The mitochondrial calcium uniporter is a multimer that can include a dominant-negative pore-forming subunit
2013; Springer Nature; Volume: 32; Issue: 17 Linguagem: Inglês
10.1038/emboj.2013.157
ISSN1460-2075
AutoresAnna Raffaello, Diego De Stefani, Davide Sabbadin, Enrico Teardo, Giulia Merli, Anne Picard, Vanessa Checchetto, Stefano Moro, Ildikò Szabó, Rosario Rizzuto,
Tópico(s)MicroRNA in disease regulation
ResumoArticle30 July 2013free access The mitochondrial calcium uniporter is a multimer that can include a dominant-negative pore-forming subunit Anna Raffaello Anna Raffaello Department of Biomedical Sciences, University of Padua and CNR Neuroscience Institute, Padua, Italy Search for more papers by this author Diego De Stefani Diego De Stefani Department of Biomedical Sciences, University of Padua and CNR Neuroscience Institute, Padua, Italy Search for more papers by this author Davide Sabbadin Davide Sabbadin Molecular Modeling Section, Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Padua, Italy Search for more papers by this author Enrico Teardo Enrico Teardo Department of Biology, University of Padua, Padua, Italy Search for more papers by this author Giulia Merli Giulia Merli Department of Biomedical Sciences, University of Padua and CNR Neuroscience Institute, Padua, Italy Search for more papers by this author Anne Picard Anne Picard Department of Biomedical Sciences, University of Padua and CNR Neuroscience Institute, Padua, Italy Search for more papers by this author Vanessa Checchetto Vanessa Checchetto Department of Biomedical Sciences, University of Padua and CNR Neuroscience Institute, Padua, Italy Search for more papers by this author Stefano Moro Stefano Moro Molecular Modeling Section, Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Padua, Italy Search for more papers by this author Ildikò Szabò Ildikò Szabò Department of Biology, University of Padua, Padua, Italy Search for more papers by this author Rosario Rizzuto Corresponding Author Rosario Rizzuto Department of Biomedical Sciences, University of Padua and CNR Neuroscience Institute, Padua, Italy Search for more papers by this author Anna Raffaello Anna Raffaello Department of Biomedical Sciences, University of Padua and CNR Neuroscience Institute, Padua, Italy Search for more papers by this author Diego De Stefani Diego De Stefani Department of Biomedical Sciences, University of Padua and CNR Neuroscience Institute, Padua, Italy Search for more papers by this author Davide Sabbadin Davide Sabbadin Molecular Modeling Section, Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Padua, Italy Search for more papers by this author Enrico Teardo Enrico Teardo Department of Biology, University of Padua, Padua, Italy Search for more papers by this author Giulia Merli Giulia Merli Department of Biomedical Sciences, University of Padua and CNR Neuroscience Institute, Padua, Italy Search for more papers by this author Anne Picard Anne Picard Department of Biomedical Sciences, University of Padua and CNR Neuroscience Institute, Padua, Italy Search for more papers by this author Vanessa Checchetto Vanessa Checchetto Department of Biomedical Sciences, University of Padua and CNR Neuroscience Institute, Padua, Italy Search for more papers by this author Stefano Moro Stefano Moro Molecular Modeling Section, Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Padua, Italy Search for more papers by this author Ildikò Szabò Ildikò Szabò Department of Biology, University of Padua, Padua, Italy Search for more papers by this author Rosario Rizzuto Corresponding Author Rosario Rizzuto Department of Biomedical Sciences, University of Padua and CNR Neuroscience Institute, Padua, Italy Search for more papers by this author Author Information Anna Raffaello1,‡, Diego De Stefani1,‡, Davide Sabbadin2, Enrico Teardo3, Giulia Merli1, Anne Picard1, Vanessa Checchetto1, Stefano Moro2, Ildikò Szabò3 and Rosario Rizzuto 1 1Department of Biomedical Sciences, University of Padua and CNR Neuroscience Institute, Padua, Italy 2Molecular Modeling Section, Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Padua, Italy 3Department of Biology, University of Padua, Padua, Italy ‡These authors contributed equally to this work. *Corresponding author. Department of Biomedical Sciences, University of Padova, Via G. Colombo 3, 35131 Padua, Italy. Tel.:+39 0498276061; Fax:+39 0498276049; E-mail: [email protected] The EMBO Journal (2013)32:2362-2376https://doi.org/10.1038/emboj.2013.157 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Mitochondrial calcium uniporter (MCU) channel is responsible for Ruthenium Red-sensitive mitochondrial calcium uptake. Here, we demonstrate MCU oligomerization by immunoprecipitation and Förster resonance energy transfer (FRET) and characterize a novel protein (MCUb) with two predicted transmembrane domains, 50% sequence similarity and a different expression profile from MCU. Based on computational modelling, MCUb includes critical amino-acid substitutions in the pore region and indeed MCUb does not form a calcium-permeable channel in planar lipid bilayers. In HeLa cells, MCUb is inserted into the oligomer and exerts a dominant-negative effect, reducing the [Ca2+]mt increases evoked by agonist stimulation. Accordingly, in vitro co-expression of MCUb with MCU drastically reduces the probability of observing channel activity in planar lipid bilayer experiments. These data unveil the structural complexity of MCU and demonstrate a novel regulatory mechanism, based on the inclusion of dominant-negative subunits in a multimeric channel, that underlies the fine control of the physiologically and pathologically relevant process of mitochondrial calcium homeostasis. Introduction The notion that energized mitochondria accumulate Ca2+ in the matrix dates half a century ago, even before the chemiosmotic theory postulated the generation by the mitochondrial respiratory chain of an electrochemical gradient, negative inside, that provides the thermodynamic basis for cation accumulation into the matrix (Deluca and Engstrom, 1961). In the following years, the fundamental transport mechanisms were characterized (for a review, see Berridge et al, 2003 and Carafoli, 2010). Ca2+ uptake was shown to occur through a low-affinity electrogenic mechanism (hence the name mitochondrial calcium uniporter, MCU), inhibited by Ruthenium Red and lanthanides and was likely to be a channel (as directly demonstrated in 2004 by patch-clamp measurements; Kirichok et al, 2004). In the last two decades, the direct measurement of [Ca2+] in the matrix ([Ca2+]mt) with recombinant targeted indicators (Rizzuto et al, 1992) demonstrated that mitochondria, upon cell stimulation, rapidly accumulate Ca2+ up to concentrations >100 μM (Montero et al, 2000). The apparent discrepancy with the low affinity of MCU was solved by the demonstration that mitochondria are located in close proximity to the Ca2+ source and exposed to microdomains of high [Ca2+] (Rizzuto et al, 1993, 1998; Csordas et al, 1999, 2010; Giacomello et al, 2010). As to the role of mitochondrial Ca2+ homeostasis, it became soon clear that the [Ca2+]mt changes modulate key cellular processes, such as aerobic metabolism (through Ca2+-sensitive dehydrogenases; McCormack et al, 1990; Denton, 2009) and the release of pro-apoptotic factors (Szalai et al, 1999 Pinton et al, 2001; Scorrano et al, 2003). On the cytosolic side, mitochondria act as large-capacity Ca2+ buffers that are responsible for compartmentalization of Ca2+ increases (Tinel et al, 1999) as well as local modulation of the activity of channels and enzymes (Boitier et al, 1999; Hajnoczky et al, 1999; Gilabert and Parekh, 2000; Hoth et al, 2000). These observations restored a great interest in mitochondria in the signalling field that was however frustrated by the lack of molecular insight into the process. The past 2 years have witnessed the molecular unveiling of mitochondrial Ca2+ homeostasis. In 2010, the main efflux pathway (the NCX) (Palty et al, 2010), as well as a protein (named MICU1), that although not being itself a channel, appeared necessary for mitochondrial Ca2+ uptake (Perocchi et al, 2010) were identified. In 2011, a protein (named MCU) was identified (Baughman et al, 2011; De Stefani et al, 2011) and was shown, in intact cells and in reconstitution experiments, to be necessary and sufficient for channel activity with electrophysiological properties and inhibitor sensitivity of the MCU (De Stefani et al, 2011). Thus, the long awaited molecules corresponding to the uniporter and the exchangers characterized in the sixties and seventies were set in place, but fundamental issues remained to be solved, that in the case of MCU appeared truly critical. How can MCU, that possesses only two predicted transmembrane domains, form a functional channel? Genomic analysis identified a gene closely related to MCU: is this gene functional? And does the encoded protein play a role in MCU function? In this contribution, we addressed all these issues, with the aim of obtaining a comprehensive understanding of this novel channel that has no similarity to all other known calcium channels. Overall, the data show that the physiological activity of MCU is compatible with the interaction of functional and inactive pore-forming subunits, a unique regulatory mechanism allowing a great plasticity in the control of the fundamental process of mitochondrial Ca2+ uptake. Results The MCU isogene Sequence analysis of MCU (i.e., the gene originally named Ccdc109a) identified a related gene (Ccdc109b, NCBI GeneID 66815) located on Mus musculus chromosome 3 (chromosome 4 for the Homo sapiens orthologue). The gene is present in vertebrates but absent in other organisms in which MCU is present (e.g., plants, kinetoplastids, Nematoda, and Arthropoda). The encoded protein (∼330 amino acids long) is highly conserved among all species and shares a 50% similarity with MCU. It has two predicted transmembrane domains similar in sequence to MCU, although some conserved differences in the primary sequence are present (Figure 1A). RT–PCR analysis of HeLa cells and of a panel of mouse tissues reveals that CCDC109B has a lower expression level and a different expression profile from MCU (Figure 1B–D). Indeed, the mRNA encoded by the CCDC109B gene (hereafter named MCUb) is expressed at a ratio with MCU (MCU/MCUb) that, based on the RT–PCR data, varies from ∼3:1 (e.g., heart or lung) to >40:1 (skeletal muscle). We thus cloned and expressed the protein in HeLa cells. Immunofluorescence of transfected cells shows a complete overlap with MCU and the mitochondrial marker HSP60 (Figure 1E). However, the lack of any structural data about the native structure of the channel seriously limits all hypotheses on ion permeation through the channel. To circumvent this problem, we developed an in silico comparative model of the pore domain of the MCU. Figure 1.The MCU isogene. (A) Multiple alignment of the TM1, L1, and TM2 regions of MCU (red) and MCUb (green) in seven different species. Blue boxes show the two critical conserved substitutions. (B–D) Quantitative real-time PCR analysis of HeLa cells and mouse tissues of MCU and MCUb. (B) MCU and MCUb relative expression in HeLa cells. (C) MCU and (D) MCUb relative expression in the indicated mouse tissues as described in Materials and methods. All values are normalized to the indicated housekeeping genes. (E) Immunolocalization of MCUb. HeLa cells were transfected with MCUb-6 × His and MCU-Flag. After 24 h, the cells were fixed and immunocytochemistry was performed with α-Flag, α-6 × His, and α-HSP60 antibodies followed by incubation with Alexa488-, Alexa555-, and Alexa647-conjugated secondary antibodies as described in Materials and methods. Confocal images were taken (scale bar: 10 μm), and a region is expanded to appreciate co-localization (scale bar: 1 μm). Download figure Download PowerPoint Predicted quaternary structure of the MCU The combination of structural bioinformatics techniques and molecular dynamics (MD) simulations provides hypothesis of ion channel topologies for which the three-dimensional structure is yet unknown and of their behaviour in a lipid bilayer environment, in particular regarding the mechanism of ion permeation. We thus developed a comparative model of the pore domain of the MCU, and used it for membrane MD simulations on a nanosecond scale, as described in Materials and methods. Briefly, a multiple-template approach has been used to identify the possible structural organization. Four-fold rotational symmetry was imposed to the oligomer construction, as suggested by most of the available crystallographic data. A refinement procedure to optimize the quaternary assembly was carried out evaluating the best surface complimentary among each subunit using a protein–protein docking approach. The three-dimensional averaged structure obtained from the last 5 ns of MD simulation of membrane-embedded MCU model and the starting conformation of the channel protein have been used to investigate the effect of a membrane-like environment on modulating tetramer packing and its effect on the circumscribed aqueous pore topology evolution. The sequence identity between MCU and all crystallized ion channels is rather low, so their initial alignment was adjusted to maximize overlap between the predicted locations of the TM helices in MCU and their locations in the X-ray structure of different templates. The final hypothetical model of the MCU pore domain linked to its C-terminus (residues 224–334) includes four identical subunits (Figure 2A), composed of two helical membrane spanning domains, connected by a short loop containing a DIME motif (Figure 2B). In particular, the region between R226 and W255 constitutes the first membrane spanning domain (TM1), whereas residues from Y267 to Y290 are part of the second helical segment (TM2), which protrudes outside the membrane region forming a long water exposed helical tail, as shown in Figure 2B. Finally, the region between E256 and T266 constitutes the water exposed loop (L1) where the DIME motif is located. This region includes a few negatively charged amino acids (such as D260 and E263) that have been shown to play an essential role in MCU-mediated mitochondrial Ca2+ uptake (Figure 2C) (Baughman et al, 2011; De Stefani et al, 2011). The three-dimensional averaged structure obtained from the last 5 ns of MD simulation of a membrane-embedded MCU model reveals the presence of a narrow selectivity filter constituted by the conserved acidic residues cited above which are able to locate a single Ca2+ above the channel pore region (Figure 2D). This feature is connected to a progressively widening chamber (3–5 Å in diameter), which extends underneath the channel mouth, through all the MCU membrane spanning domains. Surprisingly, the C-terminal tails, which are TM2 extensions directed through the solvent, define a pore of ∼1 Å wide in diameter suggesting that our MCU model is likely to stably adopt a closed state conformation, at least in the nanoseconds time scale. Such a pore topology is common among Ca2+ channels (Corry et al, 2001). Figure 2.Predicted quaternary structure of MCU. (A) Top view of the pore region of the predicted MCU tetramer. (B) Representation of the MCU model inserted into a POPC lipid bilayer. Indicated amino acids locate the boundaries among TM1, L1, and TM2. Zoomed region: E263 and D260 side chains face the pore region of the channel. E256 and T266 interaction is critical for loop conformation and dynamics. W255 and Y267 locate the upper boundaries of TM1 and TM2, respectively. N and C-terminal portion of the MCU monomers is highlighted according to the reported colour gradient bar. Chlorine and calcium ions are depicted as green and yellow spheres, respectively. (C) Electrostatic properties surface distribution of MCU. (D) Comparison of the pore width before (left) and after (right) insertion and equilibration into a lipid bilayer. The central panel shows the calculated width along the pore (before, grey trace; after, purple trace). Predicted MCU pore surface is depicted using red, green, and violet marks. Red: pore radius (R) is below 0.6 Å, green: 0.6 Å<R<1.15 Å and blue marks place where R is above 1.15 Å. Download figure Download PowerPoint We then looked for experimental confirmation of the proposed oligomeric structure. Three approaches were followed. In the first, two different tags were added to MCU (MCU-GFP and MCU-HA) and the modified MCU constructs were recombinantly expressed in HeLa cells. Thirty-six hours after transfection, the cells were lysed and immunoprecipitation was carried out with the α-HA antibody. When MCU-GFP and MCU-HA were co-expressed, the α-HA antibody immunoprecipitated MCU-GFP (Figure 3A), thus revealing the interaction in situ of MCU monomers in a higher order complex. Then, we looked for confirmation of this result in a Förster resonance energy transfer (FRET) experiment in living cells (Figure 3B). For this purpose, an MCU-GFP (donor) and an MCU-mCherry (acceptor) chimera were generated and compared with two non-interacting fluorophores (GFP and mCherry). These two chimeras proved to be properly folded and functional (Supplementary Figure S1A). FRET was evaluated by emission spectrum analysis and acceptor photobleaching. In the first set of experiments, HeLa cells were transfected with the MCU-GFP or MCU-mCherry expression plasmids. Fluorescence emission spectra were recorded in the 470–700 nm range by exciting the donor (GFP) with the 458-nm laser line, in order to minimize (<1%) the cross-excitation of the acceptor (mCherry), while donor-only transfected cells show the expected spectrum of the GFP (Figure 3B). In contrast, cells cotransfected with both MCU-GFP and MCU-mCherry clearly show a secondary emission peak at 615 nm, due to the energy transfer between donor and acceptor molecules (Figure 3B). We confirmed the occurrence of FRET by monitoring fluorescence acceptor photobleaching in MCU-GFP and MCU-mCherry expressing HeLa cells. mCherry was bleached in a defined region with a 592-nm high power laser, and the changes in MCU-GFP (excitation at 488 nm) and MCU-mCherry (excitation at 543 nm) fluorescence were measured. When FRET occurs, acceptor photobleaching leads to the de-quenching and the consequent increase in donor fluorescence. Thus, FRET was calculated as the normalized increase in donor fluorescence after acceptor bleaching. Figure 3C shows a representative experiment: when MCU-GFP and MCU-mCherry were co-expressed, a significant FRET occurred (9.333±3.256%), whereas minimal FRET was detected when GFP and mCherry were not fused to MCU (0.763±0.980%). The calculated efficiency is in line with other reports using the same FRET pair (van der Krogt et al, 2008; Goh et al, 2011) and does not correlate with the expression level of the fluorescent proteins. Finally, we loaded in a native gel and immunoblotted in vitro translated MCU (De Stefani et al, 2011), detecting a band at the expected molecular weight of the monomer (40 kDa) and a higher band at 170 kDa (reactive with 6 × His antibody), which is compatible with a tetramer (Figure 4C, left panel), indicating that MCU monomers oligomerize both in vitro and in vivo in higher order complexes, and thus support the tetrameric model of the computational analysis. Figure 3.MCU forms oligomers in vitro and in vivo. (A) Co-immunoprecipitation experiments. HeLa cells were transfected with the indicated constructs. HA-tagged MCU was immunoprecipitated from cell extracts with a specific α-HA antibody. The precipitated proteins were immunoblotted with α-HA and α-GFP antibodies. (B) Emission spectra analysis of HeLa cells transfected with MCU-GFP or MCU-GFP and MCU-mCherry and analysed after 24 h. (C) FRET analysis. HeLa cells were transfected with GFP and mCherry or MCU-GFP and MCU-mCherry and analysed after 24 h. Images of donor and acceptor were taken before and after photobleaching the indicated region (white box). FRET was calculated as detailed in Materials and methods. Histogram bar diagram shows FRET efficiency of the indicated donor and acceptor pairs. Descriptive statistics can be found in Supplementary Table S1. Download figure Download PowerPoint Figure 4.MCU and MCUb form hetero-oligomers. (A) Co-immunoprecipitation experiments. HeLa cells were infected with the indicated adenoviruses. Flag-tagged MCU was immunoprecipitated from cell extracts with a specific α-Flag antibody. The co-immunoprecipitated proteins were immunoblotted with α-Flag and α-6 × His antibodies. (B) FRET analysis. HeLa cells were transfected with MCU-GFP and MCUb-mCherry and analysed after 24 h. Images of donor and acceptor were taken before and after photobleaching of the indicated region (white box). FRET was calculated as detailed in Materials and methods. Histogram bar diagram shows FRET efficiency of the indicated donor and acceptor pairs. Descriptive statistics can be found in Supplementary Table S1. (C) In vitro expression. wheat germ lysate expressing MCU-6 × His or MCUb-StrepTag alone and co-expressing MCU-6 × His/MCUb-StrepTag (2:2 ratio) was loaded on a native polyacrylamide gel without denaturing the samples. Blots were developed with anti-6 × His and anti-StrepTag antibodies. Download figure Download PowerPoint MCU and MCUb form hetero-oligomers In view of the proposed oligomeric structure of MCU and given the predicted structural similarity with MCUb, we investigated whether the two proteins interact within the MCU oligomer with the same approach employed in Figure 3. At first, MCU-Flag and MCUb-6 × His were expressed in HeLa cells, and the α-Flag antibody immunoprecipitated also MCUb-6 × His, thus revealing the in situ interaction of MCU and MCUb (Figure 4A). We then carried out FRET analysis of the interaction, by generating and imaging different combinations of GFP- and mCherry-tagged MCU and MCUb proteins. As for MCU, also MCUb chimeras proved to be functional as they affected mitochondrial calcium uptake in intact cells (Supplementary Figure S1B). FRET was evaluated by acceptor photobleaching as in Figure 3. Representative fluorescence images of the MCU-GFP (donor) and MCUb-mCherry (acceptor) pair are shown in Figure 4B. A significant FRET was observed (8.090±3.700%), with an efficiency very similar to that generated by the MCU self-oligomerization. Similar results were obtained by switching donor and acceptor (with MCUb-GFP as a donor and MCU-mCherry as an acceptor, FRET efficiency is 9.029±4.151%). Importantly, a detectable, but lower FRET was measured also between MCUb monomers, using an MCUb-GFP and MCUb-mCherry pair (3.831±1.660%), thus indicating that MCUb also can self-oligomerize (Figure 4B). This experiment was repeated in cells silenced for MCU in order to exclude the indirect interaction due to the endogenous MCU (Supplementary Figure S2). Finally, wheat germ lysate expressing MCU or MCUb alone and co-expressing MCU/MCUb was loaded on a native polyacrylamide gel without denaturing the samples, clearly showing MCU and MCUb monomers at the expected molecular weight (40 kDa) and a higher band (170 kDa) compatible with a tetramer and reactive with both anti-6 × His (for MCU) and anti-StrepTag (for MCUb) antibodies (Figure 4C). MCUb acts as an endogenous dominant-negative MCU subunit We then investigated the function of MCUb. First, we obtained indication of an altered ion permeation pathway from molecular modelling. From a structural point of view, crucial differences between MCU and MCUb are located in the ‘DIME motif’ such as the replacement with a valine of one of the three conserved negatively charged residues of the N-terminal portion of the loop region (V251, corresponding to E256 in MCU as also depicted in Figure 1A, blue boxes). This crucial E256V substitution in MCUb might have an important impact on the kinetic of Ca2+ permeation as expected by the comparison of the surface electrostatic potentials between MCU (more negatively charged) and MCUb (less negatively charged). Based on the computational model, MCUb was thus expected to be poorly permeable to Ca2+. We tested this prediction by two different approaches: (i) in vitro analysis of MCUb channel properties and (ii) in situ investigation of the role of MCUb in mitochondrial Ca2+ handling. In the first case, the MCUb protein was either produced in vitro or expressed in E. coli (Figure 5A and B), purified and inserted in planar lipid bilayers, then electrophysiological recordings were carried out (Figure 5C). Under our recording conditions, in 100 mM calcium-gluconate, no channel activity was detected upon addition of purified MCUb into the cis chamber, whereas the subsequent addition of MCU to the same membrane gave rise to channel activity with a conductance of 7 pS, typical of MCU (Figure 5C). The lack of channel activity might have been due to misfolding of MCUb. To prove that this was not the case, we recorded the activity of the same protein preparation in a sodium-gluconate low divalent solution (10 pM calculated [Ca2+]), given the known characteristic of calcium channels (Hess and Tsien, 1984; Lepple-Wienhues and Cahalan, 1996; Talavera and Nilius, 2006) and of MCU (Kirichok et al, 2004) to allow the passage of Na+ upon removal of Ca2+ (Supplementary Figure S3). Indeed, an Na+ current was observed indicating that MCUb gives rise to a functional channel, albeit incapable of significant Ca2+ permeation. Figure 5.MCUb has no channel activity in planar lipid bilayer. (A) In vitro expression of MCUb. Empty wheat germ lysate (WGL) and WGL after expression of MCUb-StrepTag were loaded on SDS–PAGE and blotted with α-StrepTag antibody. (B) Induction and purification of MCUb in E. coli. Bacteria were harvested after induction (T24) to check for the expression of the protein. Solubilized membranous fraction was passed through Strep-Tactin column; after washing (W1–W4), protein was eluted with 2.5 mM desthiobiotin (E1–E4). All samples were blotted and developed with α-StrepTag antibody. In all, 30 μl of eluted fractions/lane was loaded. (C) Electrophysiological recordings: in vitro expressed MCUb was added to the cis side (middle panel) and current was recorded for at least 10 min (n=5) without observing channel activity in 100 mM calcium-gluconate solution. Amplitude histograms, obtained from analysis of 50 s current traces recorded at −80 mV Vcis before (left panel) and 15 min after addition of MCUb (middle panel). Following addition of excess MCU (not incorporated into liposome) to the same experiment (right panel), spiky channel activity with a conductance of 7 pS has appeared (n=3). In the lower current trace, representative channel activity is shown in an extended time scale. The open probability of MCU was compatible with that previously reported for the channel recorded in the same condition (De Stefani et al, 2011). Lack of channel activity for MCUb in calcium-gluconate was also observed using the protein incorporated into liposomes (n=4). Download figure Download PowerPoint The lack of MCUb channel activity in calcium is compatible with MCUb being a dominant-negative form of MCU, similarly to the silent mutant subunits observed for various ion channels (Lafreniere et al, 2010; Jeanguenin et al, 2011). Addition of MCUb to active homomeric MCU, already incorporated into the bilayer, did not change either conductance or open probability and kinetic behaviour of the MCU channel (as expected, given that subunit switch is unlikely to occur in lipid bilayer experiments) (Figure 6A). Therefore, we co-expressed in vitro MCU and MCUb using ratios of plasmid DNA yielding different protein expression levels (Supplementary Figure S4A), selecting a plasmid ratio (MCUb:MCU=2:2 or 3:1) that gave near equimolar or 2:1 amounts of the two proteins. MCU-only, MCUb-only or the co-expressed proteins were incorporated into liposomes (Supplementary Figure S4B) and their activity was assessed in electrophysiological experiments (Figure 6B). When the two proteins were co-expressed, the number of experiments in which we observed MCU activity in calcium (due to the presence of homomeric MCU, statistically expected to be present in the co-expressed preparation) became drastically reduced to 13% compared to MCU alone (89%) under the same recording conditions (Figure 6C). In 13% of the experiments, we observed activity with the same conductance of the MCU homomer (7 pS). These data thus indicate that MCUb subunits, when forming heteromers with MCU alter calcium permeation across the heteromeric channel, thus acting as a bona fide dominant-negative subunit. Figure 6.MCU activity in the presence of MCUb. (A) Addition of excess MCUb during the same experiment does not alter the electrophysiological properties of MCU activity. Current traces recorded at −100 mV before and 6 min after addition are shown. Conductance values are 6.7 and 6.4 pS, respectively. Mean open time constants (280 ms for MCU and 360 ms after addition of MCUb) were similar. Below: respective amplitude histograms are shown. The open probability was 0.498 before and 0.513 after addition of MCUb. Similar results were obtained in two other experiments. (B) Activities observed with homomeric MCU (upper trace, representative of 8 experiments) or heteromeric MCU/MCUb (representative of 13 experiments) in liposome recorded at −140 mV are shown (middle trace). Lower current trace: in 2 cases out of 15 we recorded the activity shown using the heteromer preparation (3:1 ratio), which displayed the same characteristics as homomeric MCU. (C) Histogram showing the percenta
Referência(s)