Shared structural determinants for the calcium-independent liposome membrane permeabilization and sarcolemma depolarization in Bothropstoxin-I, a LYS49-PLA(2) from the venom of Bothrops jararacussu.
The structural determinants of myotoxicity of bothropstoxin-I (BthTX-I), a Lys49 phospholipase A(2) from Bothrops jararacussu venom, were studied by measuring the resting membrane potential in the mouse phrenic nerve-diaphragm preparation. This method proved to be around 100-fold more sensitive than the creatine kinase release assay, and was used to evaluate a total of 31 site-directed BthTX-I alanine scanning mutants. Mutants that reduced the resting membrane potential were located in a surface patch defined by residues in the C-terminal loop (residues 115-129), positions 37-39 in the membrane interfacial recognition surface (Y46 and K54), and residue K93. These results expand the known structural determinants of the biological activity as evaluated by previous creatine kinase release experiments. Furthermore, a strong correlation is observed between the structural determinants of sarcolemma depolarization and calcium-independent disruption of liposome membranes, suggesting that a common mechanism of action underlies the permeabilization of the biological and model membranes.
Aragão
Elisangela Aparecida
EA
Departamento de Química, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, Universidade de São Paulo (USP), Avenida Bandeirantes 3900, CEP 14040-901, Ribeirão Preto, SP, Brazil.
Randazzo-Moura
Priscila
P
Rostelato-Ferreira
Sandro
S
Rodrigues-Simioni
Léa
L
Ward
Richard J
RJ
D000818:Animals; D017837:Bothrops; D002118:Calcium / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology; D003435:Crotalid Venoms / Q000633:toxicity; D054501:Group II Phospholipases A2 / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism*; D008297:Male; D008564:Membrane Potentials; D051379:Mice; D050505:Mutant Proteins / Q000378:metabolism*; D009924:Organ Culture Techniques; D010791:Phrenic Nerve / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D012508:Sarcolemma / Q000187:drug effects / Q000502:physiology
D003435:Crotalid Venoms; D008081:Liposomes; D050505:Mutant Proteins; C078097:bothropstoxin; D054501:Group II Phospholipases A2; D002118:Calcium