Channel formation by antiapoptotic protein Bcl-2.
Bcl-2 is the prototypical member of a large family of apoptosis-regulating proteins, consisting of blockers and promoters of cell death. The three-dimensional structure of a Bcl-2 homologue, Bcl-XL, suggests striking similarity to the pore-forming domains of diphtheria toxin and the bacterial colicins, prompting exploration of whether Bcl-2 is capable of forming pores in lipid membranes. Using chloride efflux from KCl-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 mutant of Bcl-2 lacking the two core hydrophobic alpha-helices (helices 5 and 6), predicted to be required for membrane insertion and channel formation, 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 (Deltah5, 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 structure 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 evidence that Bcl-2 forms channels in lipid membranes, suggesting a novel function for this antiapoptotic protein.
Schendel
S L
SL
Program on Apoptosis and Cell Death Research, The Burnham Institute, 10901 N. Torrey Pines Road, La Jolla, CA 92037, USA.
Xie
Z
Z
Montal
M O
MO
Matsuyama
S
S
Montal
M
M
Reed
J C
JC
D001427:Bacterial Toxins; D002712:Chlorides / Q000378:metabolism; D003001:Cloning, Molecular; D017931:DNA Primers; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007473:Ion Channels; D007700:Kinetics; D008051:Lipid Bilayers; D008081:Liposomes; D008564:Membrane Potentials; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D016133:Polymerase Chain Reaction; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000737:chemistry* / Q000302:isolation & purification / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016324:Sequence Tagged Sites
D001427:Bacterial Toxins; D002712:Chlorides; D017931:DNA Primers; D007473:Ion Channels; D008051:Lipid Bilayers; D008081:Liposomes; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins