Ca2+/Calmodulin Binding to STIM1 Hydrophobic Residues Facilitates Slow Ca2+-Dependent Inactivation of the Orai1 Channel.

BACKGROUND/AIMS: Store-operated Ca2+ entry (SOCE) through plasma membrane Ca2+ channel Orai1 is essential for many cellular processes. SOCE, activated by ER Ca2+ store-depletion, relies on the gating function of STIM1 Orai1-activating region SOAR of the ER-anchored Ca2+-sensing protein STIM1. Electrophysiologically, SOCE is characterized as Ca2+ release-activated Ca2+ current (ICRAC). A major regulatory mechanism that prevents deleterious Ca2+ overload is the slow Ca2+-dependent inactivation (SCDI) of ICRAC. Several studies have suggested a role of Ca2+/calmodulin (Ca2+/CaM) in triggering SCDI. However, a direct contribution of STIM1 in regulating Ca2+/CaM-mediated SCDI of ICRAC is as yet unclear. METHODS: The Ca2+/CaM binding to STIM1 was tested by pulling down recombinant GFP-tagged human STIM1 C-terminal fragments on CaM sepharose beads. STIM1 was knocked out by CRISPR/Cas9 technique in HEK293 cells stably overexpressing human Orai1. Store-operated Ca2+ influx was measured using Fluorometric Imaging Plate Reader and whole-cell patch clamp in cells transfected with STIM1 CaM binding mutants. The involvement of Ca2+/CaM in SCDI was investigated by including recombinant human CaM in patch pipette in electrophysiology. RESULTS: Here we identified residues Leu374/Val375 (H1) and Leu390/Phe391 (H2) within SOAR that serve as hydrophobic anchor sites for Ca2+/CaM binding. The bifunctional H2 site is critical for both Orai1 activation and Ca2+/CaM binding. Single residue mutations of Phe391 to less hydrophobic residues significantly diminished SOCE and ICRAC, independent of Ca2+/CaM. Hence, the role of H2 residues in Ca2+/CaM-mediated SCDI cannot be precisely evaluated. In contrast, the H1 site controls exclusively Ca2+/CaM binding and subsequently SCDI, but not Orai1 activation. V375A but not V375W substitution eliminated SCDI of ICRAC caused by Ca2+/CaM, proving a direct role of STIM1 in coordinating SCDI. CONCLUSION: Taken together, we propose a mechanistic model, wherein binding of Ca2+/CaM to STIM1 hydrophobic anchor residues, H1 and H2, triggers SCDI by disrupting the functional interaction between STIM1 and Orai1. Our findings reveal how STIM1, Orai1, and Ca2+/CaM are functionally coordinated to control ICRAC.

Single-Channel Properties of the ROMK-Pore-Forming Subunit of the Mitochondrial ATP-Sensitive Potassium Channel.

An increased flux of potassium ions into the mitochondrial matrix through the ATP-sensitive potassium channel (mitoKATP) has been shown to provide protection against ischemia-reperfusion injury. Recently, it was proposed that the mitochondrial-targeted isoform of the renal outer medullary potassium channel (ROMK) protein creates a pore-forming subunit of mitoKATP in heart mitochondria. Our research focuses on the properties of mitoKATP from heart-derived H9c2 cells. For the first time, we detected single-channel activity and describe the pharmacology of mitoKATP in the H9c2 heart-derived cells. The patch-clamping of mitoplasts from wild type (WT) and cells overexpressing ROMK2 revealed the existence of a potassium channel that exhibits the same basic properties previously attributed to mitoKATP. ROMK2 overexpression resulted in a significant increase of mitoKATP activity. The conductance of both channels in symmetric 150/150 mM KCl was around 97 ± 2 pS in WT cells and 94 ± 3 pS in cells overexpressing ROMK2. The channels were inhibited by 5-hydroxydecanoic acid (a mitoKATP inhibitor) and by Tertiapin Q (an inhibitor of both the ROMK-type channels and mitoKATP). Additionally, mitoKATP from cells overexpressing ROMK2 were inhibited by ATP/Mg2+ and activated by diazoxide. We used an assay based on proteinase K to examine the topology of the channel in the inner mitochondrial membrane and found that both termini of the protein localized to the mitochondrial matrix. We conclude that the observed activity of the channel formed by the ROMK protein corresponds to the electrophysiological and pharmacological properties of mitoKATP.

Mitochondrial BK Channel Openers CGS7181 and CGS7184 Exhibit Cytotoxic Properties.

Potassium channel openers (KCOs) have been shown to play a role in cytoprotection through the activation of mitochondrial potassium channels. Recently, in several reports, a number of data has been described as off-target actions for KCOs. In the present study, we investigated the effects of BKCa channel openers CGS7181, CGS7184, NS1619, and NS004 in neuronal cells. For the purpose of this research, we used a rat brain, the mouse hippocampal HT22 cells, and the human astrocytoma U-87 MG cell line. We showed that CGS7184 activated the mitochondrial BKCa (mitoBKCa) channel in single-channel recordings performed on astrocytoma mitoplasts. Moreover, when applied to the rat brain homogenate or isolated rat brain mitochondria, CGS7184 increased the oxygen consumption rate, and can thus be considered a potentially cytoprotective agent. However, experiments on intact neuronal HT22 cells revealed that both CGS7181 and CGS7184 induced HT22 cell death in a concentration- and time-dependent manner. By contrast, we did not observe cell death when NS1619 or NS004 was applied. CGS7184 toxicity was not abolished by BKCa channel inhibitors, suggesting that the observed effects were independent of a BKCa-type channel activity. CGS7184 treatment resulted in an increase of cytoplasmic Ca2+ concentration that likely involved efflux from internal calcium stores and the activation of calpains (calcium-dependent proteases). The cytotoxic effect of the channel opener was partially reversed by a calpain inhibitor. Our data show that KCOs under study not only activate mitoBKCa channels from brain tissue, but also induce cell death when used in cellular models.

What do we not know about mitochondrial potassium channels?

In this review, we summarize our knowledge about mitochondrial potassium channels, with a special focus on unanswered questions in this field. The following potassium channels have been well described in the inner mitochondrial membrane: ATP-regulated potassium channel, Ca(2+)-activated potassium channel, the voltage-gated Kv1.3 potassium channel, and the two-pore domain TASK-3 potassium channel. The primary functional roles of these channels include regulation of mitochondrial respiration and the alteration of membrane potential. Additionally, they modulate the mitochondrial matrix volume and the synthesis of reactive oxygen species by mitochondria. Mitochondrial potassium channels are believed to contribute to cytoprotection and cell death. In this paper, we discuss fundamental issues concerning mitochondrial potassium channels: their molecular identity, channel pharmacology and functional properties. Attention will be given to the current problems present in our understanding of the nature of mitochondrial potassium channels. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

A large-conductance calcium-regulated K+ channel in human dermal fibroblast mitochondria.

Potassium channels have been found in the inner mitochondrial membrane of various cells. These channels regulate the mitochondrial membrane potential, respiration and production of reactive oxygen species. In the present study, we identified the activity of a mitochondrial large-conductance Ca2+-regulated potassium channel (mitoBKCa channel) in mitoplasts isolated from a primary human dermal fibroblast cell line. A potassium selective current was recorded with a mean conductance of 280 ± 2 pS in a symmetrical 150 mM KCl solution. The mitoBKCa channel was activated by the Ca2+ and by potassium channel opener NS1619. The channel activity was irreversibly inhibited by paxilline, a selective inhibitor of the BKCa channels. In isolated fibroblast mitochondria NS1619 depolarized the mitochondrial membrane potential, stimulated nonphosphorylating respiration and decreased superoxide formation. Additionally, the α- and ß-subunits (predominantly the ß3-form) of the BKCa channels were identified in fibroblast mitochondria. Our findings indicate, for the first time, the presence of a large-conductance Ca2+-regulated potassium channel in the inner mitochondrial membrane of human dermal fibroblasts.

Guide to the Pharmacology of Mitochondrial Potassium Channels.

This chapter provides a critical overview of the available literature on the pharmacology of mitochondrial potassium channels. In the first part, the reader is introduced to the topic, and eight known protein contributors to the potassium permeability of the inner mitochondrial membrane are presented. The main part of this chapter describes the basic characteristics of each channel type mentioned in the introduction. However, the most important and valuable information included in this chapter concerns the pharmacology of mitochondrial potassium channels. Several available channel modulators are critically evaluated and rated by suitability for research use. The last figure of this chapter shows the results of this evaluation at a glance. Thus, this chapter can be very useful for beginners in this field. It is intended to be a time- and resource-saving guide for those searching for proper modulators of mitochondrial potassium channels.

Carbon monoxide released by CORM-401 uncouples mitochondrial respiration and inhibits glycolysis in endothelial cells: A role for mitoBKCa channels.

Carbon monoxide (CO), a product of heme degradation by heme oxygenases, plays an important role in vascular homeostasis. Recent evidence indicates that mitochondria are among a number of molecular targets that mediate the cellular actions of CO. In the present study we characterized the effects of CO released from CORM-401 on mitochondrial respiration and glycolysis in intact human endothelial cells using electron paramagnetic resonance (EPR) oximetry and the Seahorse XF technology. We found that CORM-401 (10-100µM) induced a persistent increase in the oxygen consumption rate (OCR) that was accompanied by inhibition of glycolysis (extracellular acidification rate, ECAR) and a decrease in ATP-turnover. Furthermore, CORM-401 increased proton leak, diminished mitochondrial reserve capacity and enhanced non-mitochondrial respiration. Inactive CORM-401 (iCORM-401) neither induced mitochondrial uncoupling nor inhibited glycolysis, supporting a direct role of CO in the endothelial metabolic response induced by CORM-401. Interestingly, blockade of mitochondrial large-conductance calcium-regulated potassium ion channels (mitoBKCa) with paxilline abolished the increase in OCR promoted by CORM-401 without affecting ECAR; patch-clamp experiments confirmed that CO derived from CORM-401 activated mitoBKCa channels present in mitochondria. Conversely, stabilization of glycolysis by MG132 prevented CORM-401-mediated decrease in ECAR but did not modify the OCR response. In summary, we demonstrated in intact endothelial cells that CO induces a two-component metabolic response: uncoupling of mitochondrial respiration dependent on the activation of mitoBKCa channels and inhibition of glycolysis independent of mitoBKCa channels.

[What we don't know about mitochondrial potassium channels?] / Czego nie wiemy o mitochondrialnych kanalach potasowych?

In the current work the authors present the most interesting, yet not fully understood issues regarding origin, function and pharmacology of the mitochondrial potassium channels. There are eight potassium channels known to contribute to the potassium permeability of the inner mitochondrial membrane: ATP-regulated channel, calcium-regulated channels of large, intermediate and small conductance, voltage-regulated Kv1.3 and Kv7.4 channels, two-pore-domain TASK-3 channel and SLO2 channel. The primary function of the mitochondrial potassium channels is regulation of the mitochondrial membrane potential. Additionally, mitochondrial potassium channels alter cellular respiration, regulation of the mitochondrial volume and ROS synthesis. However, mechanisms underlying these processes are not fully understood yet. In this work, the authors not only present available knowledge about this topic, but also put certain hypotheses that may set the direction for the future research on these proteins.

Influence of kinin peptides on monocyte-endothelial cell adhesion.

Adhesion of leukocytes to vascular endothelium in response to proinflammatory mediators is an important component of the overall inflammatory reaction. In the current work, we used a retinoic acid-differentiated human promonocytic cell line, U937 and a human microvascular endothelial cell line, HMEC-1 to analyze the effect of the potent pro-inflammatory bradykinin-related peptides (kinins) on cell adhesion. Bradykinin (BK) and kinin metabolites without the C-terminal arginine residue enhanced adhesion of the monocyte-like cells to fibronectin and to the HMEC-1 cells. Expression of adhesion proteins on the surface of both cell types was altered by the kinin peptides. In the monocyte-like cells, expression of CD11b, a subunit of Mac-1 integrin, was significantly increased whilst in the endothelial cells, a strong increase in the production of intercellular adhesion molecule 1 (ICAM-1) was observed. The positive bradykinin-induced effect on the cell-cell interaction was reversed by a carboxypeptidase inhibitor (MGTA), hence we suspected a significant role of the des-Arg kinin metabolites, which acted through the kinin receptor type 1. Indeed, the expression of this receptor was up-regulated not only by agonists but also by interferon-γ and bradykinin. Kinin peptides also regulated signal transducer and activator of transcription proteins (STATs) activated by cytokines. Taken together, the above observations support our hypothesis that kinins stimulate monocyte adhesion to the vessel wall, especially during pathological states of the circulatory system accompanied by proinflammatory cytokine release.

Functional Characterization of the Lysosomal Peptide/Histidine Transporter PHT1 (SLC15A4) by Solid Supported Membrane Electrophysiology (SSME).

The peptide/histidine transporter PHT1 (SLC15A4) is expressed in the lysosomal membranes of immune cells where it plays an important role in metabolic and inflammatory signaling. PHT1 is an H+-coupled/histidine symporter that can transport a wide range of oligopeptides, including a variety of bacterial-derived peptides. Moreover, it enables the scaffolding of various metabolic signaling molecules and interacts with key regulatory elements of the immune response. Not surprisingly, PHT1 has been implicated in the pathogenesis of autoimmune diseases such as systemic lupus erythematosus (SLE). Unfortunately, the pharmacological development of PHT1 modulators has been hampered by the lack of suitable transport assays. To address this shortcoming, a novel transport assay based on solid-supported membrane-based electrophysiology (SSME) is presented. Key findings of the present SSME studies include the first recordings of electrophysiological properties, a pH dependence analysis, an assessment of PHT1 substrate selectivity, as well as the transport kinetics of the identified substrates. In contrast to previous work, PHT1 is studied in its native lysosomal environment. Moreover, observed substrate selectivity is validated by molecular docking. Overall, this new SSME-based assay is expected to contribute to unlocking the pharmacological potential of PHT1 and to deepen the understanding of its functional properties.

The contribution of thioredoxin-2 reductase and glutathione peroxidase to H(2)O(2) detoxification of rat brain mitochondria.

Brain mitochondria are not only major producers of reactive oxygen species but they also considerably contribute to the removal of toxic hydrogen peroxide by the glutathione (GSH) and thioredoxin-2 (Trx2) antioxidant systems. In this work we estimated the relative contribution of both systems and catalase to the removal of intrinsically produced hydrogen peroxide (H(2)O(2)) by rat brain mitochondria. By using the specific inhibitors auranofin and 1-chloro-2,4-dinitrobenzene (DNCB), the contribution of Trx2- and GSH-systems to reactive oxygen species (ROS) detoxification in rat brain mitochondria was determined to be 60±20% and 20±15%, respectively. Catalase contributed to a non-significant extent only, as revealed by aminotriazole inhibition. In digitonin-treated rat hippocampal homogenates inhibition of Trx2- and GSH-systems affected mitochondrial hydrogen peroxide production rates to a much higher extent than the endogenous extramitochondrial hydrogen peroxide production, pointing to a strong compartmentation of ROS metabolism. Imaging experiments of hippocampal slice cultures showed on single cell level substantial heterogeneity of hydrogen peroxide detoxification reactions. The strongest effects of inhibition of hydrogen peroxide removal by auranofin or DNCB were detected in putative interneurons and microglial cells, while pyramidal cells and astrocytes showed lower effects. Thus, our data underline the important contribution of the Trx2-system to hydrogen peroxide detoxification in rat hippocampus. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).

Discovery of novel gating checkpoints in the Orai1 calcium channel by systematic analysis of constitutively active mutants of its paralogs and orthologs.

In humans, there are three paralogs of the Orai Ca2+ channel that form the core of the store-operated calcium entry (SOCE) machinery. While the STIM-mediated gating mechanism of Orai channels is still under active investigation, several artificial and natural variants are known to cause constitutive activity of the human Orai1 channel. Surprisingly, little is known about the conservation of the gating checkpoints among the different human Orai paralogs and orthologs in other species. In our work, we show that the mutation corresponding to the activating mutation H134A in transmembrane helix 2 (TM2) of human Orai1 also activates Orai2 and Orai3, likely via a similar mechanism. However, this cross-paralog conservation does not apply to the "ANSGA" nexus mutations in TM4 of human Orai1, which is reported to mimic the STIM1-activated state of the channel. In investigating the mechanistic background of these differences, we identified two positions, H171 and F246 in human Orai1, that are not conserved among paralogs and that seem to be crucial for the channel activation triggered by the "ANSGA" mutations in Orai1. However, mutations of the same residues still allow gating of Orai1 by STIM1, suggesting that the ANSGA mutant of Orai1 may not be a surrogate for the STIM1-activated state of the Orai1 channel. Our results shed new light on these important gating checkpoints and show that the gating mechanism of Orai channels is affected by multiple factors that are not necessarily conserved among orai homologs, such as the TM4-TM3 coupling.

Mitochondrial Potassium Channels as Druggable Targets.

Mitochondrial potassium channels have been described as important factors in cell pro-life and death phenomena. The activation of mitochondrial potassium channels, such as ATP-regulated or calcium-activated large conductance potassium channels, may have cytoprotective effects in cardiac or neuronal tissue. It has also been shown that inhibition of the mitochondrial Kv1.3 channel may lead to cancer cell death. Hence, in this paper, we examine the concept of the druggability of mitochondrial potassium channels. To what extent are mitochondrial potassium channels an important, novel, and promising drug target in various organs and tissues? The druggability of mitochondrial potassium channels will be discussed within the context of channel molecular identity, the specificity of potassium channel openers and inhibitors, and the unique regulatory properties of mitochondrial potassium channels. Future prospects of the druggability concept of mitochondrial potassium channels will be evaluated in this paper.

Hemin inhibits the large conductance potassium channel in brain mitochondria: a putative novel mechanism of neurodegeneration.

Intracerebral hemorrhage (ICH) is a pathological condition that accompanies certain neurological diseases like hemorrhagic stroke or brain trauma. Its effects are severely destructive to the brain and can be fatal. There is an entire spectrum of harmful factors which are associated with the pathogenesis of ICH. One of them is a massive release of hemin from the decomposed erythrocytes. It has been previously shown, that hemin can inhibit the large-conductance Ca(2+)-regulated potassium channel in the plasma membrane. However, it remained unclear whether this phenomenon applies also to the mitochondrial large-conductance Ca(2+)-regulated potassium channel. The aim of the present study was to determine the impact of hemin on the activity of the large conductance Ca(2+)-regulated potassium channel in the brain mitochondria (mitoBKCa). In order to do so, we have used a patch-clamp technique and shown that hemin inhibits mitoBKCa in human astrocytoma U-87 MG cell line mitochondria. Since opening of the mitochondrial potassium channels is known to be cytoprotective, we have elucidated whether hemin can attenuate some of the beneficiary effects of potassium channel opening. We have studied the effect of hemin on reactive oxygen species synthesis, and mild mitochondrial uncoupling in isolated rat brain mitochondria. Taken together, our data show that hemin inhibits mitoBKCa and partially abolishes some of the cytoprotective properties of potassium channel opening. Considering the role of the mitoBKCa in cytoprotection, it can be presumed that its inhibition by hemin may be a novel mechanism contributing to the severity of the ICH symptoms. However, the validity of the presented results shall be further verified in an experimental model of ICH.