CHANNEL FORMATION BY ANTIAPOPTOTIC PROTEIN BCL-2

Bcl-2 is the prototypical member of a targe family of apoptosis-regula ting proteins, consisting of blockers and promoters of cell death. The three-dimensional structure of a Bcl-2 homologue, Bcl-X-L, suggests s triking similarity to the pore-forming domains of diphtheria toxin and the bacterial colicins, prompting exploration of whether Bcl-2 is cap able of forming pores in lipid membranes. Using chloride efflux from K Cl-loaded unilamellar lipid vesicles as an assay, purified recombinant Bcl-2 protein exhibited pore-forming activity with properties similar to those of the bacterial toxins, diphtheria toxin, and colicins, i.e ., dependence on low pH and acidic lipid membranes. In contrast, a mut ant of Bcl-2 lacking the two core hydrophobic alpha-helices (helices 5 and 6), predicted to be required for membrane insertion and channel f ormation, produced only nonspecific effects. In planar lipid bilayers, where detection of single channels is possible, Bcl-2 formed discrete ion-conducting, cation-selective channels, whereas the Bcl-2 (Delta h 5, 6) mutant did not, The most frequent conductance observed (18 +/- 2 pS in 0.5 M KCl at pH 7.4) is consistent with a four-helix bundle str ucture arising from Bcl-2 dimers. However, larger channel conductances (41 +/- 2 pS and 90 +/- 10 pS) also were detected with progressively lower occurrence, implying the step-wise formation of larger oligomers of Bcl-2 in membranes. These findings thus provide biophysical eviden ce that Bcl-2 forms channels in lipid membranes, suggesting a novel fu nction for this antiapoptotic protein.