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Kv1.3 is a voltage gated potassium channel located in the plasma membrane, as well as at intracellular levels, such as mitochondria (mitoKv1.3), nucleus and Golgi apparatus. The plasma membrane channel has been shown to be important for cell proliferation, while the mitochondrial counterpart has been related to modulation of cell death. Moreover, altered expression of Kv1.3 was observed in various tumors and Kv1.3 seems to be involved in development and progression of various cancerous forms. Recent experimental evidences have proved that pharmacological inhibition of the mitoKv1.3 succeeded in reducing up to 90% of tumor volume in vivo in orthotopic mouse model. Furthermore, mitoKv1.3 modulation could impact on cell proliferation as well as on regulation of intracellular signaling pathways. Indeed, the treatment with sub-lethal doses of mitoKv1.3 inhibitors can downregulate Wnt-β catenin signaling by reducing mitochondrial ATP production and triggering ER-stress. In this review, we describe the role of the mitoKv1.3 in cell death, cancer and intracellular signaling. We will discuss how pharmacological modulation of mitochondrial potassium fluxes impact on mitochondrial membrane potential, reactive oxygen species production and ATP synthesis. All these changes in mitochondrial fitness are related to cell proliferation as well as to cell death and finally on cancer development and progression, so Kv1.3 (and mitoKv1.3) could be now considered a new oncological target.

Potassium ions can cross both the outer and inner mitochondrial membranes by means of multiple routes. A few potassium-permeable ion channels exist in the outer membrane, while in the inner membrane, a multitude of different potassium-selective and potassium-permeable channels mediate K+ uptake into energized mitochondria. In contrast, potassium is exported from the matrix thanks to an H+/K+ exchanger whose molecular identity is still debated. Among the K+ channels of the inner mitochondrial membrane, the most widely studied is the ATP-dependent potassium channel, whose pharmacological activation protects cells against ischemic damage and neuronal injury. In this review, we briefly summarize and compare the different hypotheses regarding the molecular identity of this patho-physiologically relevant channel, taking into account the electrophysiological characteristics of the proposed components. In addition, we discuss the characteristics of the other channels sharing localization to both the plasma membrane and mitochondria.

Mitochondria provide chemical energy for endoergonic reactions in the form of ATP, and their activity must meet cellular energy requirements, but the mechanisms that link organelle performance to ATP levels are poorly understood. Here we confirm the existence of a protein complex localized in mitochondria that mediates ATP-dependent potassium currents (that is, mitoKATP). We show that-similar to their plasma membrane counterparts-mitoKATP channels are composed of pore-forming and ATP-binding subunits, which we term MITOK and MITOSUR, respectively. In vitro reconstitution of MITOK together with MITOSUR recapitulates the main properties of mitoKATP. Overexpression of MITOK triggers marked organelle swelling, whereas the genetic ablation of this subunit causes instability in the mitochondrial membrane potential, widening of the intracristal space and decreased oxidative phosphorylation. In a mouse model, the loss of MITOK suppresses the cardioprotection that is elicited by pharmacological preconditioning induced by diazoxide. Our results indicate that mitoKATP channels respond to the cellular energetic status by regulating organelle volume and function, and thereby have a key role in mitochondrial physiology and potential effects on several pathological processes.

Whether the mitochondrial permeability transition pore (PTP), also called mitochondrial megachannel (MMC), originates from the F-ATP synthase is a matter of controversy. This hypothesis is supported both by site-directed mutagenesis of specific residues of F-ATP synthase affecting regulation of the PTP/MMC and by deletion of specific subunits causing dramatic changes in channel conductance. In contrast, human cells lacking an assembled F-ATP synthase apparently display persistence of the PTP. We discuss recent data that shed new light on this controversy, supporting the conclusion that the PTP/MMC originates from a Ca2+ -dependent conformational change in F-ATP synthase allowing its reversible transformation into a high-conductance channel.

Background/Aims: The permeability transition pore (PTP) is an unselective, Ca2+-dependent high conductance channel of the inner mitochondrial membrane whose molecular identity has long remained a mystery. The most recent hypothesis is that pore formation involves the F-ATP synthase, which consistently generates Ca2+-activated channels. Available structures do not display obvious features that can accommodate a channel; thus, how the pore can form and whether its activity can be entirely assigned to F-ATP synthase is the matter of debate. In this study, we investigated the role of F-ATP synthase subunits e, g and b in PTP formation. Methods: Yeast null mutants for e, g and the first transmembrane (TM) α-helix of subunit b were generated and evaluated for mitochondrial morphology (electron microscopy), membrane potential (Rhodamine123 fluorescence) and respiration (Clark electrode). Homoplasmic C23S mutant of subunit a was generated by in vitro mutagenesis followed by biolistic transformation. F-ATP synthase assembly was evaluated by BN-PAGE analysis. Cu2+ treatment was used to induce the formation of F-ATP synthase dimers in the absence of e and g subunits. The electrophysiological properties of F-ATP synthase were assessed in planar lipid bilayers. Results: Null mutants for the subunits e and g display dimer formation upon Cu2+ treatment and show PTP-dependent mitochondrial Ca2+ release but not swelling. Cu2+ treatment causes formation of disulfide bridges between Cys23 of subunits a that stabilize dimers in absence of e and g subunits and favors the open state of wild-type F-ATP synthase channels. Absence of e and g subunits decreases conductance of the F-ATP synthase channel about tenfold. Ablation of the first TM of subunit b, which creates a distinct lateral domain with e and g, further affected channel activity. Conclusion: F-ATP synthase e, g and b subunits create a domain within the membrane that is critical for the generation of the high-conductance channel, thus is a prime candidate for PTP formation. Subunits e and g are only present in eukaryotes and may have evolved to confer this novel function to F-ATP synthase.

A developing family of chemotherapeutics – derived from 5-(4-phenoxybutoxy)psoralen (PAP-1) – target mitochondrial potassium channel mtKv1.3 to selectively induce oxidative stress and death of diseased cells. The key to their effectiveness is the presence of a positively charged triphenylphosphonium group which drives their accumulation in the organelles. These compounds have proven their preclinical worth in murine models of cancers such as melanoma and pancreatic adenocarcinoma. In in vitro experiments they also efficiently killed glioblastoma cells, but in vivo they were powerless against orthotopic glioma because they were completely unable to overcome the blood-brain barrier. In an effort to improve brain delivery we have now coupled one of these promising compounds, PAPTP, to well-known cell-penetrating and brain-targeting peptides TAT48–61 and Angiopep-2. Coupling has been obtained by linking one of the phenyl groups of the triphenylphosphonium to the first amino acid of the peptide via a reversible carbamate ester bond. Both TAT48–61 and Angiopep-2 allowed the delivery of 0.3–0.4 nmoles of construct per gram of brain tissue upon intravenous (i.v.) injection of 5 µmoles/kg bw to mice. This is the first evidence of PAPTP delivery to the brain, the chemical strategy described here opens the possibility to conjugate PAPTP to small peptides in order to fine-tune tissue distribution of this interesting compound.

The field of mitochondrial ion channels underwent a rapid development during the last decade, thanks to the molecular identification of some of the nuclear-encoded organelle channels and to advances in strategies allowing specific pharmacological targeting of these proteins. Thereby, genetic tools and specific drugs aided definition of the relevance of several mitochondrial channels both in physiological as well as pathological conditions. Unfortunately, in the case of mitochondrial K

Mitochondria are intracellular organelles involved in several processes from bioenergetics to cell death. In the latest years, ion channels are arising as new possible targets in controlling several cellular functions. The discovery that several plasma membrane located ion channels have intracellular counterparts, has now implemented this consideration and the number of studies enforcing the understanding of their role in different metabolic pathways. In this review, we will discuss the recent updates in the field, focusing our attention on the involvement of potassium channels during mitochondrial mediated apoptotic cell death. Since mitochondria are one of the key organelles involved in this process, it is not surprising that potassium channels located in their inner membrane could be involved in modulating mitochondrial membrane potential, ROS production, and respiratory chain complexes functions. Eventually, these events lead to changes in the mitochondrial fitness that prelude to the cytochrome c release and apoptosis. In this scenario, both the inhibition and the activation of mitochondrial potassium channels could cause cell death, and their targeting could be a novel pharmacological way to treat different human diseases.

Cancer is the consequence of aberrations in cell growth or cell death. In this scenario, mitochondria and ion channels play a critical role in regard to cell proliferation, malignant angiogenesis, migration, and metastasis. In this review, we focus on Kv1.3 and specifically on mitoKv1.3, which showed an aberrant expression in cancer cells compared with healthy tissues and which is involved in the apoptotic pathway. In recent years, mitoKv1.3 has become an oncological target since its pharmacological modulation has been demonstrated to reduce tumor growth and progression both in vitro and in vivo using preclinical mouse models of different types of tumors.

The mitochondrial calcium uniporter (MCU) and the mitochondrial calcium uniporter dominant negative b- subunit (MCUb) are pore-forming components of the uniporter complex. We expressed these MCU subunits in cell-free transcription/translation systems, and we studied them, at the single molecule level, using the electrophysiological technique of planar lipid bilayer.We showed that MCU gives rise to single-channel Ca

Numerous researchers tried to identify the key players of calcium signaling in mitochondria using molecular and cell biology techniques for more than five decades. However, only an integrated approach involving also electrophysiological techniques has finally allowed to define the components of the protein complex responsible for the uptake of this ion into mitochondria.Here we describe the protocol used for the electrophysiological characterization of the mitochondrial calcium uniporter (MCU) complex: the following outline indicates step-by-step the setup of planar lipid bilayer experiments.

Numerous researchers tried to identify the key players of calcium signaling in mitochondria using molecular and cell biology techniques for more than five decades. However, only an integrated approach involving also electrophysiological techniques has finally allowed to define the components of the protein complex responsible for the uptake of this ion into mitochondria.Here we describe the protocol used for the electrophysiological characterization of the mitochondrial calcium uniporter (MCU) complex: the following outline indicates step-by-step the setup of planar lipid bilayer experiments.

The mitochondrial calcium uniporter (MCU) and the mitochondrial calcium uniporter dominant negative b- subunit (MCUb) are pore-forming components of the uniporter complex. We expressed these MCU subunits in cell-free transcription/translation systems, and we studied them, at the single molecule level, using the electrophysiological technique of planar lipid bilayer.We showed that MCU gives rise to single-channel Ca2+ currents. In contrast, MCUb alone does not display calcium-permeable channel activity, while the co-expression of MCUb:MCU drastically alters the calcium permeation mediated by MCU subunit.

In striking analogy with Saccharomyces cerevisiae, etiolated pea stem mitochondria did not show appreciable Ca2+ uptake. Only treatment with the ionophore ETH129 (which allows electrophoretic Ca2+ equilibration) caused Ca2+ uptake followed by increased inner membrane permeability, membrane depolarization and Ca2+ release. Like the permeability transition (PT) of mammals, yeast and Drosophila, the PT of pea stem mitochondria was stimulated by diamide and phenylarsine oxide and inhibited by Mg-ADP and Mg-ATP, suggesting a common underlying mechanism; yet, the plant PT also displayed distinctive features: (i) as in mammals it was desensitized by cyclosporin A, which does not affect the PT of yeast and Drosophila; (ii) similarly to S. cerevisiae and Drosophila it was inhibited by Pi, which stimulates the PT of mammals; (iii) like in mammals and Drosophila it was sensitized by benzodiazepine 423, which is ineffective in S. cerevisiae; (iv) like what observed in Drosophila it did not mediate swelling and cytochrome c release, which is instead seen in mammals and S. cerevisiae. We find that cyclophilin D, the mitochondrial receptor for cyclosporin A, is present in pea stem mitochondria. These results indicate that the plant PT has unique features and suggest that, as in Drosophila, it may provide pea stem mitochondria with a Ca2+ release channel.

International audience; 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.

// Simona Reina 1,2* , Vanessa Checchetto 3,4,5,* , Rosaria Saletti 6 , Ankit Gupta 7,* , Deepti Chaturvedi 7,* , Carlo Guardiani 8 , Francesca Guarino 1,2 , Mariano Andrea Scorciapino 9 , Andrea Magri 1,2 , Salvatore Foti 6 , Matteo Ceccarelli 8,10,** , Angela Anna Messina 11,12,** , Radhakrishnan Mahalakshmi 7,** , Ildiko Szabo 3,4,** and Vito De Pinto 1,2 1 Department of Biomedicine and Biotechnology BIOMETEC, Section of Biology and Genetics, University of Catania, Catania, Italy 2 National Institute for Biomembranes and Biosystems, Section of Catania, Catania, Italy 3 Department of Biology, University of Padova, Padova, Italy 4 CNR Institute of Neurosciences, Padova, Italy 5 Department of Biomedical Sciences, University of Padova, Padova, Italy 6 Department of Chemical Sciences, Mass Spectrometry Unit, University of Catania, Catania, Italy 7 Molecular Biophysics Laboratory, Department of Biological Sciences, Indian Institute of Science Education and Research, Bhopal, India 8 Department of Physics, University of Cagliari, Cagliari, Italy 9 Department of Biomedical Sciences, Biochemistry Unit, University of Cagliari, Cagliari, Italy 10 Istituto Officina dei Materiali del Consiglio Nazionale delle Ricerche (IOM-CNR), UOS, Trieste, Italy 11 Department of Biological, Geological and Environmental Sciences, Section of Molecular Biology, University of Catania, Catania, Italy 12 National Institute for Biomembranes and Biosystems, Section of Catania, Catania, Italy * These authors have contributed equally to this work ** Co-corresponding author Correspondence to: Vito De Pinto, email: // Keywords : VDACs, cysteine oxidation, disulfide bridge, mitochondrial intermembrane space, mass spectrometry Received : October 21, 2015 Accepted : December 23, 2015 Published : January 08, 2016 Abstract 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.

In living organisms different developmental and environmental stimuli can trigger Ca2+ transients of specific signature that can modulate gene expression and metabolism. Mitochondria play an important role in shaping intracellular calcium dynamics. Recently, several molecular players involved in mitochondrial Ca2+ signalling have been identified both in animals and plants, including those of the mitochondrial Ca2+ uniporter complex, MCUC. In he present review the significance of mitochondrial Ca2+ control is discussed in the light of the cytosolic calcium signalling and of specific metabolic and energetic needs of different organisms.

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., Graphical Abstract, Highlights • MICU1 and MICU2 form an obligate heterodimer • MICU1 enhances MCU opening • MICU2 acts as an MCU gatekeeper, Mitochondrial calcium uptake is rapid in response to calcium signaling. Patron et al. demonstrate that this response is not intrinsic to the mitochondrial calcium uniporter (MCU) but rather is dependent on a disulfide-mediated dimer of MCU interactors (MICU1 and MICU2). At low [Ca2+], MICU2 shuts down MCU activity, but after Ca2+ stimulation, MICU2 inhibition is released and MICU1 enhances MCU opening.

Skeletal muscle is a dynamic organ, characterized by an incredible ability to rapidly increase its rate of energy consumption to sustain activity. Muscle mitochondria provide most of the ATP required for contraction via oxidative phosphorylation. Here we found that skeletal muscle mitochondria express a unique MCU complex containing an alternative splice isoform of MICU1, MICU1.1, characterized by the addition of a micro-exon that is sufficient to greatly modify the properties of the MCU. Indeed, MICU1.1 binds Ca

Mitochondria are essential organelles that control a plethora of cellular functions, including ATP production, synthesis of intermediate metabolites, cellular signaling and regulation of cell fate. Their proper functioning is necessary to the health of the whole organism, and indeed a huge number of human pathologies are associated to mitochondrial dysfunctions. The fine control of ion homeostasis across the inner mitochondrial membrane is crucial for their activity and it is guaranteed by a large number of channels, pumps and exchangers that act together to maintain the correct organelle volume, shape and function. Here we identify a novel protein complex composed by a pore-forming core and a regulatory subunit located in the inner mitochondrial membrane. The overexpression of the channel-forming subunit alone in HeLa cells leads to uncoupling of mitochondria and organelle swelling. Conversely, its ablation causes mitochondrial dysfunction characterized by instability of mitochondrial membrane potential accompanied by a decrease of oxidative performance. Overall, our data suggest this is a novel player in the control of mitochondrial ion homeostasis.

Plasma membrane potassium channels importantly contribute to maintain ion homeostasis across the cell membrane. The view is emerging that also those residing in intracellular membranes play pivotal roles for the coordination of correct cell function. In this review we critically discuss our current understanding of the nature and physiological tasks of potassium channels in organelle membranes in both animal and plant cells, with a special emphasis on their function in the regulation of photosynthesis and mitochondrial respiration. In addition, the emerging role of potassium channels in the nuclear membranes in regulating transcription will be discussed. The possible functions of endoplasmic reticulum-, lysosome- and plant vacuolar membrane-located channels are also referred to. Altogether, experimental evidence obtained with distinct channels in different membrane systems points to a possible unifying function of most intracellular potassium channels in counterbalancing the movement of other ions including protons and calcium and modulating membrane potential, thereby fine-tuning crucial cellular processes. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-7, 2016', edited by Prof. Paolo Bernardi.

Skeletal muscle is a dynamic organ, characterized by an incredible ability to rapidly increase its rate of energy consumption to sustain activity. Muscle mitochondria provide most of the ATP required for contraction via oxidative phosphorylation. Here we found that skeletal muscle mitochondria express a unique MCU complex containing an alternative splice isoform of MICU1, MICU1.1, characterized by the addition of a micro-exon that is sufficient to greatly modify the properties of the MCU. Indeed, MICU1.1 binds Ca2+ one order of magnitude more efficiently than MICU1 and, when heterodimerized with MICU2, activates MCU current at lower Ca2+ concentrations than MICU1-MICU2 heterodimers. In skeletal muscle in vivo, MICU1.1 is required for sustained mitochondrial Ca2+ uptake and ATP production. These results highlight a novel mechanism of the molecular plasticity of the MCU Ca2+ uptake machinery that allows skeletal muscle mitochondria to be highly responsive to sarcoplasmic [Ca2+] responses.

The accumulation in mitochondria of oxidative agents must be signaled to the cell. Neverthless the organelle is the source of redox species. The accumulation in mitochondria of oxidative agents must be signaled to the cell and an organelle heavily loaded with ROS evidenced. We addressed specific structural questions related to the function of VoltageDependentAnion-selectiveChannel isoform 3 (VDAC3) cysteines. We show that VDAC3 (1–3) may be relevant for signaling the redox potential existing in the mitochondrial intermembrane space. We found that VDAC3 can be progressively modified by an accumulation of ROS, resulting in the oxidation at different extents of the exposed cysteine residues. We discovered that each single VDAC3 molecule in the membrane can contain a differently oxidated set of cysteine residues, thus giving rise to what we call “redox isomers” (4). A disulfide bridge was evidenced by mass spectrometry (5). A recent paper indicated a putative disulfide bridge that we did not find by MassSpec (6), neither the authirs detected intermediate oxidation states. Since this complex oxidation pattern is a consequence of the ROS level in the IMS, VDAC3 monitores the redox homeostasis. In our opinion this work represents a pathbreaking finding in the field of mitochondrial redox sensing.Acknowledgements MIUR-PRIN 2010-2011 n. 2010CSJX4F[1] Messina A. et al, 2012, BiochimBiophysActa 1818, 1466-1476[2] Checchetto V. et al, 2014, Cell Physiol. Biochem. 34, 842-53[3] Messina A. et al, 2014, Mol. Biosyst. 10, 2134-45[4] Reina S. et al 2015, SUBMITTED[5] Saletti R. et al, 2015, SUBMITTED[6] Okazaki M. et al, 2015, BiochimBiophysActa, in press

Mitochondrial ion channels are of great importance to ensure the proper function of this bioenergetic organelle and to regulate cell fate. However, in many cases our knowledge concerning their molecular identity and regulation is still limited. Here we describe a novel regulatory mechanism of the voltage-dependent anion channel VDAC3 and a new channel-forming protein. 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 been assigned to the VDAC3 isoform only recently (Checchetto et al, Cell Phys Biochem, 2014). This protein forms an ion channel with small conductance under standard conditions, but our study identifies the amino acid residues whose oxidation state impacts on channel activity and conductance. Furthermore, we characterize from electrophysiological point of view a novel protein complex composed by pore-forming core and regulatory subunits located in the inner mitochondrial membrane. Channel activity can be observed with the reconstituted recombinant core protein, but for its regulation the co-expression of both the core and the regulatory subunits are necessary. Overall, our data suggest that VDAC3 and the novel protein complex are new players in the control of mitochondrial ion homeostasis and might contribute to the plasticity of mitochondrial function in intact cells.

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.

Ion channels mediate ion flux across biological membranes and regulate important organellar and cellular tasks. A recent study revealed the presence of four new proteins, the MIM complex (composed by Mim1 and Mim2), Ayr1, OMC7, and OMC8, that are able to form ion-conducting channels in the outer mitochondria membrane (OMM). These findings strongly indicate that the OMM is endowed with many solute-specific channels, in addition to porins and known channels mediating protein import into mitochondria. These solute-specific channels provide essential pathways for the controlled transport of ions and metabolites and may thus add a further layer of specificity to the regulation of mitochondrial function at the organelle-cytosol and/or inter-organellar interface. Future studies will be required to fully understand the way(s) of regulation of these new channels and to integrate them into signaling pathways within the cells.

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.

The unicellular photosynthetic cyanobacterium, able to survive in varying environments, is the only prokaryote that directly converts solar energy and CO2 into organic material and is thus relevant for primary production in many ecosystems. To maintain the intracellular and intrathylakoid ion homeostasis upon different environmental challenges, the concentration of potassium as a major intracellular cation has to be optimized by various K(+)uptake-mediated transport systems. We reveal here the specific and concerted physiological function of three K(+)transporters of the plasma and thylakoid membranes, namely of SynK (K(+)channel), KtrB (Ktr/Trk/HKT) and KdpA (Kdp) in Synechocystis sp. strain PCC 6803, under specific stress conditions. The behavior of the wild type, single, double and triple mutants was compared, revealing that only Synk contributes to heavy metal-induced stress, while only Ktr/Kdp is involved in osmotic and salt stress adaptation. With regards to pH shifts in the external medium, the Kdp/Ktr uptake systems play an important role in the adaptation to acidic pH. Ktr, by affecting the CO2 concentration mechanism via its action on the bicarbonate transporter SbtA, might also be responsible for the observed effects concerning high-light stress and calcification. In the case of illumination with high-intensity light, a synergistic action of Kdr/Ktp and SynK is required in order to avoid oxidative stress and ensure cell viability. In summary, this study dissects, using growth tests, measurement of photosynthetic activity and analysis of ultrastructure, the physiological role of three K(+)transporters in adaptation of the cyanobacteria to various environmental changes.