unclear. In a recent study using confocal and electron microscopy, Bax was found to
form large clusters protruding from the mitochondrial outer membrane during
apoptosis (Nechushtan et al., 2001). The clusters were estimated to be composed of
thousands of Bax molecules. In addition, Bak was found to redistribute from its
dispersed membrane localization into the Bax clusters, effectively leaving the
mitochondrial membrane. The cluster formation was prevented by coexpression of
Bcl-XL. The physiologic function of the clusters has not been determined.
In a recent study, a new high-conductance channel was identified in mitochondria
from apoptotic cells (Pavlov et al., 2001). The channel was localized in the outer
mitochondrial membrane and its activity correlated with the onset of apoptosis. The
mitochondrial apoptosis-induced channel (MAC) has a conductance of 2.5 nS and
shows cation selectivity, and its pore size was estimated to be 4 nm. This size would
allow the passage of cytochrome c and even larger proteins. The electrophysiologic
properties of the channel resemble those of the high-conductance Bax channel but
are clearly distinct from the main outer membrane channels, VDAC and TOM.
These results provide the first proof of a specific apoptosis-related channel in the
outer mitochondrial membrane.
Several studies have shown that the mitochondrial structures are not damaged
during the release of proteins from the intermembrane space. When Bax is activated
through NGF deprivation in SCG neurons, cytochrome c release is induced without
mitochondria swelling. On the contrary, the mitochondria are smaller than in
untreated cells. Furthermore, in the presence of caspase inhibitors, the cells are
rescued, and after re-addition of growth factor, the mitochondria regain their normal
cytochrome c content and size, indicating that no irreversible damage, such as rupture
of the outer membrane, has been inflicted (Martinou et al., 1999). Similar results
were obtained by the addition of active recombinant Bax to isolated mouse-liver
mitochondria. Finucane et al. (1999) showed that Bax induced cytochrome c release
both when overexpressed in cells and when added to isolated mitochondria. No
mitochondrial swelling was detected, demonstrating that opening of PTP was not
involved. The release of cytochrome c was inhibited by Bcl-XL and resulted in
decreased cell death.
The permeabilizing activity of Bax is not inhibited by the PTP inhibitors
cyclosporin A, EDTA, or Mg2+. In fact, Mg2+ enhances the cytochrome c-releasing
activity of Bax (Eskes et al., 1998). However, some studies have suggested that Bax-
induced cytochrome c release could be inhibited by cyclosporin A (Narita et al., 1998).
In these studies, Bax was active only in the presence of Ca2+, a PTP opener.
Furthermore, the quaternary structure of Bax was not defined. In isolated
mitochondria, Ca2+ can induce cytochrome c release in a Bax-independent manner
(Eskes et al., 1998). Thus, under the conditions used in this experiment, it might be
difficult to distinguish the effect of Bax and of Ca2+, which acts as a PTP opener.
In a study by von Ahsen et al. (2000), isolated mitochondria treated with Bax or
Bid were depleted of cytochrome c; however, the mitochondria retained a fully intact
protein-import machinery. Similar results were obtained when cells were treated with
UV-irradiation or staurosporine. Mitochondrial protein import is dependent on ∆m
134 GENETICS OF APOPTOSIS