Equinatoxin II permeabilizing activity depends on the presence of sphingomyelin and lipid phase coexistence

¹ TU Dresden
² University of Ljubljana

^* To whom correspondence should be addressed. E-mail: schwille{at}biotec.tu-dresden.de.

Submitted on January 21, 2008
Revised on February 18, 2008
Accepted on 18 March 2008

	Abstract

Equinatoxin II is a pore forming protein of the actinoporin family. After membrane binding, it inserts its N-terminal -helix and forms a protein/lipid pore. Equinatoxin II activity depends on the presence of sphingomyelin in the target membrane; however the role of this specificity is unknown. On the other hand, sphingomyelin is considered to be an essential ingredient of lipid rafts and promotes liquid-ordered/liquid-disordered phase separation in model membranes that mimic raft composition. Here we have used giant unilamellar vesicles to simultaneously investigate the effect of sphingomyelin and phase separation on the membrane binding and permeabilizing activity of Equinatoxin II. Our results show that Equinatoxin II binds preferentially to the liquid ordered phase over the liquid disordered one, and that it tends to concentrate at domain interfaces. In addition, sphingomyelin strongly enhanced membrane binding of the toxin, but was not sufficient for membrane permeabilization. Under the same experimental conditions, Equinatoxin II formed pores in giant unilamellar vesicles containing sphingomyelin only when liquid ordered and disordered phases coexisted. Our observations demonstrate the importance of phase boundaries for Equinatoxin II activity and suggest a double role of sphingomyelin as a specific receptor for the toxin and as a promoter of the membrane organization necessary for Equinatoxin II action.

Key Words: equinatoxin, giant unilamellar vesicle, lipid raft, phase separation, pore forming toxin, toroidal pore