* Department of Physiology and Cell Biology, Washington University School of Medicine, St. Louis, Missouri 63110, USA† Dana Farber Cancer Institute, Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02115, USA‡ e-mail: paul@ cellbio. wustl. edu elected death signals activate pro-apoptotic BAX, resulting in translocation to mitochondria where it is inserted as an integral, oligomeric membrane protein1–3. Mitochondrial dysfunction follows, with release of cytochrome c and activation of caspases4–7. BAX forms ion-conducting channels in planar lipid bilayers and releases chloride or carboxyfluorescein from artificial liposomes8, 9. The ion-transmitting pore formed by BAX can progress to a conductance of~ 1.5 nS with low ion selectivity. In some proposed models10, 11, BAX interacts with other pore-forming molecules or, alternatively, generally disrupts the outer mitochondrial membrane10, 11. Here we show that nanomolar BAX rapidly forms membrane pores12, 13 that release intravesicular carboxyfluorescein, fluorescein-isothiocyanate-conjugated dextran (FITC–dextran) or FITC–cytochrome c, and that are blocked by dextran molecules of defined size. We also demonstrate concentrationdependent progression from a pore of 10.7±3.2 Å, consisting of two BAX molecules, to one of 22.1±3.9 Å, requiring four BAX molecules, and show that this larger BAX pore is capable of transporting cytochrome c.

We analyzed BAX-mediated release of 6-carboxyfluorescein from 200-nm unilamellar vesicles, using recombinant, purified, functionally identical BAX (∆ C19) molecules that were monodispersed at pH 7.2 (refs 9, 14–17). Previous studies of the melittin pore have shown that exponential dequenching reflects concentration-dependent activation of oligomeric pores12, 16–19. The exponential kinetics of BAX-pore activation are well described by equation (1), and the fit was not improved by further exponential terms (see Methods):