Large-conductance Ca2+-activated potassium channel in mitochondria of endothelial EA.hy926 cells

2013 ◽  
Vol 304 (11) ◽  
pp. H1415-H1427 ◽  
Author(s):  
Piotr Bednarczyk ◽  
Agnieszka Koziel ◽  
Wieslawa Jarmuszkiewicz ◽  
Adam Szewczyk
Keyword(s):  
Potassium Channel ◽  
Mitochondrial Membrane ◽  
Single Channel ◽  
Dependent Manner ◽  
Channel Activity ◽  
Inner Mitochondrial Membrane ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Α Subunit ◽  
Ea.Hy926 Cells

In the present study, we describe the existence of a large-conductance Ca2+-activated potassium (BKCa) channel in the mitochondria of the human endothelial cell line EA.hy926. A single-channel current was recorded from endothelial mitoplasts (i.e., inner mitochondrial membrane) using the patch-clamp technique in the mitoplast-attached mode. A potassium-selective current was recorded with a mean conductance equal to 270 ± 10 pS in a symmetrical 150/150 mM KCl isotonic solution. The channel activity, which was determined as the open probability, increased with the addition of calcium ions and the potassium channel opener NS1619. Conversely, the activity of the channel was irreversibly blocked by paxilline and iberiotoxin, BKCa channel inhibitors. The open-state probability was found to be voltage dependent. The substances known to modulate BKCa channel activity influenced the bioenergetics of mitochondria isolated from human endothelial EA.hy926 cells. In isolated mitochondria, 100 μM Ca2+, 10 μM NS1619, and 0.5 μM NS11021 depolarized the mitochondrial membrane potential and stimulated nonphosphorylating respiration. These effects were blocked by iberiotoxin and paxilline in a potassium-dependent manner. Under phosphorylating conditions, NS1619-induced, iberiotoxin-sensitive uncoupling diverted energy from ATP synthesis during the phosphorylating respiration of the endothelial mitochondria. Immunological analysis with antibodies raised against proteins of the plasma membrane BKCa channel identified a pore-forming α-subunit and an auxiliary β2-subunit of the channel in the endothelial mitochondrial inner membrane. In conclusion, we show for the first time that the inner mitochondrial membrane in human endothelial EA.hy926 cells contains a large-conductance calcium-dependent potassium channel with properties similar to those of the surface membrane BKCa channel.

Modulation of ATP-sensitive K+ channels by internal acidification in insulin-secreting cells

1994 ◽  
Vol 267 (4) ◽  
pp. C1036-C1044 ◽  
Author(s):  
Z. Fan ◽  
Y. Tokuyama ◽  
J. C. Makielski
Keyword(s):  
Insulin Secretion ◽  
Cell Line ◽  
Single Channel ◽  
Inhibitory Effect ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Activation Effect ◽  
Insulin Secreting Cells ◽  
Single Channel Currents

The effect of intracellular acidification (low pHi) on open probability of the ATP-sensitive K+ (KATP) channel was examined in insulin-secretion cells using an inside-out configuration of the patch-clamp technique. In an insulin-secreting cell line beta-TC3, KATP single-channel currents (IKATP) were readily recorded in the absence of internal ATP. ATP (50 microM and 0.5 mM) dramatically decreased the channel activity. A step decrease of intracellular pH (pHi) from 7.4 to 6.7 or 6.3 in the presence of ATP gradually increased the channel activity. In addition, low pHi in the presence of ATP could partially restore channel activity lost in a process called "rundown." Kinetic analysis revealed a change in channel gating at low pHi with ATP. The bursting durations of IKATP at pHi 6.3 in the presence of ATP were significantly longer than those at pHi 7.4 in the absence of ATP. These results suggest that the increased channel activity at low pHi might have resulted from a mechanism involving an alteration of channel conformation. We also observed an inhibitory effect of low pHi on channel activity. However, the inhibitory effect was much more apparent at pHi 5.7 and was only partially reversible. The activation effect of low pHi on IKATP in the presence of ATP was also observed in acutely isolated rat islet cells and in another insulin-secretion cell line RINm5F, although the effect was weaker and was variable among experiments. We conclude that, as in frog skeletal muscle and cardiac muscle, an increase in channel activity at low pHi is one of the mechanisms underlying proton modulation of IKATP in insulin-secreting cells.

scholarly journals Characterization of the calcium-activated chloride channel in isolated guinea-pig hepatocytes.

1994 ◽  
Vol 104 (2) ◽  
pp. 357-373 ◽  
Author(s):  
S Koumi ◽  
R Sato ◽  
T Aramaki
Keyword(s):  
Guinea Pig ◽  
Single Channel ◽  
Hill Coefficient ◽  
Disulfonic Acid ◽  
Dependent Manner ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Cl Channels ◽  
Inside Out

Macroscopic and unitary currents through Ca(2+)-activated Cl- channels were examined in enzymatically isolated guinea-pig hepatocytes using whole-cell, excised outside-out and inside-out configurations of the patch-clamp technique. When K+ conductances were blocked and the intracellular Ca2+ concentration ([Ca2+]i) was set at 1 microM (pCa = 6), membrane currents were observed under whole-cell voltage-clamp conditions. The reversal potential of the current shifted by approximately 60 mV per 10-fold change in the external Cl- concentration. In addition, the current did not appear when Cl- was omitted from the internal and external solutions, indicating that the current was Cl- selective. The current was activated by increasing [Ca2+]i and was inactivated in Ca(2+)-free, 5 mM EGTA internal solution (pCa > 9). The current was inhibited by bath application of 9-anthracenecarboxylic acid (9-AC) and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) in a voltage-dependent manner. In single channel recordings from outside-out patches, unitary current activity was observed, whose averaged slope conductance was 7.4 +/- 0.5 pS (n = 18). The single channel activity responded to extracellular Cl- changes as expected for a Cl- channel current. The open time distribution was best described by a single exponential function with mean open lifetime of 97.6 +/- 10.4 ms (n = 11), while at least two exponentials were required to fit the closed time distributions with a time constant for the fast component of 21.5 +/- 2.8 ms (n = 11) and that for the slow component of 411.9 +/- 52.0 ms (n = 11). In excised inside-out patch recordings, channel open probability was sensitive to [Ca2+]i. The relationship between [Ca2+]i and channel activity was fitted by the Hill equation with a Hill coefficient of 3.4 and the half-maximal activation was 0.48 microM. These results suggest that guinea-pig hepatocytes possess Ca(2+)-activated Cl- channels.

scholarly journals BK in Double-Membrane Organelles: A Biophysical, Pharmacological, and Functional Survey

2021 ◽  
Vol 12 ◽  
Author(s):  
Naileth González-Sanabria ◽  
Felipe Echeverría ◽  
Ignacio Segura ◽  
Rosangelina Alvarado-Sánchez ◽  
Ramon Latorre
Keyword(s):  
Skeletal Muscle ◽  
Plasma Membrane ◽  
Potassium Channel ◽  
Lipid Bilayers ◽  
Bk Channels ◽  
Bk Channel ◽  
Inner Mitochondrial Membrane ◽  
Modular Architecture ◽  
Open Probability ◽  
Α Subunit

In the 1970s, calcium-activated potassium currents were recorded for the first time. In 10years, this Ca2+-activated potassium channel was identified in rat skeletal muscle, chromaffin cells and characterized in skeletal muscle membranes reconstituted in lipid bilayers. This calcium- and voltage-activated potassium channel, dubbed BK for “Big K” due to its large ionic conductance between 130 and 300 pS in symmetric K+. The BK channel is a tetramer where the pore-forming α subunit contains seven transmembrane segments. It has a modular architecture containing a pore domain with a highly potassium-selective filter, a voltage-sensor domain and two intracellular Ca2+ binding sites in the C-terminus. BK is found in the plasma membrane of different cell types, the inner mitochondrial membrane (mitoBK) and the nuclear envelope’s outer membrane (nBK). Like BK channels in the plasma membrane (pmBK), the open probability of mitoBK and nBK channels are regulated by Ca2+ and voltage and modulated by auxiliary subunits. BK channels share common pharmacology to toxins such as iberiotoxin, charybdotoxin, paxilline, and agonists of the benzimidazole family. However, the precise role of mitoBK and nBK remains largely unknown. To date, mitoBK has been reported to play a role in protecting the heart from ischemic injury. At the same time, pharmacology suggests that nBK has a role in regulating nuclear Ca2+, membrane potential and expression of eNOS. Here, we will discuss at the biophysical level the properties and differences of mitoBK and nBK compared to those of pmBK and their pharmacology and function.

scholarly journals Mitochondrial potassium channels: A novel calcitriol target

2022 ◽  
Vol 27 (1) ◽  
Author(s):  
Anna M. Olszewska ◽  
Adam K. Sieradzan ◽  
Piotr Bednarczyk ◽  
Adam Szewczyk ◽  
Michał A. Żmijewski
Keyword(s):  
Vitamin D ◽  
Potassium Channels ◽  
Potassium Channel ◽  
Mitochondrial Membrane ◽  
Human Keratinocytes ◽  
Inner Mitochondrial Membrane ◽  
Open Probability ◽  
Expression Of Genes ◽  
High Calcium ◽  
Genes Encoding

Abstract Background Calcitriol (an active metabolite of vitamin D) modulates the expression of hundreds of human genes by activation of the vitamin D nuclear receptor (VDR). However, VDR-mediated transcriptional modulation does not fully explain various phenotypic effects of calcitriol. Recently a fast non-genomic response to vitamin D has been described, and it seems that mitochondria are one of the targets of calcitriol. These non-classical calcitriol targets open up a new area of research with potential clinical applications. The goal of our study was to ascertain whether calcitriol can modulate mitochondrial function through regulation of the potassium channels present in the inner mitochondrial membrane. Methods The effects of calcitriol on the potassium ion current were measured using the patch-clamp method modified for the inner mitochondrial membrane. Molecular docking experiments were conducted in the Autodock4 program. Additionally, changes in gene expression were investigated by qPCR, and transcription factor binding sites were analyzed in the CiiiDER program. Results For the first time, our results indicate that calcitriol directly affects the activity of the mitochondrial large-conductance Ca2+-regulated potassium channel (mitoBKCa) from the human astrocytoma (U-87 MG) cell line but not the mitochondrial calcium-independent two-pore domain potassium channel (mitoTASK-3) from human keratinocytes (HaCaT). The open probability of the mitoBKCa channel in high calcium conditions decreased after calcitriol treatment and the opposite effect was observed in low calcium conditions. Moreover, using the AutoDock4 program we predicted the binding poses of calcitriol to the calcium-bound BKCa channel and identified amino acids interacting with the calcitriol molecule. Additionally, we found that calcitriol influences the expression of genes encoding potassium channels. Such a dual, genomic and non-genomic action explains the pleiotropic activity of calcitriol. Conclusions Calcitriol can regulate the mitochondrial large-conductance calcium-regulated potassium channel. Our data open a new chapter in the study of non-genomic responses to vitamin D with potential implications for mitochondrial bioenergetics and cytoprotective mechanisms.

Activation by Membrane Stretch and Depolarization of an Epithelial Monovalent Cation Channel from Teleost Intestine

1992 ◽  
Vol 169 (1) ◽  
pp. 87-104
Author(s):  
WENHAN CHANG ◽  
CHRISTOPHER A. LORETZ
Keyword(s):  
Single Channel ◽  
Applied Pressure ◽  
Cell Volume Regulation ◽  
Monovalent Cation ◽  
Dependent Manner ◽  
Membrane Tension ◽  
Channel Activity ◽  
Open Probability ◽  
Membrane Stretch ◽  
Inside Out

The intestine of euryhaline teleosts is an important osmoregulatory organ which actively absorbs Na+, Cl− and water from the lumen. This ion-transporting epithelium experiences a variety of physical stimuli resulting from variations in luminal osmolality and distension and from peristaltic contractions. Using patchclamp techniques in the inside-out configuration, single stretch-activated channels (SA channels) were identified and characterized. These SA channels had a conductance of about 67 pS in symmetrical solutions containing 140 mmoll−1 NaCl and were permeable to both Na+ and K+ (PNa/PK≈0.83) but not to anions. In excised, inside-out membrane patches, channel activity could be enhanced in the absence of membrane tension by strong depolarization of the membrane potential (Vm) to between 0 mV and + 90 mV, with Vo [Vm at which the single-channel open probability (Po)=0.5] at + 25.7 mV. In the presence of membrane tension, the voltage-dependence of channel activity was shifted into the physiological range of Vm. Each kPa (10 cmH2O) of applied pressure (ΔP) generated the same effect on Po as a membrane depolarization of 49 mV. Membrane tension also increased the single-channel current and single-channel conductance in a dose-dependent manner. The kinetic data suggest that this channel has two open states and three closed states. Both stretch- and depolarization-induced increases in Po were attributed to prolongation of the lifetime of the longer open state. Possible physiological roles for this channel include the cellular uptake of Na+ from the lumen as part of the salt and water absorptive process or a yet undefined involvement in cell volume regulation.

Isoflurane Decreases ATP Sensitivity of Guinea Pig Cardiac Sarcolemmal KATPChannel at Reduced Intracellular pH

2003 ◽  
Vol 98 (2) ◽  
pp. 396-403 ◽  
Author(s):  
Anna Stadnicka ◽  
Zeljko J. Bosnjak
Keyword(s):  
Guinea Pig ◽  
Intracellular Ph ◽  
Single Channel ◽  
Volatile Anesthetics ◽  
Dependent Manner ◽  
Channel Activity ◽  
Open Probability ◽  
Concentration Dependent Manner ◽  
Channel Interaction ◽  
Channel Opening

Background Volatile anesthetics can protect the myocardium against ischemic injury by opening the adenosine triphosphate (ATP)-sensitive potassium (K(atp)) channels. However, direct evidence for anesthetic-channel interaction is still limited, and little is known about the role K(atp) channel modulators play in this effect. Because pH is one of the regulators of K(atp) channels, the authors tested the hypothesis that intracellular pH (pHi) modulates the direct interaction of isoflurane with the cardiac K(atp) channel. Methods The effects of isoflurane on sarcolemmal K(atp) channels were investigated at pHi 7.4 and pHi 6.8 in excised inside-out membrane patches from ventricular myocytes of guinea pig hearts. Results At pHi 7.4, intracellular ATP (1-1,000 microm) inhibited K(atp) channels and decreased channel open probability (Po) in a concentration-dependent manner with an IC(50) of 8 +/- 1.5 microm, and isoflurane (0.5 mm) either had no effect or decreased channel activity. Lowering pHi from 7.4 to 6.8 enhanced channel opening by increasing Po and reduced channel sensitivity to ATP, with IC shifting from 8 +/- 1.2 to 45 +/- 5.6 microm. When applied to the channels activated at pHi 6.8, isoflurane (0.5 mm) increased Po and further reduced ATP sensitivity, shifting IC(50) to 110 +/- 10.0 microm. Conclusions Changes in pHi appear to modulate isoflurane interaction with the cardiac K(atp) channel. At pHi 6.8, which itself facilitates channel opening, isoflurane enhances channel activity by increasing Po and reduces sensitivity to inhibition by ATP without changing the unitary amplitude of single channel current or the conductance. These results support the hypothesis of direct isoflurane-K(atp) channel interaction that may play a role in cardioprotection by volatile anesthetics.

scholarly journals A large-conductance calcium-activated potassium channel in potato (Solanum tuberosum) tuber mitochondria

2009 ◽  
Vol 424 (2) ◽  
pp. 307-316 ◽  
Author(s):  
Izabela Koszela-Piotrowska ◽  
Karolina Matkovic ◽  
Adam Szewczyk ◽  
Wieslawa Jarmuszkiewicz
Keyword(s):  
Solanum Tuberosum ◽  
Potassium Channel ◽  
Potato Tuber ◽  
Mitochondrial Membrane ◽  
Dependent Manner ◽  
Channel Activity ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane ◽  
Calcium Activated Potassium Channel ◽  
Potassium Channel Activity

In the present study, we describe the existence of a novel potassium channel in the plant [potato (Solanum tuberosum) tuber] mitochondrial inner membrane. We found that substances known to modulate large-conductance calcium-activated potassium channel activity influenced the bioenergetics of potato tuber mitochondria. In isolated mitochondria, Ca2+ and NS1619 {1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-ben-zimidazole-2-one; a potassium channel opener} were found to depolarize the mitochondrial membrane potential and to stimulate resting respiration. These effects were blocked by iberiotoxin (a potassium channel inhibitor) in a potassium-dependent manner. Additionally, the electrophysiological properties of the large-conductance potassium channel present in the potato tuber inner mitochondrial membrane are described in a reconstituted system, using planar lipid bilayers. After incorporation in 50/450 mM KCl gradient solutions, we recorded large-conductance potassium channel activity with conductance from 502±15 to 615±12 pS. The probability of channel opening was increased by Ca2+ and reduced by iberiotoxin. Immunological analysis with antibodies raised against the mammalian plasma-membrane large-conductance Ca2+-dependent K+ channel identified a pore-forming α subunit and an auxiliary β2 subunit of the channel in potato tuber mitochondrial inner membrane. These results suggest that a large-conductance calcium-activated potassium channel similar to that of mammalian mitochondria is present in potato tuber mitochondria.

scholarly journals Characterization of human cardiac mitochondrial ATP-sensitive potassium channel and its regulation by phorbol ester in vitro

2006 ◽  
Vol 290 (5) ◽  
pp. H1770-H1776 ◽  
Author(s):  
Ming Tao Jiang ◽  
Marko Ljubkovic ◽  
Yuri Nakae ◽  
Yang Shi ◽  
Wai-Meng Kwok ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Mitochondrial Membrane ◽  
Alternative Pathway ◽  
Dependent Manner ◽  
Inner Mitochondrial Membrane ◽  
Open Probability ◽  
Pharmaceutical Agents ◽  
Inactive Analog

Activation of the mitochondrial ATP-sensitive K+ channel (mitoKATP) and its regulation by PKC are critical events in preconditioning induced by ischemia or pharmaceutical agents in animals and humans. The properties of the human cardiac mitoKATP channel are unknown. Furthermore, there is no evidence that cytosolic PKC can directly regulate the mitoKATP channel located in the inner mitochondrial membrane (IMM) due to the physical barrier of the outer mitochondrial membrane. In the present study, we characterized the human cardiac mitoKATP channel and its potential regulation by PKC associated with the IMM. IMM fractions isolated from human left ventricles were fused into lipid bilayers in symmetrical potassium glutamate (150 mM). The conductance of native mitoKATP channels was usually below 80 pS (∼70%), which was reduced by ATP and 5-hydroxydecanoic acid (5-HD) in a dose- and time-dependent manner. The native mitoKATP channel is activated by diazoxide and inhibited by ATP and 5-HD. The PKC activator phorbol 12-myristate 13-acetate (2 μM) increased the cumulative open probability of the mitoKATP channel previously inhibited by ATP ( P < 0.05), but its inactive analog 4α-phorbol 12,13-didecanoate had no effect. Western blot analysis detected an inward rectifying K+ channel (Kir6.2) immunoreactive protein at 56 kDa and PKC-δ in the IMM. These data provide the first characterization of the human cardiac mitoKATP channel and its regulation by PKC(s) in IMM. This local PKC control mechanism may represent an alternative pathway to that proposed previously for cytosolic PKC during ischemic/pharmacological preconditioning.

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

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shur Gałecka ◽  
Bogusz Kulawiak ◽  
Piotr Bednarczyk ◽  
Harpreet Singh ◽  
Adam Szewczyk
Keyword(s):  
Plasma Membrane ◽  
Potassium Channel ◽  
Splice Variant ◽  
Single Channel ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Cardiac Cells ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Functional Channel

AbstractThe 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.

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