The C terminus of Bax inhibitor-1 forms a Ca2+-permeable channel pore

Abstract

Bax inhibitor-1 (BI-1) is a multitransmembrane domain-spanning endoplasmic reticulum (ER)-located protein that is evolutionarily conserved and protects against apoptosis and ER stress. Furthermore, BI-1 is proposed to modulate ER Ca(2+) homeostasis by acting as a Ca(2+)-leak channel. Based on experimental determination of the BI-1 topology, we propose that its C terminus forms a Ca(2+) pore responsible for its Ca(2+)-leak properties. We utilized a set of C-terminal peptides to screen for Ca(2+) leak activity in unidirectional (45)Ca(2+)-flux experiments and identified an α-helical 20-amino acid peptide causing Ca(2+) leak from the ER. The Ca(2+) leak was independent of endogenous ER Ca(2+)-release channels or other Ca(2+)-leak mechanisms, namely translocons and presenilins. The Ca(2+)-permeating property of the peptide was confirmed in lipid-bilayer experiments. Using mutant peptides, we identified critical residues responsible for the Ca(2+)-leak properties of this BI-1 peptide, including a series of critical negatively charged aspartate residues. Using peptides corresponding to the equivalent BI-1 domain from various organisms, we found that the Ca(2+)-leak properties were conserved among animal, but not plant and yeast orthologs. By mutating one of the critical aspartate residues in the proposed Ca(2+)-channel pore in full-length BI-1, we found that Asp-213 was essential for BI-1-dependent ER Ca(2+) leak. Thus, we elucidated residues critically important for BI-1-mediated Ca(2+) leak and its potential channel pore. Remarkably, one of these residues was not conserved among plant and yeast BI-1 orthologs, indicating that the ER Ca(2+)-leak properties of BI-1 are an added function during evolution.

FIGURE 1.

BI-1 contains six transmembrane domains and a C-terminal loop domain. A, bioinformatical prediction of the topology of BI-1 in the ER membrane according to TMpred and TMHMM. The arrowhead in model B indicates the beginning of the C-terminal region of BI-1, including the CTP1, CTP2, and pH-sensor domains. B, amino acid-sequence of the BI-1 C terminus according to model B, indicating CTP1, CTP2, a potential loop domain with homology to SCN5A (indicated in blue), and the C terminus according to model A (indicated in red). Mapped interacting sites are indicated. C, selective permeabilization of the plasma membrane allows staining of HA-BI-1 and BI-1-HA but not of luminal ERp44. N2a cells were transiently transfected with the indicated plasmids and permeabilized with digitonin, which only permeabilizes the plasma membrane, or Triton X-100, which permeabilizes all cellular membranes. Cells were then stained with an FITC-labeled anti-HA antibody and fluorescence was quantitated by flow cytometry as shown in the representative plots. Bar graphs represent mean ± S.E. of six pooled independent experiments done in triplicates.

FIGURE 2.

A peptide derived from the C-terminal end of BI-1 (CTP1), corresponding to amino acids 198–217 induces Ca²⁺ release from the ER. A, permeabilized MEF cells loaded to steady-state with ⁴⁵Ca²⁺ were incubated in Ca²⁺-free efflux medium and their Ca²⁺ content, expressed in counts per min (cpm), monitored as a function of time. In the time period indicated by the line, vehicle, CTP1, and CTP2 were added for 8 min. CTP1, but not CTP2, led to a decrease in the Ca²⁺ content. B, results obtained from the experiment described in A were plotted as “fractional loss” (%/2 min) as a function of time. CTP1, but not vehicle or CTP2, provoked an increase in the fractional loss. C, different concentrations of CTP1 were added during the efflux phase for 6 min. Results were plotted as fractional loss as a function of time. D, CTP1-induced Ca²⁺ release from ⁴⁵Ca²⁺-loaded permeabilized cells was quantified as the difference in fractional loss between 2 min after application of CTP1 and before CTP1, i.e. the fractional loss at 10 min minus the fractional loss at 8 min. Concentration-response curve for CTP1-induced Ca²⁺ release was obtained from three independent experiments. All results were normalized to the Ca²⁺ released by the Ca²⁺ ionophore, A23187, and expressed as % of total A23187-induced Ca²⁺ release.

FIGURE 3.

CTP1-induced Ca²⁺ release is independent of BI-1, IP₃Rs, RyRs, presenilins, and translocon. A, CTP1-induced Ca²⁺ release from ⁴⁵Ca²⁺-loaded permeabilized wild-type and BI-1^−/− MEF cells. CTP1 (40 μm) was added during the efflux as indicated. B, CTP1-induced Ca²⁺ release from ⁴⁵Ca²⁺-loaded permeabilized MEF cells in the presence of inhibitors of the IP₃R (100 μm 2-aminoethoxydiphenyl borate (2-APB)), the RyR (100 μm ryanodine), and the translocon (100 μm anisomycin). C, CTP1-induced Ca²⁺ release from ⁴⁵Ca²⁺-loaded permeabilized PS1^−/− PS2^−/− MEF cells (PS, presenilin). Different concentrations of CTP1 were added during the efflux as indicated. D, CTP1-induced Ca²⁺ release from ⁴⁵Ca²⁺-loaded permeabilized Lvec and IP₃R1-overexpressing L15 fibroblasts. E and F, CTP1-induced Ca²⁺ release from Mag-Fluo4-loaded ER of permeabilized DT40 TKO cells and TKO cells ectopically expressing IP₃R1. The MagFluo4 signal monitored over time is related to the [Ca²⁺]_ER. Ca²⁺ uptake in the ER is initiated by addition of ATP. After reaching steady-state, thapsigargin was added to follow unidirectional Ca²⁺ flux from the ER. After 90 s, different concentrations of CTP1 were added. Ionomycin (10 μm) was added to determine the maximal releasable Ca²⁺. The vehicle (DMSO, dimethyl sulfoxide) was added to determine the passive Ca²⁺ leak.

FIGURE 4.

CTP1-induced Ca²⁺ release originates from the IP₃-sensitive store. A, CTP1-induced Ca²⁺ release from ⁴⁵Ca²⁺-loaded permeabilized MEF cells was measured without (■) and with (●) preincubation of a supramaximal concentration of IP₃ (50 μm). B, CTP1-induced Ca²⁺ release, provoked by different concentrations of CTP1, was quantified relative to a supramaximal concentration of IP₃ (50 μm). The maximal CTP1-induced Ca²⁺ release amounted to about 80% of the Ca²⁺ release provoked by a saturating concentration of IP₃.

FIGURE 5.

CTP1-induced Ca²⁺ release depends on hydrophobic residues Leu-210 and Phe-211 and on its α-helical properties. A, ⁴⁵Ca²⁺ release from permeabilized MEF cells provoked by CTP1 and its truncated forms. Truncated forms of CTP1 did not provoke Ca²⁺ release from the ER. A representative experiment is shown for 3 independent experiments. B, I-TASSER predictions for CTP1, CTP1-L210A,F211A, CTP1-L210G,F211G, CTP1-C207G,I208G, and CTP1-C207A. C, a plot showing the hydrophobicity score of the CTP1 and the different mutants following the Kyte-Doolittle model (50). Positive values indicate higher hydrophobicity, whereas negative values indicate higher hydrophilicity. D, Ca²⁺ release from permeabilized MEF cells provoked by CTP1 and mutant forms. A representative experiment is shown for 4 independent experiments. DMSO, dimethyl sulfoxide.

FIGURE 6.

CTP1 displays ion channel-like activity in planar lipid bilayers. A, traces of currents from two representative experiments attempting incorporation of the CTP1 (top) and CTP1-L210A,F211A (bottom) peptides into the artificial lipid bilayers. The peptide-concentration increments are indicated by arrows. Breaks within traces correspond to 1-min cis chamber solution stirring intervals after peptide additions. B, higher resolution (4-s long) fragments of the CTP1-generated currents at the time points corresponded to the numbered circles in A. C, current sublevel-probability histograms obtained from continuous 60-s long fragments of traces presented in A during the first minute of the channel activity appearance for CTP1 (40 μm) and during prolonged incubation with CTP1-L210A,F211A (50 μm). D, values of the mean current (I_mean ± S.E.) calculated from 60-s long recordings and averaged for four experiments for each peptide. For CTP1, the 60-s recordings were taken immediately after appearance of the initial series of spikes indicating formation of the CTP1-assembled channels (*, p < 0.05). E, representative 4-s long recordings obtained at various membrane potentials (shown on the right) from the same planar lipid bilayer within an interval of quasistable basal current carried by CTP1-induced (40 μm) ion channels in asymmetric bilayer ionic conditions (cis, HEPES/Tris; trans, HEPES/Ba²⁺, 50 mm, see ”Experimental Procedures“).

FIGURE 7.

CTP1 peptides corresponding to sequences of animal orthologs of BI-1, but not plant and yeast orthologs, provoked Ca²⁺ release. A, amino acid sequence of different animal and plant BI-1 orthologs. B, application of 40 μm CTP1 peptides corresponding to different animal, plant, and yeast orthologs of BI-1 during the efflux phase of ⁴⁵Ca²⁺-loaded permeabilized MEF cells.

FIGURE 8.

Asp-209 and Asp-213 are essential residues of the Ca²⁺-channel pore of CTP1. A, double Asp/Arg mutants of CTP1 and CTP1-D213R were not able to provoke ⁴⁵Ca²⁺ mobilization from the ER of permeabilized MEF cells. A typical experiment, representative for three independent experiments, is shown. B, mutation of each of the four Asp residues into Ala in the CTP1 peptide reveals a critical role for Asp-209 and Asp-213. CTP1-D209T represents the change in Asp residues between animal and plant CTP1 orthologs. A typical experiment, representative for four independent experiments, is shown. C, quantification of the amount of Ca²⁺ released by CTP1 and its respective aspartate mutations.

FIGURE 9.

Asp-213 is essential for the function of full-length BI-1. A, Western blot analysis using anti-Myc antibodies showing the expression of Myc-BI-1 and Myc-BI-1-D213R in HeLa cell lysates. B, representative Ca²⁺ signals obtained from calibrated Fura-2-loaded HeLa cells co-transfected with mCherry plasmid and mock (plasmid only), myc-BI-1, and myc-BI-1-D213R, respectively. C, mean maximal peak height of Ca²⁺ signals (n = 61 to 68, five biological replicates). Two-tailed t test shows significant difference between mock- and myc-BI-1 (p < 0.005) as well as myc-BI-1 and myc-BI-1-D213R-transfected cells (p < 0.005), but not between mock- and BI-1-D213R-transfected cells (p = 0.80).