mitoBKCa Channel Research Articles

Deficiency of the BKCa potassium channel displayed significant implications for the physiology of the human bronchial epithelium

Plasma membrane large-conductance calcium-activated potassium (BKCa) channels are important players in various physiological processes, including those mediated by epithelia. Like other cell types, human bronchial epithelial (HBE) cells also express BKCa in the inner mitochondrial membrane (mitoBKCa). The genetic relationships between these mitochondrial and plasma membrane channels and the precise role of mitoBKCa in epithelium physiology are still unclear. Here, we tested the hypothesis that the mitoBKCa channel is encoded by the same gene as the plasma membrane BKCa channel in HBE cells. We also examined the impact of channel loss on the basic function of HBE cells, which is to create a tight barrier. For this purpose, we used CRISPR/Cas9 technology in 16HBE14o- cells to disrupt the KCNMA1 gene, which encodes the α-subunit responsible for forming the pore of the plasma membrane BKCa channel. Electrophysiological experiments demonstrated that the disruption of the KCNMA1 gene resulted in the loss of BKCa-type channels in the plasma membrane and mitochondria. We have also shown that HBE ΔαBKCa cells exhibited a significant decrease in transepithelial electrical resistance which indicates a loss of tightness of the barrier created by these cells. We have also observed a decrease in mitochondrial respiration, which indicates a significant impairment of these organelles.In conclusion, our findings indicate that a single gene encodes both populations of the channel in HBE cells. Furthermore, this channel is critical for maintaining the proper function of epithelial cells as a cellular barrier.

Loss of the large conductance calcium-activated potassium channel causes an increase in mitochondrial reactive oxygen species in glioblastoma cells.

Mitochondrial potassium (mitoK) channels play an important role in cellular physiology. These channels are expressed in healthy tissues and cancer cells. Activation of mitoK channels can protect neurons and cardiac tissue against injury induced by ischemia-reperfusion. In cancer cells, inhibition of mitoK channels leads to an increase in mitochondrial reactive oxygen species, which leads to cell death. In glioma cell activity of the mitochondrial, large conductance calcium-activated potassium (mitoBKCa) channel is regulated by the mitochondrial respiratory chain. In our project, we used CRISPR/Cas9 technology in human glioblastoma U-87 MG cells to generate knockout cell lines lacking the α-subunit of the BKCa channel encoded by the KCNMA1 gene, which also encodes cardiac mitoBKCa. Mitochondrial patch-clamp experiments showed the absence of an active mitoBKCa channel in knockout cells. Additionally, the absence of this channel resulted in increased levels of mitochondrial reactive oxygen species. However, analysis of the mitochondrial respiration rate did not show significant changes in oxygen consumption in the cell lines lacking BKCa channels compared to the wild-type U-87 MG cell line. These observations were reflected in the expression levels of selected mitochondrial genes, organization of the respiratory chain, and mitochondrial morphology, which did not show significant differences between the analyzed cell lines. In conclusion, we show that in U-87 MG cells, the pore-forming subunit of the mitoBKCa channel is encoded by the KCNMA1 gene. Additionally, the presence of this channel is important for the regulation of reactive oxygen species levels in mitochondria.

Effect of Quercetin on mitoBKCa Channel and Mitochondrial Function in Human Bronchial Epithelial Cells Exposed to Particulate Matter.

Particulate matter (PM) exposure increases reactive oxygen species (ROS) levels. It can lead to inflammatory responses and damage of the mitochondria thus inducing cell death. Recently, it has been shown that potassium channels (mitoK) located in the inner mitochondrial membrane are involved in cytoprotection, and one of the mechanisms involves ROS. To verify the cytoprotective role of mitoBKCa, we performed a series of experiments using a patch-clamp, transepithelial electrical resistance assessment (TEER), mitochondrial respiration measurements, fluorescence methods for the ROS level and mitochondrial membrane potential assessment, and cell viability measurements. In the human bronchial epithelial cell model (16HBE14σ), PM < 4 μm in diameter (SRM-PM4.0) was used. We observed that PM decreased TEER of HBE cell monolayers. The effect was partially abolished by quercetin, a mitoBKCa opener. Consequently, quercetin decreased the mitochondrial membrane potential and increased mitochondrial respiration. The reduction of PM-induced ROS level occurs both on cellular and mitochondrial level. Additionally, quercetin restores HBE cell viability after PM administration. The incubation of cells with PM substantially reduced the mitochondrial function. Isorhamnetin had no effect on TEER, the mitoBKCa activity, respiratory rate, or mitochondrial membrane potential. Obtained results indicate that PM has an adverse effect on HBE cells at the cellular and mitochondrial level. Quercetin is able to limit the deleterious effect of PM on barrier function of airway epithelial cells. We show that the effect in HBE cells involves mitoBKCa channel-activation. However, quercetin's mechanism of action is not exclusively determined by modulation of the channel activity.

External Hemin as an Inhibitor of Mitochondrial Large-Conductance Calcium-Activated Potassium Channel Activity.

The mitochondrial large-conductance calcium-activated potassium channel (mitoBKCa) is located in the inner mitochondrial membrane and seems to play a crucial role in cytoprotection. The mitoBKCa channel is regulated by many modulators, including activators, such as calcium ions and inhibitors, such as heme and its oxidized form hemin. Heme/hemin binds to the heme-binding motif (CXXCH) located between two RCK domains present in the mitochondrial matrix. In the present study, we used the patch-clamp technique in the outside-out configuration to record the activity of mitoBKCa channels. This allowed for the application of channel modulators to the intermembrane-space side of the mitoBKCa. We found that hemin applied in this configuration inhibits the activity of mitoBKCa. In addition, we proved that the observed hemin effect is specific and it is not due to its interaction with the inner mitochondrial membrane. Our data suggest the existence of a new potential heme/hemin binding site in the structure of the mitoBKCa channel located on the mitochondrial intermembrane space side, which could constitute a new way for the regulation of mitoBKCa channel activity.

Luteolin-Induced Activation of Mitochondrial BKCa Channels: Undisclosed Mechanism of Cytoprotection.

Luteolin (LUT) is a well-known flavonoid that exhibits a number of beneficial properties. Among these, it shows cardioprotective effects, as confirmed by numerous studies. However, its effect on mitochondrial potassium channels, the activation of which is related to cytoprotection, as well as on heart ischemia/reperfusion (I/R) damage prevention, has not yet been investigated. The large conductance calcium-regulated potassium channel (mitoBKCa) has been identified in both the mitochondria of the vascular endothelial cells, which plays a significant role in the functioning of the cardiovascular system under oxidative stress-related conditions, and in the mitochondria of cardiomyocytes, where it is deeply involved in cardiac protection against I/R injury. Therefore, the aim of this study was to explore the role of the mitoBKCa channel in luteolin-induced cytoprotection. A number of in vitro, in vivo, ex vivo and in silico studies have confirmed that luteolin activates this channel in the mitochondria of cardiomyocytes and endothelial cells, which in turn leads to the protection of the endothelium and a significant reduction in the extent of damage resulting from myocardial infarction, where this effect was partially abolished by the mitoBKCa channel blocker paxilline. In conclusion, these results suggest that luteolin has cardioprotective effects, at least in part, through the activation of the mitoBKCa channel, shedding light on a new putative mechanism of action.

The Effect of 40-Hz White LED Therapy on Structure-Function of Brain Mitochondrial ATP-Sensitive Ca-Activated Large-Conductance Potassium Channel in Amyloid Beta Toxicity.

Photobiomodulation therapy has become the focus of medical research in many areas such as Alzheimer's disease (AD), because of its modulatory effect on cellular processes through light energy absorption via photoreceptors/chromophores located in the mitochondria. However, there are still many questions around the underlying mechanisms. This study was carried out to unravel whether the function-structure of ATP-sensitive mitoBKCa channels, as crucial components for maintenance of mitochondrial homeostasis, can be altered subsequent to light therapy in AD. Induction of Aβ neurotoxicity in male Wistar rats was done by intracerebroventricular injection of Aβ1-42. After a week, light-treated rats were exposed to 40-Hz white light LEDs, 15min for 7days. Electrophysiological properties of mitoBKCa channel were investigated using a channel incorporated into the bilayer lipid membrane, and mitoBKCa-β2 subunit expression was determined using western blot analysis in Aβ-induced toxicity and light-treated rats. Our results describe that conductance and open probability (Po) of mitoBKCa channel decreased significantly and was accompanied by a Po curve rightward shift in mitochondrial preparation in Aβ-induced toxicity rats. We also showed a significant reduction in expression of mitoBKCa-β2 subunit, which is partly responsible for a leftward shift in BKCa Po curve in low calcium status. Interestingly, we provided evidence of a significant improvement in channel conductance and Po after light therapy. We also found that light therapy improved mitoBKCa-β2 subunit expression, increasing it close to saline group. The current study explains a light therapy improvement in brain mitoBKCa channel function in the Aβ-induced neurotoxicity rat model, an effect that can be linked to increased expression of β2 subunit.

Flavonoid quercetin abolish paxilline inhibition of the mitochondrial BKCa channel

Large-conductance calcium-regulated potassium channel (BKCa) is known to play an important role in physiological and pathological processes. Despite the BKCa channel being encoded by one gene, this channel has been found to be located not only in the cell membrane but also in the membranes of intracellular compartments, such as in the inner mitochondrial membrane. With some differences, the mitochondrial BKCa (mitoBKCa) channel has been shown to be activated or inhibited by both synthetic and natural compounds. One of them, paxilline, has been considered to be a canonical blocker of this channel.In the previous study, we showed that the natural origin substance quercetin activates the mitoBKCa channel at ten times lower the concentration compared to channel present in the plasma membrane. Here, using the patch-clamp technique, we report that after inhibition of mitoBKCa channels by paxilline, quercetin activates these channels, indicating a paxilline and quercetin binding competition in the regulation of the mitoBKCa channel. To support our hypothesis, we used an analog of quercetin – isorhamnetin, a substance with one substituent changed. Isorhamnetin has no effect on the mitoBKCa channel activity, and after its application, paxilline fully inhibits the channel. Additionally, the molecular modeling studies were used. The results of docking quercetin and paxilline to the BKCa channel suggest that paxilline cannot bind after activation of the channel with quercetin. It seems that the likely mechanism of this phenomenon is the formation of spatial hindrance by quercetin. The results obtained shed a completely new, groundbreaking in the paxilline context, light on the current knowledge about mitochondrial potassium channel regulation.

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.

Kv7.4 channels regulate potassium permeability in neuronal mitochondria

Mitochondrial K+ permeability regulates neuronal apoptosis, energy metabolism, autophagy, and protection against ischemia–reperfusion injury. Kv7.4 channels have been recently shown to regulate K+ permeability in cardiac mitochondria and exert cardioprotective effects. Here, the possible expression and functional role of Kv7.4 channels in regulating membrane potential, radical oxygen species (ROS) production, and Ca2+ uptake in neuronal mitochondria was investigated in both clonal (F11 cells) and native brain neurons.In coupled mitochondria isolated from F11 cells, K+-dependent changes of mitochondrial membrane potential (ΔΨ) were unaffected by the selective mitoBKCa channel blocker iberiotoxin and only partially inhibited by the mitoKATP blockers glyburide or ATP. Interestingly, K+-dependent ΔΨ decrease was significantly reduced by the Kv7 blocker XE991 and enhanced by the Kv7 activator retigabine. Among Kv7s, western blot experiments showed the expression of only Kv7.4 subunits in F11 mitochondrial fractions; immunocytochemistry experiments showed a strong overlap between the Kv7.4 fluorescent signal and that of the mitochondrial marker Mitotracker. Silencing of Kv7.4 expression significantly suppressed retigabine-dependent decrease in ΔΨ in intact F11 cells. Expression of Kv7.4 subunits was also detected by western blot in isolated mitochondria from total mouse brain and by immunofluorescence in mouse primary cortical neurons. Pharmacological experiments revealed a relevant functional role for Kv7.4 channels in regulating membrane potential and Ca2+ uptake in isolated neuronal mitochondria, as well as ΔΨ and ROS production in intact cortical neurons. In conclusion, these findings provide the first experimental evidence for the expression of Kv7.4 channels and their contribution in regulating K+ permeability of neuronal mitochondria.

Mitochondrial BKCa Mediates the Protective Effect of Low-Dose Ethanol Preconditioning on Oxygen-Glucose Deprivation and Reperfusion-Induced Neuronal Apoptosis.

Ischemia-reperfusion (I/R) injury contributes to the morbidity and mortality of ischemic strokes. As an in vitro model, oxygen-glucose deprivation and reperfusion (OGD/R) exposure induces neuronal injury. Low-dose ethanol preconditioning (EtOH-PC) was reported to alleviate neuronal apoptosis during OGD/R. However, whether the mitochondrial BKCa (mitoBKCa) channel is involved in the neuroprotective effect of EtOH-PC during OGD/R is not clearly defined. This study attempts to explore the mediation of the mitoBKCa channel in the neuroprotective effect of EtOH-PC on OGD/R-induced neuronal apoptosis and the underlying mechanisms. OGD/R model was established using primary cortical neurons that were preincubated with ethanol. Subsequently, the cell viability was measured by CCK-8 assay, and the apoptotic cells were determined by TUNEL assay. Annexin V/7-AAD staining and mitochondrial membrane potential using JC-10 were detected by flow cytometry. Western blot analysis was performed to check the apoptosis-related proteins. In the mixed primary culture, 95% neurofilament-positive cells were cortical neurons. Low-dose EtOH-PC (10 mmol/L) for 24 h significantly attenuated the OGD2h/R24h-induced neuronal apoptosis through activating the BKCa channel. Further investigations suggested that ethanol pretreatment increased the mitochondrial membrane potential (MMP) and downregulated the production of cleaved caspase 3 in OGD/R-injured neurons by activating the mitoBKCa channel. Low-dose ethanol pretreatment significantly attenuated the OGD/R-induced neuronal apoptosis mediated by the mitoBKCa channel which modulated the mitochondrial function by impeding the uncontrolled opening of mitochondrial permeability transition pore (MPTP).

Cytoprotective effects of the flavonoid quercetin by activating mitochondrial BKCa channels in endothelial cells.

Mitochondrial potassium channels have been implicated in cytoprotective mechanisms. Activation of the mitochondrial large-conductance Ca2+-regulated potassium (mitoBKCa) channel is important for protecting brain tissue against stroke damage as well as heart tissue against ischemia damage. In this paper, we examine the effect of the natural flavonoid quercetin as an activator of the mitoBKCa channel. Quercetin has a beneficial effect on many processes in the human body and interacts with many receptors and signaling pathways. We found that quercetin acts on mitochondria as a mitoBKCa channel opener. The activation observed with the patch-clamp technique was potent and increased the channel open probability from approximately 0.35 to 0.95 at + 40 mV in the micromolar concentration range. Moreover, quercetin at a concentration of 10 µM protected cells by reducing damage from treatment factors (tumor necrosis factor α and cycloheximide) by 40%, enhancing cellular migration and depolarizing the mitochondrial membrane. Moreover, the presence of quercetin increased the gene expression and protein level of the mitoBKCa β3 regulatory subunit. The observed cytoprotective effects suggested the involvement of BKCa channel activation. Additionally, the newly discovered mitoBKCa activator quercetin elucidates a new mitochondrial pathway that is beneficial for vascular endothelial cells.

Identification of the Large-Conductance Ca2+-Regulated Potassium Channel in Mitochondria of Human Bronchial Epithelial Cells

Mitochondria play a key role in energy metabolism within the cell. Potassium channels such as ATP-sensitive, voltage-gated or large-conductance Ca2+-regulated channels have been described in the inner mitochondrial membrane. Several hypotheses have been proposed to describe the important roles of mitochondrial potassium channels in cell survival and death pathways. In the current study, we identified two populations of mitochondrial large-conductance Ca2+-regulated potassium (mitoBKCa) channels in human bronchial epithelial (HBE) cells. The biophysical properties of the channels were characterized using the patch-clamp technique. We observed the activity of the channel with a mean conductance close to 285 pS in symmetric 150/150 mM KCl solution. Channel activity was increased upon application of the potassium channel opener NS11021 in the micromolar concentration range. The channel activity was completely inhibited by 1 µM paxilline and 300 nM iberiotoxin, selective inhibitors of the BKCa channels. Based on calcium and iberiotoxin modulation, we suggest that the C-terminus of the protein is localized to the mitochondrial matrix. Additionally, using RT-PCR, we confirmed the presence of α pore-forming (Slo1) and auxiliary β3-β4 subunits of BKCa channel in HBE cells. Western blot analysis of cellular fractions confirmed the mitochondrial localization of α pore-forming and predominately β3 subunits. Additionally, the regulation of oxygen consumption and membrane potential of human bronchial epithelial mitochondria in the presence of the potassium channel opener NS11021 and inhibitor paxilline were also studied. In summary, for the first time, the electrophysiological and functional properties of the mitoBKCa channel in a bronchial epithelial cell line were described.

Single channel properties of mitochondrial large conductance potassium channel formed by BK-VEDEC splice variant

The activation of mitochondrial large conductance calcium-activated potassium (mitoBKCa) channels increases cell survival during ischemia/reperfusion injury of cardiac cells. The basic biophysical and pharmacological properties of mitoBKCa correspond to the properties of the BKCa channels from the plasma membrane. It has been suggested that the VEDEC splice variant of the KCNMA1 gene product encoding plasma membrane BKCa is targeted toward mitochondria. However there has been no direct evidence that this protein forms a functional channel in mitochondria. In our study, we used HEK293T cells to express the VEDEC splice variant and observed channel activity in mitochondria using the mitoplast patch-clamp technique. For the first time, we found that transient expression with the VEDEC isoform resulted in channel activity with the conductance of 290 ± 3 pS. The channel was voltage-dependent and activated by calcium ions. Moreover, the activity of the channel was stimulated by the potassium channel opener NS11021 and inhibited by hemin and paxilline, which are known BKCa channel blockers. Immunofluorescence experiments confirmed the partial colocalization of the channel within the mitochondria. From these results, we conclude that the VEDEC isoform of the BKCa channel forms a functional channel in the inner mitochondrial membrane. Additionally, our data show that HEK293T cells are a promising experimental model for expression and electrophysiological studies of mitochondrial potassium channels.

Intranasal insulin improves the structure–function of the brain mitochondrial ATP–sensitive Ca2+ activated potassium channel and respiratory chain activities under diabetic conditions

Although it is well established that diabetes impairs mitochondrial respiratory chain activity, little is known of the effects of intranasal insulin (INI) on the mitochondrial respiratory chain and structure-function of mitoBKCa channel in diabetes. We have investigated this mechanism in an STZ-induced early type 2 diabetic model. Single ATP-sensitive mitoBKCa channel activity was considered in diabetic and INI-treated rats using a channel incorporated into the bilayer lipid membrane. Because mitoBKCa channels have been involved in mitochondrial respiratory chain activity, a study was undertaken to investigate whether the NADH, complexes I and IV, mitochondrial ROS production, and ΔΨm are altered in an early diabetic model. In this work, we provide evidence for a significant decrease in channel open probability and conductance in diabetic rats. Evidence has been shown that BKCa channel β2 subunits induce a left shift in the BKCa channel voltage dependent curve in low Ca2+ conditions,; our results indicated a significant decrease in mitoBKCa β2 subunits using Western blot analysis. Importantly, INI treatment improved mitoBKCa channel behaviors and β2 subunits expression up to ~70%. We found that early diabetes decreased activities of complex I and IV and increased NADH, ROS production, and ΔΨm. Surprisingly, INI modified the mitochondrial respiratory chain, ROS production, and ΔΨm up to ~70%. Our results thus demonstrate an INI improvement in respiratory chain activity and ROS production in brain mitochondrial preparations coming from the STZ early diabetic rat model, an effect potentially linked to INI improvement in mitoBKCa channel activity and channel β2 subunit expression.

Regulation of the Mitochondrial BKCa Channel by the Citrus Flavonoid Naringenin as a Potential Means of Preventing Cell Damage.

Naringenin, a flavanone obtained from citrus fruits and present in many traditional Chinese herbal medicines, has been shown to have various beneficial effects on cells both in vitro and in vivo. Although the antioxidant activity of naringenin has long been believed to be crucial for its effects on cells, mitochondrial pathways (including mitochondrial ion channels) are emerging as potential targets for the specific pharmacological action of naringenin in cardioprotective strategies. In the present study, we describe interactions between the mitochondrial large-conductance calcium-regulated potassium channel (mitoBKCa channel) and naringenin. Using the patch-clamp method, we showed that 10 µM naringenin activated the mitoBKCa channel present in endothelial cells. In the presence of 30 µM Ca2+, the increase in the mitoBKCa channel probability of opening from approximately 0.25 to 0.50 at −40 mV was observed. In addition, regulation of the mitoBKCa channel by naringenin was dependent on the concentration of calcium ions. To confirm our data, physiological studies on the mitochondria were performed. An increase in oxygen consumption and a decrease in membrane potential was observed after naringenin treatment. In addition, contributions of the mitoBKCa channel to apoptosis and necrosis were investigated. Naringenin protected cells against damage induced by tumor necrosis factor α (TNF-α) in combination with cycloheximide. In this study, we demonstrated that the flavonoid naringenin can activate the mitoBKCa channel present in the inner mitochondrial membrane of endothelial cells. Our studies describing the regulation of the mitoBKCa channel by this natural, plant-derived substance may help to elucidate flavonoid-induced cytoprotective mechanisms.

Heme is required for carbon monoxide activation of mitochondrial BKCa channel

Carbon monoxide (CO) is an endogenously synthesized gaseous mediator and is involved in the regulation of numerous physiological processes. Mitochondria, in which hemoproteins are abundant, are among the targets for CO action. Large-conductance calcium-activated (mitoBKCa) channels in the inner mitochondrial membrane share multiple biophysical similarities with the BKCa channels of the plasma membrane and could be a potential target for CO. To test this hypothesis, the activity of the mitoBKCa channels in human astrocytoma U-87 MG cell mitochondria was assessed with the patch-clamp technique. The effects of CO-releasing molecules (CORMs), such as CORM-2, CORM-401, and CORM-A1, were compared to the application of a CO-saturated solution to the mitoBKCa channels in membrane patches. The applied CORMs showed pleiotropic effects including channel inhibition, while the CO-containing solution did not significantly modulate channel activity. Interestingly, CO applied to the mitoBKCa channels, which were inhibited by exogenously added heme, stimulated the channel. To summarize, our findings indicate a requirement of heme binding to the mitoBKCa channel for channel modulation by CO and suggest that CORMs might have complex unspecific effects on mitoBKCa channels.

The monomers, oligomers, and fibrils of amyloid-β inhibit the activity of mitoBKCa channels by a membrane-mediated mechanism

A causative agent of Alzheimer's disease (AD) is a short amphipathic peptide called amyloid beta (Aβ). Aβ monomers undergo structural changes leading to their oligomerization or fibrillization. The monomers as well as all aggregated forms of Aβ, i.e., oligomers, and fibrils, can bind to biological membranes, thereby modulating membrane mechanical properties. It is also known that some isoforms of the large-conductance calcium-activated potassium (BKCa) channel, including the mitochondrial BKCa (mitoBKCa) channel, respond to mechanical changes in the membrane. Here, using the patch-clamp technique, we investigated the impact of full-length Aβ (Aβ1–42) and its fragment, Aβ25–35, on the activity of mitoBKCa channels. We found that all forms of Aβ inhibited the activity of the mitoBKCa channel in a concentration-dependent manner. Since monomers, oligomers, and fibrils of Aβ exhibit different molecular characteristics and structures, we hypothesized that the inhibition was not due to direct peptide-protein interactions but rather to membrane-binding of the Aβ peptides. Our findings supported this hypothesis by showing that Aβ peptides block mitoBKCa channels irrespective of the side of the membrane to which they are applied. In addition, we found that the enantiomeric peptide, D-Aβ1–42, demonstrated similar inhibitory activity towards mitoBKCa channels. As a result, we proposed a general model in which all Aβ forms i.e., monomers, oligomers, and amyloid fibrils, contribute to the progression of AD by exerting a modulatory effect on mechanosensitive membrane components.

MitoBKCa channel is functionally associated with its regulatory β1 subunit in cardiac mitochondria.

Association of plasma membrane BKCa channels with BK-β subunits shapes their biophysical properties and physiological roles; however, functional modulation of the mitochondrial BKCa channel (mitoBKCa ) by BK-β subunits is not established. MitoBKCa -α and the regulatory BK-β1 subunit associate in mouse cardiac mitochondria. A large fraction of mitoBKCa display properties similar to that of plasma membrane BKCa when associated with BK-β1 (left-shifted voltage dependence of activation, V1/2 =-55mV, 12µm matrix Ca2+ ). In BK-β1 knockout mice, cardiac mitoBKCa displayed a low Po and a depolarized V1/2 of activation (+47mV at 12µm matrix Ca2+ ) Co-expression of BKCa with the BK-β1 subunit in HeLa cells doubled the density of BKCa in mitochondria. The present study supports the view that the cardiac mitoBKCa channel is functionally modulated by the BK-β1 subunit; proper targeting and activation of mitoBKCa shapes mitochondrial Ca2+ handling. Association of the plasma membrane BKCa channel with auxiliary BK-β1-4 subunits profoundly affects the regulatory mechanisms and physiological processes in which this channel participates. However, functional association of mitochondrial BK (mitoBKCa ) with regulatory subunits is unknown. We report that mitoBKCa functionally associates with its regulatory subunit BK-β1 in adult rodent cardiomyocytes. Cardiac mitoBKCa is a calcium- and voltage-activated channel that is sensitive to paxilline with a large conductance for K+ of 300pS. Additionally, mitoBKCa displays a high open probability (Po ) and voltage half-activation (V1/2 =-55mV, n=7) resembling that of plasma membrane BKCa when associated with its regulatory BK-β1 subunit. Immunochemistry assays demonstrated an interaction between mitochondrial BKCa -α and its BK-β1 subunit. Mitochondria from the BK-β1 knockout (KO) mice showed sparse mitoBKCa currents (five patches with mitoBKCa activity out of 28 total patches from n=5 different hearts), displaying a depolarized V1/2 of activation (+47mV in 12µm matrix Ca2+ ). The reduced activity of mitoBKCa was accompanied by a high expression of BKCa transcript in the BK-β1 KO, suggesting a lower abundance of mitoBKCa channels in this genotype. Accordingly, BK-β1subunit increased the localization of BKDEC (i.e. the splice variant of BKCa that specifically targets mitochondria) into mitochondria by two-fold. Importantly, both paxilline-treated and BK-β1 KO mitochondria displayed a more rapid Ca2+ overload, featuring an early opening of the mitochondrial transition pore. We provide strong evidence that mitoBKCa associates with its regulatory BK-β1 subunit in cardiac mitochondria, ensuring proper targeting and activation of the mitoBKCa channel that helps to maintain mitochondrial Ca2+ homeostasis.

Naringenin as an opener of mitochondrial potassium channels in dermal fibroblasts.

Flavonoids belong to a large group of polyphenolic compounds that are widely present in plants. Certain flavonoids, including naringenin, have cytoprotective properties. Although the antioxidant effect has long been thought to be a crucial factor accounting for the cellular effects of flavonoids, mitochondrial channels have emerged recently as targets for cytoprotective strategies. In the present study, we characterized interactions between naringenin and the mitochondrial potassium (mitoBKCa and mitoKATP ) channels recently described in dermal fibroblasts. With the use of the patch-clamp technique and mitoplasts isolated from primary human dermal fibroblast cells, our study shows that naringenin in micromolar concentrations leads to an increase in mitoBKCa channel activity. The opening probability of the channel decreased from 0.97 in the control conditions (200μmol/L Ca2+ ) to 0.06 at a low Ca2+ level (1μmol/L) and increased to 0.85 after the application of 10μmol/L naringenin. Additionally, the activity of the mitoKATP channel increased following the application of 10μmol/L naringenin. To investigate the effects of naringenin on mitochondrial function, the oxygen consumption of dermal fibroblast cells was measured in potassium-containing media. The addition of naringenin significantly and dose-dependently increased the respiratory rate from 5.8±0.2 to 14.0±0.6nmol O2 ×min-1 ×mg protein-1 . Additionally, a Raman spectroscopy analysis of skin penetration indicated that the naringenin was distributed in all skin layers, including the epidermis and dermis. In this study, we demonstrated that a flavonoid, naringenin, can activate two potassium channels present in the inner mitochondrial membrane of dermal fibroblasts.

Single Channel Recordings of mitoBKCa Channel Formed by BK-Dec Splice Variant

Single Channel Recordings of mitoBKCa Channel Formed by BK-Dec Splice Variant