Ildikò Szabò

A forty-kilodalton protein of the inner membrane is the mitochondrial calcium uniporter

Mitochondrial Ca2+ homeostasis has a key role in the regulation of aerobic metabolism and cell survival, but the molecular identity of the Ca2+ channel, the mitochondrial calcium uniporter, is still unknown. Here we have identified in silico a protein (named MCU) that shares tissue distribution with MICU1 (also known as CBARA1), a recently characterized uniporter regulator, is present in organisms in which mitochondrial Ca2+ uptake was demonstrated and whose sequence includes two transmembrane domains. Short interfering RNA (siRNA) silencing of MCU in HeLa cells markedly reduced mitochondrial Ca2+ uptake. MCU overexpression doubled the matrix Ca2+ concentration increase evoked by inositol 1,4,5-trisphosphate-generating agonists, thus significantly buffering the cytosolic elevation. The purified MCU protein showed channel activity in planar lipid bilayers, with electrophysiological properties and inhibitor sensitivity of the uniporter. A mutant MCU, in which two negatively charged residues of the putative pore-forming region were replaced, had no channel activity and reduced agonist-dependent matrix Ca2+ concentration transients when overexpressed in HeLa cells. Overall, these data demonstrate that the 40-kDa protein identified is the channel responsible for ruthenium-red-sensitive mitochondrial Ca2+ uptake, thus providing a molecular basis for this process of utmost physiological and pathological relevance.

Dimers of mitochondrial ATP synthase form the permeability transition pore

Here we define the molecular nature of the mitochondrial permeability transition pore (PTP), a key effector of cell death. The PTP is regulated by matrix cyclophilin D (CyPD), which also binds the lateral stalk of the FOF1 ATP synthase. We show that CyPD binds the oligomycin sensitivity-conferring protein subunit of the enzyme at the same site as the ATP synthase inhibitor benzodiazepine 423 (Bz-423), that Bz-423 sensitizes the PTP to Ca2+ like CyPD itself, and that decreasing oligomycin sensitivity-conferring protein expression by RNAi increases the sensitivity of the PTP to Ca2+. Purified dimers of the ATP synthase, which did not contain voltage-dependent anion channel or adenine nucleotide translocator, were reconstituted into lipid bilayers. In the presence of Ca2+, addition of Bz-423 triggered opening of a channel with currents that were typical of the mitochondrial megachannel, which is the PTP electrophysiological equivalent. Channel openings were inhibited by the ATP synthase inhibitor AMP-PNP (γ-imino ATP, a nonhydrolyzable ATP analog) and Mg2+/ADP. These results indicate that the PTP forms from dimers of the ATP synthase.

Formation of anion‐selective channels in the cell plasma membrane by the toxin VacA of Helicobacter pylori is required for its biological activity

The vacuolating toxin VacA, a major determinant of Helicobacter pylori‐associated gastric diseases, forms anion‐selective channels in artificial planar lipid bilayers. Here we show that VacA increases the anion permeability of the HeLa cell plasma membrane and determines membrane depolarization. Electrophysiological and pharmacological approaches indicated that this effect is due to the formation of low‐conductance VacA pores in the cell plasma membrane and not to the opening of Ca2+‐ or volume‐activated chloride channels. VacA‐dependent increase of current conduction both in artificial planar lipid bilayers and in the cellular system was effectively inhibited by the chloride channel blocker 5‐nitro‐2‐(3‐phenylpropylamino) benzoic acid (NPPB), while2‐[(2‐cyclopentenyl‐6,7dichloro‐2,3‐dihydro‐2‐methyl‐1‐oxo‐1H‐inden‐5‐yl)oxy]acetic acid (IAA‐94) was less effective. NPPB inhibited and partially reversed the vacuolation of HeLa cells and the increase of ion conductivity of polarized Madine Darby canine kidney cell monolayers induced by VacA, while IAA‐94 had a weaker effect. We conclude that pore formation by VacA accounts for plasma membrane permeabilization and is required for both cell vacuolation and increase of trans‐epithelial conductivity.

Cell volume in the regulation of cell proliferation and apoptotic cell death.

Cell proliferation must – at some time point – lead to increase of cell volume and one of the hallmarks of apoptosis is cell shrinkage. At constant extracellular osmolarity those alterations of cell volume must reflect respective changes of cellular osmolarity which are hardly possible without the participation of cell volume regulatory mechanisms. Indeed, as shown for ras oncogene expressing 3T3 fibroblasts, cell proliferation is paralleled by activation of Na+/H+ exchange and Na+,K+,2Cl- cotransport, the major transport systems accomplishing regulatory cell volume increase. Conversely, as evident from CD95-induced apoptotic cell death, apoptosis is paralleled by inhibition of Na+/H+ exchanger and by activation of Cl- channels and release of the organic osmolyte taurine, major components of regulatory cell volume decrease. However, ras oncogene activation leads to activation and CD95 receptor triggering to inhibition of K+ channels. The effects counteract the respective cell volume changes. Presumably, they serve to regulate cell membrane potential, which is decisive for Ca++ entry through ICRAC and the generation of cytosolic Ca++ oscillations in proliferating cells. As a matter of fact ICRAC is activated in ras oncogene expressing cells and inhibited in CD95-triggered cells. Activation of K+ channels and Na+/H+ exchanger as well as Ca++ oscillations have been observed in a wide variety of cells upon exposure to diverse mitogenic factors. Conversely, diverse apoptotic factors have been shown to activate Cl- channels and organic osmolyte release. Inhibition of K+ channels is apparently, however, not a constant phenomenon paralleling apoptosis which in some cells may even require the operation of K+ channels. Moreover, cell proliferation may at some point require activation of Cl- channels. In any case, the alterations of cell volume are obviously important for the outcome, as cell shrinkage impedes cell proliferation and apoptosis can be elicited by increase of extracellular osmolarity. At this stage little is known about the interplay of cell volume regulatory mechanisms and the cellular machinery leading to mitosis or death of the cell. Thus, considerable further experimental effort is required in this exciting area of cell physiology.

Tyrosine Phosphorylation-dependent Suppression of a Voltage-gated K+ Channel in T Lymphocytes upon Fas Stimulation

Selective cell death plays a critical role in the development of the immune system and in the elimination of target cells expressing foreign antigens. Most of programmed cell death occurs by apoptosis. Apoptotic cell death of lymphocytes can be triggered by ligation of APO-1/Fas (CD95) antigen (Suda, T., and Nagata, S. (1994) J. Exp. Med. 179, 873-879; Nagata, S., and Golstein, P. (1995) Science 267, 1449-1456). We find that activation of Fas leads to the inhibition of the voltage-dependent n-type K+ channels (Kv1.3) studied by patch clamp technique in Jurkat T lymphocytes. Tyrosine kinases have been shown to be crucial in Fas-induced cell death (Eischen, C. M., Dick, C. J., and Leibson, P. J. (1994) J. Immunol. 153, 1947-1954). The inhibition of the current is correlated with the tyrosine phosphorylation of immunoprecipitated and blotted K+ channel protein. We show, that the Src-like protein-tyrosine kinase inhibitor herbimycin A and the deficiency of the p56lck tyrosine kinase in mutant Jurkat cells abolished the channel inhibition and phosphorylation by anti-Fas antibody, while reconstitution of the p56lck kinase partly restored these effects of Fas receptor triggering. These results suggest a regulation of n-type K+ channels by tyrosine kinases upon Fas receptor triggering, which might be important for apoptosis.

The mitochondrial megachannel is the permeability transition pore

Single-channel electrophysiological recordings from rat liver mitoplast membranes showed that the 1.3-nS mitochondrial megachannel was activated by Ca++ and inhibited by Mg++, Cyclosporin A, and ADP, probably acting at matrix-side sites. These agents are known to modulate the so-called mitochondrial permeability transition pore (Gunter, T. E., and Pfeiffer, D. R. (1990)Am. J. Physiol.258, C755–C786) in the same manner. Furthermore, the megachannel is unselective, and the minimum pore size calculated from its conductance is in agreement with independent estimates of the minimum size of the permeabilization pore. The results support the tentative identification of the megachannel with the pore believed to be involved in the permeabilization process.

The mitochondrial calcium uniporter is a multimer that can include a dominant‐negative pore‐forming subunit

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.

MICU1 and MICU2 Finely Tune the Mitochondrial Ca2+ Uniporter by Exerting Opposite Effects on MCU Activity

Summary Mitochondrial calcium accumulation was recently shown to depend on a complex composed of an inner-membrane channel (MCU and MCUb) and regulatory subunits (MICU1, MCUR1, and EMRE). A fundamental property of MCU is low activity at resting cytosolic Ca2+ concentrations, preventing deleterious Ca2+ cycling and organelle overload. Here we demonstrate that these properties are ensured by a regulatory heterodimer composed of two proteins with opposite effects, MICU1 and MICU2, which, both in purified lipid bilayers and in intact cells, stimulate and inhibit MCU activity, respectively. Both MICU1 and MICU2 are regulated by calcium through their EF-hand domains, thus accounting for the sigmoidal response of MCU to [Ca2+] in situ and allowing tight physiological control. At low [Ca2+], the dominant effect of MICU2 largely shuts down MCU activity; at higher [Ca2+], the stimulatory effect of MICU1 allows the prompt response of mitochondria to Ca2+ signals generated in the cytoplasm.

The inner mitochondrial membrane contains ion-conducting channels similar to those found in bacteria

Patch‐clamp experiments were performed on rat liver mitochondria inner membranes. Application of voltage gradients of either polarity revealed the presence of several different conductances, ranging up to 1.3 nS in symmetrical 150 mM KCl. Evidence is presented that at least those higher than 0.3 nS are substates of the highest conductance channel. Increasing matrix‐side‐positive (unphysiological) transmembrane voltage gradients favored the switch of the 1.3 nS channel to operation in lower conductance states. The size of these conductances, the presence of substates and the channel behavior are strongly reminiscent on one hand of the observations on the membrane of protoplasts from the gram‐positive bacterium Streptococcus faecalis, [Zoratti M. and Petronilli V. (1988) FEBS Lett. 240, 105‐109], and on the other of some properties of previously described channels of mitochondrial origin.

Light and oxygenic photosynthesis: energy dissipation as a protection mechanism against photo‐oxidation

Efficient photosynthesis is of fundamental importance for plant survival and fitness. However, in oxygenic photosynthesis, the complex apparatus responsible for the conversion of light into chemical energy is susceptible to photodamage. Oxygenic photosynthetic organisms have therefore evolved several protective mechanisms to deal with light energy. Rapidly inducible non‐photochemical quenching (NPQ) is a short‐term response by which plants and eukaryotic algae dissipate excitation energy as heat. This review focuses on recent advances in the elucidation of the molecular mechanisms underlying this protective quenching pathway in higher plants.