Pharmacological and structural characterization of a novel phospholipase A 2 from Micrurus dumerilii carinicauda venom

Abstract

We have isolated a new phospholipase A 2 (MiDCA1) from the venom of the coral snake Micrurus dumerilii carinicauda. This toxin, which had a molecular mass of 15,552 Da, shared high sequence homology with the PLA 2 toxins MICNI A and B from Micrurus nigrocinctus venom (77.7% and 73.1%, respectively). In chick biventer cervicis preparations, MiDCA1 produced concentration- and time-dependent neuromuscular blockade that reached 100% after 120 min (2.4 μM, n=6); contractures to exogenously applied carbachol (8 μM) and KCl (13 mM) were still seen after complete blockade. In mouse phrenic-nerve diaphragm preparations, MiDCA1 (2.4 μM; n=6) caused triphasic changes followed by partial neuromuscular blockade. Intracellular recordings of end-plate potentials (EPPs) and miniature end-plate potentials (MEPPs) from mouse diaphragm preparations showed that MiDCA1 increased the quantal content by 386±12% after 10 min ( n=14; p<0.05) and caused a triphasic change in the frequency of MEPPs. MiDCA1 also decreased the resting membrane potential, an effect that was prevented by tetrodotoxin and/or low extracellular calcium, but not by d-tubocurarine. The toxin increased the amplitude of mouse sciatic-nerve compound action potentials by 30±9% (0.6 μM; p<0.05). Potassium currents elicited in freshly dissociated dorsal root ganglia neurones were blocked by 31±1% ( n=4; p<0.05) in the presence of 2.4 μM MiDCA1. These results show that MiDCA1 is a new presynaptic phospholipase A 2 that produces neuromuscular blockade in vertebrate nerve-muscle preparations. The triphasic effects seen in mammalian preparations and the facilitatory response were probably caused mainly by the activation of sodium channels, complemented by the blockade of nerve terminal potassium channels. The inability of d-turocurarine to prevent the depolarization by MiDCA1 indicated that cholinergic nicotinic receptors were not involved in this phenomenon.