Vanessa Checchetto

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.

Thylakoid potassium channel is required for efficient photosynthesis in cyanobacteria

A potassium channel (SynK) of the cyanobacterium Synechocystis sp. PCC 6803, a photoheterotrophic model organism for the study of photosynthesis, has been recently identified and demonstrated to function as a potassium selective channel when expressed in a heterologous system and to be located predominantly to the thylakoid membrane in cyanobacteria. To study its physiological role, a SynK-less knockout mutant was generated and characterized. Fluorimetric experiments indicated that SynK-less cyanobacteria cannot build up a proton gradient as efficiently as WT organisms, suggesting that SynK might be involved in the regulation of the electric component of the proton motive force. Accordingly, measurements of flash-induced cytochrome b6f turnover and respiration pointed to a reduced generation of ΔpH and to an altered linear electron transport in mutant cells. The lack of the channel did not cause an altered membrane organization, but decreased growth and modified the photosystem II/photosystem I ratio at high light intensities because of enhanced photosensitivity. These data shed light on the function of a prokaryotic potassium channel and reports evidence, by means of a genetic approach, on the requirement of a thylakoid ion channel for optimal photosynthesis.

F-ATPase of Drosophila melanogaster forms 53-picosiemen (53-pS) channels responsible for mitochondrial Ca2+-induced Ca2+ release.

Background: The Ca2+-induced Ca2+ release channel (mCrC) of Drosophila mitochondria is similar to the permeability transition pore (PTP). Results: mCrC is modulated by PTP effectors and Drosophila F-ATPase forms 53-pS channels. Conclusion: F-ATPase mediates Ca2+-induced Ca2+ release in Drosophila mitochondria. Significance: Channel formation by F-ATPases has been conserved in evolution, but species-specific features exist that may underscore different roles in different organisms. Mitochondria of Drosophila melanogaster undergo Ca2+-induced Ca2+ release through a putative channel (mCrC) that has several regulatory features of the permeability transition pore (PTP). The PTP is an inner membrane channel that forms from F-ATPase, possessing a conductance of 500 picosiemens (pS) in mammals and of 300 pS in yeast. In contrast to the PTP, the mCrC of Drosophila is not permeable to sucrose and appears to be selective for Ca2+ and H+. We show (i) that like the PTP, the mCrC is affected by the sense of rotation of F-ATPase, by Bz-423, and by Mg2+/ADP; (ii) that expression of human cyclophilin D in mitochondria of Drosophila S2R+ cells sensitizes the mCrC to Ca2+ but does not increase its apparent size; and (iii) that purified dimers of D. melanogaster F-ATPase reconstituted into lipid bilayers form 53-pS channels activated by Ca2+ and thiol oxidants and inhibited by Mg2+/γ-imino ATP. These findings indicate that the mCrC is the PTP of D. melanogaster and that the signature conductance of F-ATPase channels depends on unique structural features that may underscore specific roles in different species.

VDAC3 as a sensor of oxidative state of the intermembrane space of mitochondria: the putative role of cysteine residue modifications

Voltage-Dependent Anion selective Channels (VDAC) are pore-forming mitochondrial outer membrane proteins. In mammals VDAC3, the least characterized isoform, presents a set of cysteines predicted to be exposed toward the intermembrane space. We find that cysteines in VDAC3 can stay in different oxidation states. This was preliminary observed when, in our experimental conditions, completely lacking any reducing agent, VDAC3 presented a pattern of slightly different electrophoretic mobilities. This observation holds true both for rat liver mitochondrial VDAC3 and for recombinant and refolded human VDAC3. Mass spectroscopy revealed that cysteines 2 and 8 can form a disulfide bridge in native VDAC3. Single or combined site-directed mutagenesis of cysteines 2, 8 and 122 showed that the protein mobility in SDS-PAGE is influenced by the presence of cysteine and by the redox status. In addition, cysteines 2, 8 and 122 are involved in the stability control of the pore as shown by electrophysiology, complementation assays and chemico-physical characterization. Furthermore, a positive correlation between the pore conductance of the mutants and their ability to complement the growth of porin-less yeast mutant cells was found. Our work provides evidence for a complex oxidation pattern of a mitochondrial protein not directly involved in electron transport. The most likely biological meaning of this behavior is to buffer the ROS load and keep track of the redox level in the inter-membrane space, eventually signaling it through conformational changes.

A Novel Potassium Channel in Photosynthetic Cyanobacteria

Elucidation of the structure-function relationship of a small number of prokaryotic ion channels characterized so far greatly contributed to our knowledge on basic mechanisms of ion conduction. We identified a new potassium channel (SynK) in the genome of the cyanobacterium Synechocystis sp. PCC6803, a photosynthetic model organism. SynK, when expressed in a K+-uptake-system deficient E.coli strain, was able to recover growth of these organisms. The protein functions as a potassium selective ion channel when expressed in Chinese Hamster Ovary cells. The location of SynK in cyanobacteria in both thylakoid and plasmamembranes was revealed by immunogold electron microscopy and Western blotting of isolated membrane fractions. SynK seems to be conserved during evolution, giving rise to a TPK (two-pore K+ channel) family member which is shown here to be located in the thylakoid membrane of Arabidopsis. Our work characterizes a novel cyanobacterial potassium channel and indicates the molecular nature of the first higher plant thylakoid cation channel, opening the way to functional studies.

Recombinant Human Voltage Dependent Anion Selective Channel Isoform 3 (hVDAC3) Forms Pores with a Very Small Conductance

Background/Aims: Voltage-dependent anion channels (VDAC), also known as eukaryotic porins, are located in the outer mitochondrial membrane and allow the flux of ions and small metabolites. While the pore-forming ability of recombinant VDAC1 and VDAC2 has been extensively studied during the last decades, a clear-cut ion conducting channel activity has not been assigned to the VDAC3 isoform. Methods: Electrophysiological characterization of the recombinant protein purified and refolded was obtained after incorporation into planar lipid bilayers. Results: Here we report for the first time that recombinant hVDAC3, upon expression in E.coli and purification-refolding, shows a channel activity with a very small conductance (90 pS in 1 M KCl) with respect to the conductance of hVDAC1 (>3500 pS in 1 M KCl). Purified hVDAC3 allowed the passage of both chloride and gluconate anions and did not distinguish between potassium, sodium and calcium used as cations. In contrast to VDAC1, the channel was active also at transmembrane voltages higher than +/-40 mV and displayed a relatively high open probability even at +/-80 mV. hVDAC3 was only slightly voltage-dependent, displaying a tendency to adopt lower-conductance states at positive voltages applied to the cis chamber. In accordance with the small conductance of the pore, expression of hVDAC3 in a porin-less yeast strain allowed only partial recovery of the growth under non-permissive conditions. Conclusion: The observed electrophysiological properties of hVDAC3 are surprisingly different from the other isoforms and are discussed in relation to the proposed physiological role of the protein in mammalian cells.

Ion Channels in Plant Bioenergetic Organelles, Chloroplasts and Mitochondria: From Molecular Identification to Function

Recent technical advances in electrophysiological measurements, organelle-targeted fluorescence imaging, and organelle proteomics have pushed the research of ion transport a step forward in the case of the plant bioenergetic organelles, chloroplasts and mitochondria, leading to the molecular identification and functional characterization of several ion transport systems in recent years. Here we focus on channels that mediate relatively high-rate ion and water flux and summarize the current knowledge in this field, focusing on targeting mechanisms, proteomics, electrophysiology, and physiological function. In addition, since chloroplasts evolved from a cyanobacterial ancestor, we give an overview of the information available about cyanobacterial ion channels and discuss the evolutionary origin of chloroplast channels. The recent molecular identification of some of these ion channels allowed their physiological functions to be studied using genetically modified Arabidopsis plants and cyanobacteria. The view is emerging that alteration of chloroplast and mitochondrial ion homeostasis leads to organelle dysfunction, which in turn significantly affects the energy metabolism of the whole organism. Clear-cut identification of genes encoding for channels in these organelles, however, remains a major challenge in this rapidly developing field. Multiple strategies including bioinformatics, cell biology, electrophysiology, use of organelle-targeted ion-sensitive probes, genetics, and identification of signals eliciting specific ion fluxes across organelle membranes should provide a better understanding of the physiological role of organellar channels and their contribution to signaling pathways in plants in the future.

Plasma Membrane Aquaporin AqpZ Protein Is Essential for Glucose Metabolism during Photomixotrophic Growth of Synechocystis sp. PCC 6803

The genome of Synechocystis PCC 6803 contains a single gene encoding an aquaporin, aqpZ. The AqpZ protein functioned as a water-permeable channel in the plasma membrane. However, the physiological importance of AqpZ in Synechocystis remains unclear. We found that growth in glucose-containing medium inhibited proper division of ΔaqpZ cells and led to cell death. Deletion of a gene encoding a glucose transporter in the ΔaqpZ background alleviated the glucose-mediated growth inhibition of the ΔaqpZ cells. The ΔaqpZ cells swelled more than the wild type after the addition of glucose, suggesting an increase in cytosolic osmolarity. This was accompanied by a down-regulation of the pentose phosphate pathway and concurrent glycogen accumulation. Metabolite profiling by GC/TOF-MS of wild-type and ΔaqpZ cells revealed a relative decrease of intermediates of the tricarboxylic acid cycle and certain amino acids in the mutant. The changed levels of metabolites may have been the cause for the observed decrease in growth rate of the ΔaqpZ cells along with decreased PSII activity at pH values ranging from 7.5 to 8.5. A mutant in sll1961, encoding a putative transcription factor, and a Δhik31 mutant, lacking a putative glucose-sensing kinase, both exhibited higher glucose sensitivity than the ΔaqpZ cells. Examination of protein expression indicated that sll1961 functioned as a positive regulator of aqpZ gene expression but not as the only regulator. Overall, the ΔaqpZ cells showed defects in macronutrient metabolism, pH homeostasis, and cell division under photomixotrophic conditions, consistent with an essential role of AqpZ in glucose metabolism.

ATP-sensitive cation-channel in wheat (Triticum durum Desf.): identification and characterization of a plant mitochondrial channel by patch-clamp.

Indirect evidence points to the presence of K+ channels in plant mitochondria. In the present study, we report the results of the first patch clamp experiments on plant mitochondria. Single-channel recordings in 150 mM potassium gluconate have allowed the biophysical characterization of a channel with a conductance of 150 pS in the inner mitochondrial membrane of mitoplasts obtained from wheat (Triticum durum Desf.). The channel displayed sharp voltage sensitivity, permeability to potassium and cation selectivity. ATP in the mM concentration range completely abolished the activity. We discuss the possible molecular identity of the channel and its possible role in the defence mechanisms against oxidative stress in plants.