). P o l i demonstrated saturability, c o m petition for specific binding sites by nonradioactive brevetoxin agonists , binding maxima i n the pmol/mg protein concentration range, reversibility o f radioactive toxin binding, half times for association and dissociation consistent w i t h specific binding, distinct brain regional and subcellular distribution, the presence o f a pharmacological response at appropriate concentrations for binding, tissue linearity, and temperature dependence (11). Dissociation constants, binding maxima, and competitive displacement curves for brevetoxin at site 5 parallel those constants derived for saxitoxin binding at site 1 (Figure 5). 2 2

2 2

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Protocols for Binding Assays Excitable tissue preparations were obtained fresh daily from live animals using the technique described by D o d d et al. (12). Protein was measured o n each synaptosome preparation using the Coomassie Brilliant B l u e dye technique described by Bradford (13); results were expressed as "toxin b o u n d per mg synaptosome protein". B i n d i n g o f tritiated PbTx-3 was measured using a rapid centrifugation technique. A l l binding experiments were conducted i n a binding medium consisting o f 50 m M H E P E S ( p H 7.4), 130 m M choline chloride, 5.5 m M glucose, 0.8 m M magnesium sulfate, 5.4 m M potassium chloride, 1 m g / m L bovine serum albumin, and 0.01% E m u l p h o r E L - 6 2 0 as an emulsifier (4). In addition, 370 m M sucrose was added to fish synaptosome experiments to maintain iso-osmolarity. Synaptosomes, suspended i n 0.1 m L o f binding medium minus B S A , were added to a reaction mixture containing [ H ] PbTx-3 and other effectors i n 0.9 m L o f binding medium i n 1.5 m L polypropylene microfuge tubes. After mixing and i n c u bating at the desired temperatures for 1 hr, samples were centrifuged at 15,000 x g for 2 m i n . Supernatant solutions were sampled for the measurement o f free toxin concentrations, and the remainder was aspirated i n each case. Pelleted synaptosomes were rapidly washed w i t h 4 drops o f a wash medium consisting o f 5 m M H E P E S ( p H 7.4), 163 m M choline chloride, 1.8 m M calcium chloride, 0.8 m M magnesium sulfate, and 1 m g / m L B S A Pellets were transferred to liquid scintillation vials containing 3 m L o f liquid scintillant, and the bound radioactivity was measured using liquid scintillation techniques. Nonspecific binding was measured i n the presence o f a saturating concentration o f unlabeled PbTx-3 and was subtracted from total binding to yield specific binding. 3

Dissociation Constants and Binding Maxima for Brevetoxin PbTx-3 Brevetoxins b i n d w i t h high specificity to synaptosomes o f fish (TUapia sp.\ turtles (P. scripta), and rats (Table I). In all cases, the K was i n the nanomolar d


TRAINER ET AL.

Brevetoxins and Voltage-Sensitive Sodium Channels

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PbTx-3 Concentration

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Brevetoxins: Unique Activators of Voltage-Sensitive Sodium Channels

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