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

Abstract

Brevetoxins and ciguatoxins are two classes of phycotoxins which exert their toxic effect by binding to site-5 of voltage-gated 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 (Na_v1.4), or the cardiac (Na_v1.5) sodium channel α-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.

Introduction

Excitable tissues such as nerve, skeletal muscle and heart contain voltage-gated sodium channels that mediate the rapidly activating and inactivating inward Na⁺ 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 Na_v1.1, 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 Na_v1.4 (or μ1) (Trimmer et al., 1989) is expressed primarily, and the predominant form in the heart is the type Na_v1.5 (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 lipid-soluble 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 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 Na⁺ 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 Na_v1.1 and 1.2 subtypes) and B104 (expressing type Na_v1.3 and Na_v1.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 μM) 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 Na⁺ 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.