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Comparative Study
. 1997 Oct 14;94(21):11357-62.
doi: 10.1073/pnas.94.21.11357.

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

P H Schlesinger  1 , A Gross, X M Yin, K Yamamoto, M Saito, G Waksman, S J Korsmeyer
Affiliations
Comparative Study

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

P H Schlesinger et al. Proc Natl Acad Sci U S A. .

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-XL 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 efflux. Release is maximum at approximately pH 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 (PK/PCl = 0.3) 1.5-nS channel displaying mild inward rectification was noted. In contrast, BCL-2 formed mildly K+-selective (PK/PCl = 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 (PK/PCl = 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.

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Figures

Figure 1
Figure 1
BAX and BCL-2 induced release of ions from synthetic lipid vesicles. (A) An analytical gel of purified recombinant BAX and BCL-2 preparations used in this study. Recombinant murine BAXΔC19 and BCL-2ΔC21, prepared as described in Materials and Methods, were separated on a 12% SDS polyacrylamide gel and stained with Coomassie blue. Markers (Mr) are in the left lane. Lane 1, recombinant BAXΔC19; lane 2, recombinant BCL-2ΔC21. Graphs of BAX-induced (B) or BCL-2-induced (D) Cl− efflux from synthetic lipid vesicles are shown. KCl-loaded, negatively charged, unilamellar vesicles were added to 10 mM dimethylglutarate buffer at the indicated pH values, resulting in a 200-fold inside:outside KCl gradient. BAX or BCL-2 was added (time = 0) and ion efflux was measured using a Cl−-sensitive electrode. Triton X-100 was added (horizontal arrow) to release all encapsulated Cl−. Graphs show BAX-induced (C) or BCL-2-induced (E) Cl− efflux from vesicles in buffer at pH 6.0 or pH 5.0, respectively, which subsequently shifted to pH 4.0 (vertical arrow).
Figure 2
Figure 2
Insertion of soluble BAX into planar lipid bilayers. (A) Purified BAX (≈1 μg) was added to the cis chamber of a 450-/150-mM KCl gradient, and the first spontaneous current (OS; V = 0 mV) was anionic with a slope conductance of 22 ± 5 pS. (B) Tracings of a well defined intermediate stage that appeared during pH 4.0 BAX insertions. The records are at +40 mV (Upper) and −40 mV (Lower) with a 450-/150-mM KCl gradient, which accounts for the asymmetry in the current amplitudes. The difference in opening kinetics is a voltage-dependent effect seen at this intermediate stage of channel maturation. ∗ denotes direct C−OS-to-O2 transitions. (C) The current–voltage plots for the open pore at pH 4.0 and after a shift to pH 7.0 in the presence of a 450-/150-mM KCl gradient (○) or in symmetric 150-mM KCl (▪). The region of reversal potential is expanded in Inset.
Figure 3
Figure 3
Proteoliposome insertion of BAX into planar lipid bilayer membranes. (A) Proteoliposomes with incorporated BAX were added to the cis chamber after a bilayer with 0.4-μF capacitance was obtained. A large Cl−-selective pore (O) appears in the presence of a 450-/150-mM KCl gradient (V = 0 mV). The size of the current labeled O2 that appears after the initial activity (O) implies the presence of two channels. (B) Voltage dependence of the current in symmetrical 150 mM KCl. (C) Longer tracing of currents demonstrates rectification at +70 mV and closures at −70 mV.
Figure 4
Figure 4
Insertion of soluble BCL-2 into planar lipid bilayers. (A) Purified BCL-2 (≈1 μg) was added to the cis chamber of a 450-/150-mM KCl gradient, and in 5–20 min an outward K+ current was consistently observed. (B) Long recordings of the channel transitions between the open (OS) and closed (C) state. A histogram of the distribution of amplitudes over 120 sec is shown. (C) A current–voltage plot of the BCL-2 channel from B. (D) A current–voltage plot of the large pore that formed over time at pH 4.0 and its current–voltage plot following the shift to pH 7.0.
Figure 5
Figure 5
Proteoliposome insertion of BCL-2 into planar lipid bilayer membranes. (A) Proteoliposomes with incorporated BCL-2 were added to the cis chamber after a bilayer with 0.4 μF capacitance was obtained. In multiple determinations an initial K+ channel was always obtained. In the preparation shown, two BCL-2 conductance levels (O, O2) appear simultaneously in the bilayer (V = 0 mV). (B) A series of voltage steps (indicated in the lower tracing) was applied to an established single channel (indicated by O) in a 450-/150-mM KCl gradient. The resultant continuous tracing of channel current is shown in the upper tracing. (C) The open channel currents in symmetric 150-mM KCl and the current–voltage plot for the BCL-2 pore.
Figure 6
Figure 6
Comparison of the charged amino acids in the putative membrane penetrating α-5 and α-6 helices of BAX and BCL-2. (A) Views of the positively charged surface of the α-5 and α-6 helices of BAX (Left) and of the negatively charged surface of the same region of BCL-2 (Right), calculated and displayed using grasp (40). The surfaces are colored deep blue (15 kBT) in the most positively charged regions and deep red (−15 kBT) in the most negative, with linear interpolation for values in between. Both models were generated using insightii (BiosymTechnologies, San Diego) from the crystallographic model of BCL-XL (PDB entry 1MAZ). (B) Sequence alignment of α-5 and α-6 helices in two antiapoptotic molecules (BCL-XL and BCL-2) and in two proapoptotic molecules (BAX and BAK).
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