Isolation, characterization and partial purification of a transferable membrane channel (amoebapore) produced by Entamoeba histolytica

Abstract

Entamoeba histolytica kills cells by contact dependent cytolysis. The mechanism underlying this process must be of rapid onset because target cells round up and show marked zeiosis within 15 min of contact. In earlier work, we identified a remarkable ion-channel forming protein which we named amoebapore, that may contribute to the amoeba-induced target cell killing. Within the amoeba it exists as part of a supramolecular aggregate together with other proteins of unknown function. In this work we report the purification of a solubilized form of the amoebapore. Amoebapore was found to exist as an apparent dimer of the previously reported protein whose molecular weight had been determined under denaturing conditions. Two isoforms of this dimer, with p I values of 6.8 and 5.3 present at a ratio of 7 to 1, were identified and purified. Both isoforms demonstrate ion-channel forming activity in planar lipid membranes. These channels show a unit conductance of 5–20 pS and remain open for < 1 s. Upon lateral aggregation, opening becomes concerted to a greater degree with channel conductance appearing in the 500 pS range (270 mM KCl). Aggregation seems to be highly cooperative because no intermediate single channel conductances are observed. The isolated particulate form of amoebapore depolarizes cells.