Pharmacological and structural characterization of a novel phospholipase A2 from Micrurus dumerilii carinicauda venom.
We have isolated a new phospholipase A2 (MiDCA1) from the venom of the coral snake Micrurus dumerilii carinicauda. This toxin, which had a molecular mass of 15,552Da, shared high sequence homology with the PLA2 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 microM, n = 6); contractures to exogenously applied carbachol (8 microM) and KCl (13 mM) were still seen after complete blockade. In mouse phrenic-nerve diaphragm preparations, MiDCA1 (2.4 microM; 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 microM; 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 microM MiDCA1. These results show that MiDCA1 is a new presynaptic phospholipase A2 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.
Belo
Cháriston André Dal
CA
Department of Pharmacology, Faculty of Medical Sciences, State University of Campinas (UNICAMP), P.O. Box 6111, 13083-970 Campinas, SP, Brazil.
Leite
Gildo Bernardo
GB
Toyama
Marcos Hikari
MH
Marangoni
Sergio
S
Corrado
Alexandre Pinto
AP
Fontana
Marcos Dias
MD
Southan
Andy
A
Rowan
Edward G
EG
Hyslop
Stephen
S
Rodrigues-Simioni
Léa
L
D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D003964:Diaphragm / Q000294:innervation; D004546:Elapid Venoms / Q000201:enzymology* / Q000494:pharmacology; D017815:Elapidae; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D010741:Phospholipases A / Q000737:chemistry* / Q000494:pharmacology* / Q000633:toxicity; D054467:Phospholipases A2; D010791:Phrenic Nerve / Q000187:drug effects / Q000502:physiology; D017386:Sequence Homology, Amino Acid
D004546:Elapid Venoms; C053972:micrurus venom; D010741:Phospholipases A; D054467:Phospholipases A2