Proapoptotic N-truncated BCL-xL protein activates endogenous mitochondrial channels in living synaptic terminals - PubMed (original) (raw)

. 2004 Sep 14;101(37):13590-5.

doi: 10.1073/pnas.0401372101. Epub 2004 Sep 1.

John A Hickman, Mushtaque Chachar, Brian M Polster, Teresa A Brandt, Yihru Fannjiang, Iva Ivanovska, Gorka Basañez, Kathleen W Kinnally, Joshua Zimmerberg, J Marie Hardwick, Leonard K Kaczmarek

Affiliations

Proapoptotic N-truncated BCL-xL protein activates endogenous mitochondrial channels in living synaptic terminals

Elizabeth A Jonas et al. Proc Natl Acad Sci U S A. 2004.

Abstract

Neuronal death is often preceded by functional alterations at nerve terminals. Anti- and proapoptotic BCL-2 family proteins not only regulate the neuronal death pathway but also affect excitability of healthy neurons. We found that exposure of squid stellate ganglia to hypoxia, a death stimulus for neurons, causes a cysteine protease-dependent loss of full-length antiapoptotic BCL-xL, similar to previous findings in mammalian cells. Therefore, to determine the direct effect of the naturally occurring proapoptotic cleavage product of BCL-xL on mitochondria, recombinant N-truncated BCL-xL was applied to mitochondria inside the squid presynaptic terminal and to purified mitochondria isolated from yeast. N-truncated BCL-xL rapidly induced large multi-conductance channels with a maximal conductance significantly larger than those produced by full-length BCL-xL. This activity required the hydrophobic C terminus and the BH3 domain of BCL-xL. Moreover, N-truncated BCL-xL failed to produce any channel activity when applied to plasma membranes, suggesting that a component of the mitochondrial membrane is necessary for its actions. Consistent with this idea, the large channels induced by N-truncated BCL-xL are inhibited by NADH and require the presence of VDAC, a voltage-dependent anion channel present in the outer mitochondrial membrane. These observations suggest that the mitochondrial channels specific to full-length and N-truncated BCL-xL contribute to their opposite effects on synaptic transmission, and are consistent with their opposite effects on the cell death pathway.

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Figures

Fig. 1.

Fig. 1.

Mitochondrial channel activity produced by ΔN BCL-xL. (a) Channel activity recorded at –100 mV using pipettes containing 8.0 μg/ml ΔN BCL-xL. (b) I-V relations for ΔN BCL-xL-induced activity, showing examples of intermediate and large conductances. (c) Cumulative distribution of probability of channel activity of different conductances in experiments with control intracellular solution, ΔN BCL-xL, or full-length BCL-xL. Histograms combining all experiments show the probability of closed channels, activity <180 pS (Small), activity between 180 and 760 pS (Intermediate), and activity >760 pS (Large). For controls, n = 16; for ΔN BCL-xL, n = 6; for FL BCL-xL, n = 4 (probabilities relative to control *, P < 0.025; **, P < 0.005). (d) Time course of transitions of channel opening to different conductances. Shown are transitions in recordings with 8.0 μg/ml ΔN BCL-xL (Right) or full-length BCL-xL (Left) in the patch pipette solution. Openings were divided into the three groups as above. Probability of occurrence of openings in each group within successive 10-s recording periods is plotted as a function of time.

Fig. 2.

Fig. 2.

Specificity of channel formation by ΔN BCL-xL. (a) Activity on a mitochondrial membrane recorded at –100 mV using a pipette containing 8.0 μg/ml ΔN BCL-xL. C marks the closed state. (b) Lack of activity in the presence of the BH3 domain mutant ΔN Δ90-92 BCL-xL. Patch potential was –100 mV. (c) Lack of effect of ΔN BCL-xL in cell-attached plasma membrane recordings of molluscan neurons. Endogenous small-conductance plasma membrane channel activity (42) can be detected. Patch potential was –160 mV.

Fig. 3.

Fig. 3.

Effects of NADH on multiconductance channel activity produced by ΔN BCL-xL. (a) Bar graphs show distribution of openings for patches exposed to 8.0 μg/ml ΔN BCL-xL with 2 mM NADH (n = 10). (b) Lack of effect of NADH on the release of fluorescent indicator (ANTS) from artificial lipid vesicles in the presence of ΔN BCL-xL. Lipid and protein concentrations were 50 μM and 50 nM, respectively.

Fig. 4.

Fig. 4.

Actions of ΔN BCL-xL on channel activity in isolated mitochondria from wild-type yeast and those lacking the POR1 gene (ΔPOR1). (a) Typical channel activity in wild-type yeast mitochondria in the absence (Left) or presence (Right) of 8.0 μg/ml ΔN BCL-xL. (b) Channel activity during a 10-s ramp from –100 to +100 mV. Holding potential before and after the ramp was 0 mV. (Left) Response in the absence of ΔN BCL-xL. As expected for the VDAC, increased channel openings occur near the center of the ramp. The slope of the closed state is indicated, as is the slope of the subconductance state (sub) that persists at positive and negative potentials. (Right) Response with 8.0 μg/ml ΔN BCL-xL. (c) Bar graphs show the maximal channel conductance recorded in the absence or presence of 8.0 μg/ml ΔN BCL-xL, or of ΔN BCL-xL with 2 mM NADH. Shown are data for wild-type mitochondria (Left) and ΔPOR1 mutant mitochondria (Right).

Fig. 5.

Fig. 5.

zVAD prevents loss of full-length BCL-xL protein during hypoxia. (a) Immunoblot of untreated or hypoxia-treated (60 min) squid stellate ganglia incubated in the absence or presence of zVAD (100 μM) by using anti-chicken BCL-x antibody. Equal loading was verified by comparing a crossreacting band near the top of the gel. (b) Immunoblot of recombinant human BCL-xL protein and of mitochondria (mito) purified from hypoxic (20 min) squid stellate ganglia (± zVAD) by using anti-Bcl-x antibody (Top). (Middle) Immunoblots using the same antibody preadsorbed with recombinant human BCL-xL protein. (Bottom) Immunoblot analysis using an anti-VDAC antibody.

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