Comparison of the ion channel characteristics of proapoptotic BAX and antiapoptotic BCL-2

Abstract

The BCL-2 family of proteins is composed of both pro-and antiapoptotic regulators, although its most critical biochemical functions remain uncertain. The structural similarity between the BCL-X L monomer and several ion-pore-forming bacterial toxins has prompted electrophysiologic studies. Both BAX and BCL-2 insert into KCl-loaded vesicles in a pH-dependent fashion and demonstrate macroscopic ion eff lux. Release is maximum at ϷpH 4.0 for both proteins; however, BAX demonstrates a broader pH range of activity. Both purified proteins also insert into planar lipid bilayers at pH 4.0. Single-channel recordings revealed a minimal channel conductance for BAX of 22 pS that evolved to channel currents with at least three subconductance levels. The final, apparently stable BAX channel had a conductance of 0.731 nS at pH 4.0 that changed to 0.329 nS when shifted to pH 7.0 but remained mildly Cl ؊ selective and predominantly open. When BAX-incorporated lipid vesicles were fused to planar lipid bilayers at pH 7.0, a Cl ؊ -selective (P K ͞P Cl ‫؍‬ 0.3) 1.5-nS channel displaying mild inward rectification was noted. In contrast, BCL-2 formed mildly K ؉ -selective (P K ͞P Cl ‫؍‬ 3.9) channels with a most prominent initial conductance of 80 pS that increased to 1.90 nS. Fusion of BCL-2-incorporated lipid vesicles into planar bilayers at pH 7.0 also revealed mild K ؉ selectivity (P K ͞P Cl ‫؍‬ 2.4) with a maximum conductance of 1.08 nS. BAX and BCL-2 each form channels in artificial membranes that have distinct characteristics including ion selectivity, conductance, voltage dependence, and rectification. Thus, one role of these molecules may include pore activity at selected membrane sites.

Proapoptotic genes BAX and CD40L are predictors of survival in transitional cell carcinoma of the bladder

“…BAX counteracts the apoptosis-preventing effect of BCL2 and may directly induce apoptosis (Xiang et al, 1996;Zha and Reed, 1997). The proapoptotic BAX is located in the outer mitochondrial membrane (Schlesinger et al, 1997). BAX overexpression induces mitochondrial permeability transition, which leads to the release of cytochrome c (Pastorino et al, 1998).…”

The Bax pore in liposomes, Biophysics

“…[25][26][27][28] In all cases, pore formation was enhanced by acid pH. As the pIs of these proteins vary from 7 to 4.5, it seemed unlikely that this pH dependence was reflecting a common mode of activating the individual proteins.…”

“…34,[54][55][56] We determined that by shortening this helix we could generate an active form, Bax(DC19), for biophysical studies in liposomes and planar lipid bilayers. 25,49 Extensive mutation studies confirm that the Bax BH3 helix mediates binding between Bcl-2-family proteins. 19,51,[57][58][59] Although central to the regulation of apoptosis, a single binding site will provide only for specific dimerization.…”

“…This engineered preparation, Bax(DC19), was used to study the biophysics of cytochrome c-competent pore activation in define liposomes and planar lipid bilayers. 25,49 Structural Requirements for Pore Formation…”

“…This observation correctly focused interest on the membrane association and the in-membrane pore-forming activities of the Bcl-2-family proteins. [25][26][27][28] The realization that cytochrome c release from the mitochondria was the essential consequence of Bcl-2-family protein activity [29][30][31][32] has crystallized our focus on oligomerization and pore formation by this protein family. Stan Korsmeyer and his collaborators were instrumental in defining the Fas (TNF)-Bid-Bax/Bak pathway of apoptosis regulation.…”