Partial Blockade of Human Voltage-Dependent Sodium Channels by the Marine Toxins Azaspiracids

Abstract

Azaspiracid toxins were first identified at the end of the last century in Irish mussels, and during the last two decades considerable cytotoxic and neurotoxic effects caused by these toxins have been described. Azaspiracids are synthesized by dinoflagellates and accumulate in several species of filter-feeding bivalve mollusks, thereby incorporating into the food chain and causing human intoxications. Among the cellular effects of azaspiracids, inhibition of spikes in neurons and hyperpolarization of the neuronal membrane potential have been reported; however, the underlying processes leading to these effects were never elucidated. In this regard, initial studies reported no activity of the toxin in neuronal voltage-gated sodium channels, and a recent work described no effect of azaspiracid-1 on the inactivation kinetics of voltage-gated sodium channels; however, the relationship between the known alterations of the cytoskeleton caused by these toxins and their effects on ion channels has never been evaluated. In this work, the cytotoxic effect of azaspiracids was evaluated in human cells as well as their activity on voltage-gated sodium channels and in cell morphology in order to unravel the cellular targets involved in the mechanism of action of this group of marine toxins. The data reported here demonstrate, for the first time, that both azaspiracid-1 and azaspiracid-2 caused a rapid concentration-dependent inhibition of the amplitude of voltage-gated sodium currents without affecting their inactivation kinetics, an effect that was increased after long-term treatment of the cells with the toxin. Simultaneously, long-term exposure of the cells to azaspiracids caused a profound alteration of the cell cytoskeleton and decreased the metabolic activity of human cells. Altogether, the data presented here indicate that the partial blockade of voltage-gated sodium channels by these toxins is not related with their effect on the actin cytoskeleton. However, since azaspiracids are common toxins in European waters, their effect on voltage-gated sodium channels, first reported here, should be considered to avoid synergistic toxicity with other marine toxins that are known potent blockers of sodium channels such as the saxitoxins and tetrodotoxins, but further studies are needed in order to elucidate how these compounds alter ion homeostasis.

Targeting Chloride Ion Channels: New Insights into the Mechanism of Action of the Marine Toxin Azaspiracid

“…VRAC channels were initially proposed to be related with the neurotoxic effects of azaspiracids, because the toxic effect of these compounds in neurons was diminished in the presence of nonspecific chloride channel blockers . In addition to this initial observation, recently we demonstrated that long-term exposure of the cells to azaspiracids led to a dramatic cell shrinkage and to evident changes in cell morphology under light microscopy . Because, apart from osmotically challenging the cells, the only known chemical activator of the VRAC channels is the GTPγS (guanosine 5′- O -[gamma-thio]­triphosphate) which needs to be used at concentrations of about 100 μM and it is capable of activating VRAC channels when applied into the intracellular solution, we engaged in the complete pharmacological characterization of the potential role of azaspiracids in VRAC activation.…”

“…Despite the fact that ion channels were suggested to underlie some of the cellular alterations caused by this group of toxins, only few studies have been released on this subject. ,, In this sense, initial studies reported that AZA1 inhibited electrical activity in spinal cord neurons, and recently, it was demonstrated that AZA1 and AZA2 at nanomolar concentrations acted as partial blockers of voltage-gated sodium channels (Na v ). However, the blockade of sodium channels was not related with the cellular modifications elicited by azaspiracid, because low nanomolar (nM) concentrations of TTX (a selective channel blocker) did not modify the effect of AZA on cell adhesion …”

Current Trends and New Challenges in Marine Phycotoxins

“…A variety of studies have reported alteration in several pathways under AZAs treatment both in vitro and in vivo. Some of the features determined in vitro comprise cytoskeletal reorganization, apoptosis induction, and mitochondrial and nuclear impairment [ 16 , 210 , 211 ], depending on dose, experimental time, and cell line. Interestingly, AZAs effects on ion channels have also been described.…”

“…Research pointed that these phycotoxins at micromolar concentrations decreased potassium currents acting as open state blockers of ether-à-go-go potassium channel (hERG) [ 212 ]. In the same line, three VGSCs isoforms are partially blocked in vitro by AZA1, AZA2, and AZA3 [ 211 ]. A recent publication reported the interaction of azaspiracids with volume-regulated anion channels (VRAC).…”

“…A recent publication reported the interaction of azaspiracids with volume-regulated anion channels (VRAC). Chloride currents’ amplitude is not only increased under AZAs treatment but also significantly diminished when cells are exposed to a selective VRAC inhibitor [ 211 ]. Several of these reports might provide some insight to in vivo toxicology.…”