Interaction of ROMK2 channel with lipid kinases DGKE and AGK: Potential channel activation by localized anionic lipid synthesis.

In this study, we utilized enzyme-catalyzed proximity labeling with the engineered promiscuous biotin ligase Turbo-ID to identify the proxisome of the ROMK2 channel. This channel resides in various cellular membrane compartments of the cell including the plasma membrane, endoplasmic reticulum and mitochondria. Within mitochondria, ROMK2 has been suggested as a pore-forming subunit of mitochondrial ATP-regulated potassium channel (mitoKATP). We found that ROMK2 proxisome in addition to previously known protein partners included two lipid kinases: acylglycerol kinase (AGK) and diacylglycerol kinase ε (DGKE), which are localized in mitochondria and the endoplasmic reticulum, respectively. Through co-immunoprecipitation, we confirmed that these two kinases are present in complexes with ROMK2 channels. Additionally, we found that the products of AGK and DGKE, lysophosphatidic acid (LPA) and phosphatidic acid (PA), stimulated the activity of ROMK2 channels in artificial lipid bilayers. Our molecular docking studies revealed the presence of acidic lipid binding sites in the ROMK2 channel, similar to those previously identified in Kir2 channels. Based on these findings, we propose a model wherein localized lipid synthesis, mediated by channel-bound lipid kinases, contributes to the regulation of ROMK2 activity within distinct intracellular compartments, such as mitochondria and the endoplasmic reticulum.

Pharmacological Characterization of a Recombinant Mitochondrial ROMK2 Potassium Channel Expressed in Bacteria and Reconstituted in Planar Lipid Bilayers.

In the inner mitochondrial membrane, several potassium channels that play a role in cell life and death have been identified. One of these channels is the ATP-regulated potassium channel (mitoKATP). The ROMK2 potassium channel is a potential molecular component of the mitoKATP channel. The current study aimed to investigate the pharmacological modulation of the activity of the ROMK2 potassium channel expressed in Escherichia coli bacteria. ROMK2 was solubilized in polymer nanodiscs and incorporated in planar lipid bilayers. The impact of known mitoKATP channel modulators on the activity of the ROMK2 was characterized. We found that the ROMK2 channel was activated by the mitoKATP channel opener diazoxide and blocked by mitoKATP inhibitors such as ATP/Mg2+, 5-hydroxydecanoic acid, and antidiabetic sulfonylurea glibenclamide. These results indicate that the ROMK2 potassium protein may be a pore-forming subunit of mitoKATP and that the impact of channel modulators is not related to the presence of accessory proteins.

Targeting Mitochondrial Large-Conductance Calcium-Activated Potassium Channel by Hydrogen Sulfide via Heme-Binding Site.

Reperfusion together with the preceding ischemic period results in serious damage to brain and heart tissues. Activation of potassium channels from the inner mitochondrial membrane leads to cytoprotection during such events. The mitochondrial large-conductance calcium-activated potassium channel (mitoBKCa) is one of these cytoprotective channels. It was previously shown that BKCa channels are blocked by hemin, which is present in excess during hemorrhage. In the experiments described in this work, we checked whether NaHS, known as a donor of gasotransmitter hydrogen sulfide (H2S), which can play an important role in cytoprotection, interacts with mitoBKCa channels. Indeed, using the biotin-switch method, it was found that mitoBKCa channels undergo S-sulfhydration in the presence of NaHS. Although patch-clamp experiments showed that NaHS has negligible effects on the activity of mitoBKCa channels, NaHS has been shown to almost fully activate hemin-inhibited mitoBKCa channels. The effects of NaHS were mimicked by imidazole, suggesting a common mechanism of activation of mitoBKCa channels inhibited by heme/hemin by molecules able to coordinate the iron ion of porphyrin. A set of absorption spectroscopy experiments with the 23 amino acid model peptides containing the heme-binding motif CXXCH suggested previously unrecognized roles of cysteines in heme binding. SIGNIFICANCE STATEMENT: The activity of mitochondrial channels including mitoBKCa seems to play a significant role in cytoprotection during ischemia/reperfusion. Hemin, which is present in excess during hemorrhage, can potentially bind to and inhibit mitoBKCa activity. We found that hydrogen sulfide does not affect mitoBKCa activity unless it is blocked by hemin. In this case, hydrogen sulfide activates hemin-inhibited mitoBKCa by binding to hemin iron. The hydrogen sulfide effect could be mimicked in patch-clamp experiments by imidazole probably acting by a similar mechanism.

Methods of Measuring Mitochondrial Potassium Channels: A Critical Assessment.

In this paper, the techniques used to study the function of mitochondrial potassium channels are critically reviewed. The majority of these techniques have been known for many years as a result of research on plasma membrane ion channels. Hence, in this review, we focus on the critical evaluation of techniques used in the studies of mitochondrial potassium channels, describing their advantages and limitations. Functional analysis of mitochondrial potassium channels in comparison to that of plasmalemmal channels presents additional experimental challenges. The reliability of functional studies of mitochondrial potassium channels is often affected by the need to isolate mitochondria and by functional properties of mitochondria such as respiration, metabolic activity, swelling capacity, or high electrical potential. Three types of techniques are critically evaluated: electrophysiological techniques, potassium flux measurements, and biochemical techniques related to potassium flux measurements. Finally, new possible approaches to the study of the function of mitochondrial potassium channels are presented. We hope that this review will assist researchers in selecting reliable methods for studying, e.g., the effects of drugs on mitochondrial potassium channel function. Additionally, this review should aid in the critical evaluation of the results reported in various articles on mitochondrial potassium channels.

Solubilization, purification, and functional reconstitution of human ROMK potassium channel in copolymer styrene-maleic acid (SMA) nanodiscs.

Expression, purification, and functional reconstitution of mammalian ion channels are often challenging. Heterologous expression of mammalian channels in bacteria can be advantageous due to unrelated protein environment and the lack of risk of copurification of endogenous proteins, e.g., accessory channel subunits that can influence the channel activity. Also, direct recording of channel activity could be challenging due to their intracellular localization like in the case of mitochondrial channels. The activity of purified channels can be characterized at the single-molecule level by electrophysiological techniques, such as planar lipid bilayers (PLB). In this work, we describe a simple approach to accomplish PLB recording of the activity of single renal outer medullary potassium channels ROMK expressed in E. coli. We focused on the ROMK2 isoform that is present at low levels in the mitochondria and can be responsible for mitoKATP activity. We screened for the best construct to express the codon-optimized ROMK proteins with a 6xHis tag for protein purification. The strategy involved the use of optimal styrene-maleic acid (SMA) copolymer, which forms so-called polymer nanodiscs, to solubilize and purify ROMK-containing SMA lipid particles (SMALPs), which were amenable for fusion with PLB. Reconstituted ROMK channels exhibited ion selectivity, rectification, and pharmacological properties, which are in agreement with previous work on ROMK channels.

Evidence for a mitochondrial ATP-regulated potassium channel in human dermal fibroblasts.

Mitochondrial ATP-regulated potassium channels are present in the inner membrane of the mitochondria of various cells. In the present study, we show for the first time mitochondrial ATP-regulated potassium channels in human dermal fibroblast cells. Using the patch-clamp technique on the inner mitochondrial membrane of fibroblasts, we detected a potassium channel with a mean conductance equal to 100 pS in symmetric 150 mM KCl. The activity of this channel was inhibited by a complex of ATP/Mg2+ and activated by potassium channel openers such as diazoxide or BMS 191095. Channel activity was inhibited by antidiabetic sulfonylurea glibenclamide and 5-hydroxydecanoic acid. The influence of substances modulating ATP-regulated potassium channel activity on oxygen consumption and membrane potential of isolated fibroblast mitochondria was also studied. Additionally, the potassium channel opener diazoxide lowered the amount of superoxide formed in isolated fibroblast mitochondria. Using reverse transcriptase-PCR, we found an mRNA transcript for the KCNJ1(ROMK) channel. The presence of ROMK protein was observed in the inner mitochondrial membrane fraction. Moreover, colocalization of the ROMK protein and a mitochondrial marker in the mitochondria of fibroblast cells was shown by immunofluorescence. In summary, the ATP-regulated mitochondrial potassium channel in a dermal fibroblast cell line have been identified.

Mitochondrial potassium channels - an overview.

Mitochondria play a fundamental role in ATP synthesis within the majority of mammalian cells. Potassium channels present in the inner mitochondrial membrane are fine regulators of mitochondrial function, based on inner membrane K+ permeability. These channels are regulated by a plethora of factors and conditions in a way similar to plasma membrane potassium channels. Regulators of mitochondrial potassium channels include the membrane potential, calcium ions, free fatty acids and ATP levels within the cells. Recently, it was shown that these channels are regulated by the respiratory chain, stretching of the membrane and phosphorylation. The essential interest that has driven studies of mitochondrial potassium channels for nearly 25 years is their role in cytoprotection and in cell death. Mitochondrial potassium channels have been described in neurons, astrocytoma, cardiac and skeletal muscles, fibroblasts, keratinocytes and endothelial cells. In this overview, we summarize the current knowledge of mitochondrial potassium channels. This summary will be done with a special focus on studies performed over the last 20 years in the Laboratory of Intracellular Ion Channels at the Nencki Institute. These include studies on the electrophysiological and pharmacological properties of mitochondrial potassium channels and on their regulation by endogenous intracellular substances. Additionally, the regulation of mitochondrial potassium channels by the respiratory chain and by stretching of the inner mitochondrial membrane will be reviewed. Properties of mitochondrial potassium channels in various organisms will also be summarized.