One of the biochemical activities ascribed to some members of the Bcl-2 family of proteins is ion-channel activity (reviewed in ref. 42). The determination of the three-dimensional structure of Bcl-XL (43) showed that this protein shares striking similarities with the previously determined structures of the pore-forming domains of the bacterial toxins diphtheria (44) and colicins A, El, and la (45-47). These bacterial proteins share the same unique ability in that they can exist either as soluble or membrane-inserted proteins. They are able to achieve this large change in character by burying two, long, predominantly hydrophobic a-helices within a shell of amphipathic helices. Under appropriate conditions, the outer amphipathic a helices splay away, freeing the central hydrophobic a helical hairpin to insert into the membrane bilayer which initiates channel formation.
Bcl-XL consists of a bundle of seven a helices, arranged in three layers. The outer two layers of amphipathic helices shield between them two long central a-helices, each ~20 amino acids in length with a bias towards hydrophobic residues (43). The two central a helices are of sufficient length to span the hydrophobic cross-section of a membrane bilayer. The structural similarity between Bcl-XL and the pore-forming domains of the bacterial toxins suggests that the Bcl-2 protein family may possess pore-forming potential as well. Indeed, several studies have shown that Bcl-2, Bcl-XL, and Bax exhibit channel-forming activity in vitro (36,48-50).
The following section describes methods for examining Bcl-2 protein family channel formation on a macroscopic level. Macroscopic level measurements have the advantage in that in order for channel activity to be detected, a majority of the protein molecules must participate in channel formation. However, the methods described do not yield information about channel formation on a single-channel basis, particularly in terms of conductivity and ion selectivity. For quantifying these characteristics, planar bilayer measurements are more appropriate. Explanation of this technique may be found in several reviews, including refs 51 and 52.
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