Brevenal, a brevetoxin antagonist from Karenia brevis, binds to a previously unreported site on mammalian sodium channels

Abstract

Brevetoxins are a family of ladder-frame polyether toxins produced by the marine dinoflagellate Karenia brevis. During blooms of K. brevis, inhalation of brevetoxins aerosolized by wind and wave action can lead to asthma-like symptoms in persons at the beach. Consumption of either shellfish or finfish contaminated by K. brevis blooms can lead to the development of neurotoxic shellfish poisoning. The toxic effects of brevetoxins are due to binding at a defined site on, and subsequent activation of, voltage-sensitive sodium channels (VSSCs) in cell membranes (site 5). In addition to brevetoxins, K. brevis produces several other ladder-frame compounds. One of these compounds, brevenal, has been shown to antagonize the effects of brevetoxin. In an effort to further characterize to effects of brevenal, a radioactive analog ([³H]-brevenol) was produced by reducing the side-chain terminal aldehyde moiety of brevenal to an alcohol using tritiated sodium borohydride. A K_D of 67 nM and B_max of 7.1 pmol/mg protein were obtained for [³H]-brevenol in rat brain synaptosomes, suggesting a 1:1 matching with VSSCs. Brevenal and brevenol competed for [³H]-brevenol binding with K_i values of 75 nM and 56 nM, respectively. However, although both brevenal and brevenol can inhibit brevetoxin binding, brevetoxin was completely ineffective at competition for [³H]-brevenol binding. After examining other site-specific compounds, it was determined that [³H]-brevenol binds to a site that is distinct from the other known sites including the brevetoxin site (site 5) although some interaction with site 5 is apparent.

Keywords: Brevenal, Brevetoxin; Competition Binding Assay; Radioligand Assay.

Figure 1

Structure of all published brevetoxins (PbTxs) (Rein et al. 1994).

Figure 2

Structure of brevenal and synthesis brevenol and [³H]-brevenol.

Figure 3

Reduction of ³H-PbTx-3 binding as a percent of the maximum specific binding using unlabeled competitors: (▵) PbTx-2, (▴) PbTx-3, (∎) brevenal, and (◻) brevenol. Each data point represents the mean ± s.e.m. of values obtained from three independent experiments with triplicate determinations in each experiment. Nonspecific binding was 20% of the total binding.

Figure 4

Competitive inhibition of ³H-PbTx-3 binding to site 5 (VSSC) by brevenol. Synaptosomes were incubated for 1 hour at 4°C with increasing concentrations of ³H-PbTx-3 in the presence of (∎) 0 nM, (◻) 10 nM, (▴) 25 nM, or (▵) 50 nM brevenol. Each point represents the mean ± s.e.m. of values obtained from two independent experiments with triplicate determinations per experiment.

Figure 5

(A) Specific¹ saturation binding of ³H-brevenol to rat brain synaptosomes. Each data point represents the mean ± s.e.m. of triplicate determinations for each concentration. Analysis of nonlinear regression of specific binding yields an approximate K_D of 67.5 nM and a B_max of 7.2 ± 0.9 pmoles of ³H-brevenol per mg of synaptosomal protein. ¹ Total and nonspecific binding were measured by liquid scintillation techniques, the difference between the two represent specific (◻) binding.

Figure 6

Reduction of ³H-brevenol binding as a percent of the maximum specific binding using unlabeled competitors: (▵) PbTx-2 (site 5), (▴) PbTx-3 (site 5), (∎) brevenal, and (◻) brevenol. Each data point represents the mean ± s.e.m. of values obtained from three independent experiments with triplicate determinations in each experiment. Nonspecific binding was calculated as 20% of the total binding.

Figure 7

A. Competitive Inhibition of ³H-brevenol versus brevenol. Synaptosomes were incubated for 1 hour at 4°C with increasing concentrations of ³H-brevenol in the presence of (∎) 0 nM, (◻) 10 nM, or (▴) 25 nM brevenol. Each data point represents the mean ± s.e.m. of values obtained from three independent experiments with triplicate determinations in each experiment. B. Competitive Inhibition of ³H-brevenol by brevenal. Synaptosomes were incubated with increasing concentrations of ³H-brevenol in the presence of (∎) 0 nM, (◻) 10 nM, or (▴) 25 nM brevenal. Each data point represents the mean ± s.e.m. of values obtained from three independent experiments with triplicate determinations in each experiment.

Figure 8

(adapted from Segel 1968). Interactions between cell surface receptors, ligands (L) and inhibitors (I). These interactions are used to explain the binding of PbTxs to site 5 and the novel brevenal/ol receptor located on VSSCs.