Cytochrome c release induced by t-BID is dose-dependent and requires expression of Bax and/or Bak. Mitochondria isolated from the indicated cell lines were incubated with varying concentrations of t-Bid or with Bid or vehicle, and cytochrome c (Cyt c) release was quantified by ELISA. A, histograms show the dose-dependent release of cytochrome c by t-Bid after 30 min, relative to release by 80 μg/ml alamethicin. The EC₅₀ of t-Bid for cytochrome c release for Parental and Bax KO cell lines was 7 and 17 nm with correlation coefficients of 0.92 and 0.98, respectively. EC₅₀ was estimated at 250 nm t-Bid for mitochondria from Bak KO cells. B, histograms show the time-dependent release of cytochrome c from isolated mitochondria after incubation with either 20 nm t-Bid at various times or 1 μm t-Bid after 30 min relative to alamethicin. Data are mean ± S.E.

Dibucaine and cytochrome c regulate MAC activity induced by t-Bid. MAC activity was induced by patch clamping isolated mitochondria with micropipettes containing t-Bid. A, current trace of MAC-Bax shows a decrease in conductance from 2.4 to 0.62 nS after perfusion of the bath with media containing 100 μm of the MAC inhibitor dibucaine. B and C, current traces of MAC-Bax (B) and MAC (C) show decreases in conductance from 3.6 to 0.95 nS in B and from 1.9 to 0.9 nS in C after perfusion with media containing physiological levels (100 μm) cytochrome c (Cyt c).

Kinetics of MAC formation. A–E, mitochondria were isolated from Parental, Bax KO, Bak KO, and VDAC1VDAC3 KO MEFs and monitored for t-Bid-induced MAC by patch clamping as usual (see legend to Fig. 3 and “Experimental Procedures”). The arrows at right in traces A–D indicate the time elapsed due to seal formation. Current traces recorded from mitochondria of Parental (A), VDAC1VDAC3 KO (B), Bax KO (C), and Bak KO (D) MEF cells show an increase in conductance corresponding to MAC formation. Kinetics and amplitude of formation of MAC channels in mitochondria from the VDAC1VDAC3 KO cells (B) and the Bax or Bak KO cells (C and D, labeled as MAC-Bak or MAC-Bax to indicate protein present in these mitochondria) were similar. E, current trace from a membrane patch from mitochondria isolated from Bax KO cells illustrates the common ∼300-pS transitions and less common larger (∼600 and ∼1000 pS) transitions associated with MAC formation. F, current trace from a liposome patch shows an increase in conductance with time when 380 ng/μl of monomeric BAXΔC and 35 ng/μl t-Bid were included in the micropipette. The conductance increased in multiple steps of ∼300 pS until it reached about 1200 pS.

MAC forms in the mitochondrial outer membrane of apoptotic cells expressing Bax and/or Bak. A, Western blots show normal expression of Bax (5 μg of protein) and Bak (30 μg of protein) in whole cell extracts of Parental and indicated knock-out MEF cells. B, changes in Bax and Bak protein levels in mitochondria isolated from Parental and indicated knock-out cell lines were estimated by densitometry after immunoblotting. Cells were harvested 16 h after addition of vehicle (-) or 1 μm STS (+) to induce apoptosis. Pixel densities of Bax and Bak bands were normalized to those of VDAC (a constitutive mitochondrial outer membrane protein) in the same gel lanes using the Parental (-) condition as standard. Data are the average of two independent experiments. C, peak conductances of outer membranes were measured by directly patch clamping mitochondria isolated from control and STS-treated MEF cells. The increase in conductance corresponds to the difference between mean conductance of STS-treated and control patches. These permeability increases are consistent with the presence of MAC in mitochondria of apoptotic Parental, Bax KO, and Bak KO cells, but not in the STS-treated DKO cells. Data are mean of 20–23 independent patches, and error bars indicate S.E. D, cytochrome c (Cyt c) release to the cytosol was monitored by ELISA and is expressed relative to the positive control alamethicin (80 μg/10⁶ cells). Data are mean ± S.E. of at least three different preparations. E and F, current traces show single MAC-Bax (E) and MAC whose peak conductances were ∼5 nS that were recorded at +10 and -20 mV from mitochondria isolated from Bak^-/- and parental cells, respectively, after treatment with 1 μm STS for 16 h. Filtration was 2 kHz with 5-kHz sampling. Dotted lines indicate the 0 pA level. Bath solution was 150 mm KCl, 5 mm HEPES, 0.23 mm CaCl₂, 1 mm EGTA, pH 7.4.

MAC formation induced by t-Bid in normal mitochondria requires Bax or Bak expression. Isolated mitochondria were directly patch-clamped to detect MAC activity, using micropipettes whose tips contained t-Bid, Bid, or vehicle alone. A, current traces recorded from membrane patches clamped at +40 mV or -40 mV show formation of MAC in less than 10 min for mitochondria isolated from Parental cells when the micropipette contained 20 nm t-Bid, but not Bid or vehicle alone. In similar experiments, MAC activity was not detected in mitochondria isolated from DKO cells or protein-free liposomes exposed to 1 or 2.3 μm t-Bid, respectively. B, current traces recorded at 40 mV and displayed at higher time resolution than A show channel activity induced by t-Bid in mitochondria from Parental cells (labeled MAC), Bax KO cells (labeled MAC-Bak), Bak KO cells (labeled MAC-Bax), and DKO cells. Peak conductances in these traces were 3.2, 3.7, 4.5, and 0.5 nS, respectively. C, histogram shows the frequency of detecting patches with conductances ≥1.5 nS MAC formation in mitochondria from the four MEF cell lines and in liposomes (negative control) when vehicle, t-Bid, or Bid were included in the micropipette. n corresponds to the number of independent patches. Other conditions are as in Fig. 1.

VDAC1 and VDAC3 are not essential for MAC formation. A, Western blots show expression of Bax, Bak, and VDAC1 in mitochondria of VDAC1VDAC3 KO MEF cells (lane 1;7 μg) and FL5.12 cells (lane 2;2 μg). B, release of cytochrome c (Cyt c) from mitochondria isolated from VDAC1VDAC3 KO MEF cells after incubation with t-Bid, Bid, or vehicle, determined by ELISA and expressed relative to release by alamethicin (80 μg/ml). C, histogram shows the detection frequency of patches with conductances ≥1.5 nS in mitochondria isolated from VDAC1VDAC3 KO cells patch-clamped with micropipettes backfilled with t-Bid (1 μm), Bid (1.6 μm), or vehicle. D, current traces show dibucaine reversibly inhibited MAC induced by t-Bid in VDAC1VDAC3 KO mitochondria. The downward deflection in the top current trace shows MAC assembled ∼5 min after seal formation. Middle and lower current traces show the closing effect of adding (upward deflection) and opening effect (downward deflection) of washing out 200 μm dibucaine from the bath. E, current traces show MAC activity in membrane patches clamped at +20 mV in the absence (upper, 2.6 nS) and presence of 100 μm cytochrome (lower panel, +Cyt c; 2.1 nS). Immediately after perfusion, the conductance of MAC decreased ∼20%, although the noise of the current trace increased, which is consistent with the type 1 effect of cytochrome c (10). F, current trace from VDAC1VDAC3 KO mitochondria patch-clamped at 20 mV shows a large decrease in MAC conductance from 4.2 to 1.0 nS after perfusion with 100 μm cytochrome (+Cyt c) illustrating a type 2 effect (10). Other conditions were the same as in experiments of Figs. 1 and 3.

Modeling MAC formation. The relationship between the conductance of MAC and the number of α-helices that would form circular pores of equivalent conductance was modeled as explained under “Experimental Procedures.” A, plot shows the cross-sectional area inside a circular pore as a function of the number of α-helices comprising it. The line is the best fit of data to area predicted by Equation 7 under “Experimental Procedures,” with a correlation coefficient of 0.999. B, plot shows number of α-helices needed to form pores as MAC assembles in t-Bid-treated mitochondrial membrane patches from various cell types, using the method of Hille (36) that corrects for access resistance (see “Experimental Procedures”). Line is the best fit of the data correlation, which had a correlation coefficient of 1.0. Data correspond to at least five different patches for each cell line. C, plot shows the number of α-helices that are estimated to form the pore of MAC as a function of the mean conductance calculated for MAC from the indicated cell types. Data are mean ± S.E. D, curves correspond to the predicted increments in conductance (or transition size) when 1–4 or 6 α-helices are inserted into a pore, plotted as a function of conductance at the time of the transition. Data points correspond to actual conductance increments observed during MAC formation in membrane patches for the indicated cell types. Approximately 80% of the data points fall on or near the curves corresponding to 1 and 2 α-helix insertions. The data correspond to 16 different patches. E, model shows how the MAC pore might be formed by sequential addition of α-helices during oligomerization of Bax and Bak in the membrane plane. The initial MAC pore detected is presumed to be a hexamer (6 α-helices) and had a conductance of 280 pS. The conductances corresponding to the subsequent stages in assembly of the mature pore (with multiple pathways labeled 1–3) are indicated in the schematic. See also supplemental Table 1S.