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

2019 ◽  
Vol 20 (21) ◽  
pp. 5323 ◽  
Author(s):  
Michał Laskowski ◽  
Bartłomiej Augustynek ◽  
Piotr Bednarczyk ◽  
Monika Żochowska ◽  
Justyna Kalisz ◽  
...  
Keyword(s):  
Potassium Channel ◽  
Single Channel ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Proteinase K ◽  
Mitochondrial Matrix ◽  
Inner Mitochondrial Membrane ◽  
Single Channel Activity ◽  
First Time ◽  
Channel Properties

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.

scholarly journals Mitochondrial matrix K+ flux independent of large-conductance Ca2+-activated K+ channel opening

2010 ◽  
Vol 298 (3) ◽  
pp. C530-C541 ◽  
Author(s):  
Mohammed Aldakkak ◽  
David F. Stowe ◽  
Qunli Cheng ◽  
Wai-Meng Kwok ◽  
Amadou K. S. Camara
Keyword(s):  
Lipid Bilayers ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Reactive Oxygen Species Generation ◽  
K Channel ◽  
Mitochondrial Matrix ◽  
Inner Mitochondrial Membrane ◽  
Channel Opening ◽  
Matrix Volume ◽  
Change Matrix

Large-conductance Ca2+-activated K+ channels (BKCa) in the inner mitochondrial membrane may play a role in protecting against cardiac ischemia-reperfusion injury. NS1619 (30 μM), an activator of BKCa channels, was shown to increase respiration and to stimulate reactive oxygen species generation in isolated cardiac mitochondria energized with succinate. Here, we tested effects of NS1619 to alter matrix K+, H+, and swelling in mitochondria isolated from guinea pig hearts. We found that 30 μM NS1619 did not change matrix K+, H+, and swelling, but that 50 and 100 μM NS1619 caused a concentration-dependent increase in matrix K+ influx (PBFI fluorescence) only when quinine was present to block K+/H+ exchange (KHE); this was accompanied by increased mitochondrial matrix volume (light scattering). Matrix pH (BCECF fluorescence) was decreased slightly by 50 and 100 μM NS1619 but markedly more so when quinine was present. NS1619 (100 μM) caused a significant leak in lipid bilayers, and this was enhanced in the presence of quinine. The K+ ionophore valinomycin (0.25 nM), which like NS1619 increased matrix volume and increased K+ influx in the presence of quinine, caused matrix alkalinization followed by acidification when quinine was absent, and only alkalinization when quinine was present. If K+ is exchanged instantly by H+ through activated KHE, then matrix K+ influx should stimulate H+ influx through KHE and cause matrix acidification. Our results indicate that KHE is not activated immediately by NS1619-induced K+ influx, that NS1619 induces matrix K+ and H+ influx through a nonspecific transport mechanism, and that enhancement with quinine is not due to the blocking of KHE, but to a nonspecific effect of quinine to enhance current leak by NS1619.

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.

Modulation of type-1 Ins(1,4,5)P3 receptor channels by the FK506-binding protein, FKBP12

2002 ◽  
Vol 361 (2) ◽  
pp. 401-407 ◽  
Author(s):  
Sheila L. DARGAN ◽  
Edward J. A. LEA ◽  
Alan P. DAWSON
Keyword(s):  
Lipid Bilayers ◽  
Calcium Release ◽  
Single Channel ◽  
Binding Protein ◽  
Ryanodine Receptors ◽  
Fk506 Binding Protein ◽  
Single Channel Activity ◽  
And Function ◽  
First Time

FK506-binding protein (FKBP12) is highly expressed in neuronal tissue, where it is proposed to localize calcineurin to intracellular calcium-release channels, ryanodine receptors and Ins(1,4,5)P3 receptors (InsP3Rs). The effects of FKBP12 on ryanodine receptors have been well characterized but the nature and function of binding of FKBP12 to InsP3R is more controversial, with evidence for and against a tight interaction between these two proteins. To investigate this, we incorporated purified type-1 InsP3R from rat cerebellum into planar lipid bilayers to monitor the effects of exogenous recombinant FKBP12 on single-channel activity, using K+ as the current carrier. Here we report for the first time that FKBP12 causes a substantial change in single-channel properties of the type-1 InsP3R, specifically to increase the amount of time the channel spends in a fully open state. In the presence of ATP, FKBP12 can also induce co-ordinated gating with neighbouring receptors. The effects of FKBP12 were reversed by FK506. We also present data showing that rapamycin, at sub-optimal concentrations of Ins(2,4,5)P3, decreases the rate of calcium release from cerebellar microsomes. These results provide evidence for a direct functional interaction between FKBP12 and the type-1 InsP3R.

scholarly journals Disease-Linked Super-Trafficking of a Mutant Potassium Channel

2020 ◽  
Author(s):  
Hui Huang ◽  
Laura M. Chamness ◽  
Carlos G. Vanoye ◽  
Georg Kuenze ◽  
Jens Meiler ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Potassium Channel ◽  
Single Channel ◽  
Channel Activity ◽  
Wild Type ◽  
Channel Activation ◽  
Voltage Gated ◽  
Disease Mutations ◽  
Single Channel Activity

ABSTRACTGain-of-function (GOF) mutations in the KCNQ1 voltage-gated potassium channel can induce cardiac arrhythmia. We tested whether any of the known GOF disease mutations in KCNQ1 act by increasing the amount of KCNQ1 that reaches the cell surface—“super-trafficking”. We found that levels of R231C KCNQ1 in the plasma membrane are 5-fold higher than wild type KCNQ1. This arises from both enhanced translocon-mediated membrane integration of the S4 voltage-sensor helix and an energetic linkage of C231 with the V129 and F166 side chains. Whole-cell electrophysiology recordings confirmed that R231C KCNQ1 in complex with KCNE1 is constitutively active, but also revealed the single channel activity of this mutant to be only 20% that of WT. The GOF phenotype associated with R231C therefore reflects the net effects of super-trafficking, reduced single channel activity, and constitutive channel activation. These investigations document membrane protein super-trafficking as a contributing mechanism to human disease.

Abstract 204: Modification of AIFm2 By 4HNE Leads to Retrograde Stress Signaling During Ischemia- Reperfusion Injury in Heart

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Sudha Sharma ◽  
Susmita Bhattarai ◽  
Utsab Subedi ◽  
Christina Acosta ◽  
Hosne Ara ◽  
...  
Keyword(s):  
Protein Expression ◽  
Reperfusion Injury ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Adaptor Protein ◽  
Ischemic Heart ◽  
Spectrometric Analysis ◽  
Apoptosis Inducing Factor ◽  
First Time

Myocardial infarction is a leading cause of death worldwide and occurs due to blockage in blood supply to the heart. Re-establishment of blood flow after a brief period of ischemia leads to paradoxical exacerbation of the cardiomyocyte and its death. This phenomenon is known as ischemia-reperfusion injury. Major evidence in the pathogenesis of ischemia-reperfusion injury is due to oxidative stress, which is an imbalance between reactive oxygen species (ROS) and antioxidants. Membrane consisting of polyunsaturated fatty acid is attacked by ROS leading to lipid peroxidation and the generation of one of the toxic aldehydes 4-hydroxynonenal (4-HNE). Evidence suggests that 4-HNE increases during an ischemia-reperfusion injury in the heart. Apoptosis-inducing factor, mitochondrion-associated 2 (AIFM2) is a mitochondrial located oxidoreductase that participates in caspase-independent apoptosis. In this study, we sought to identify the role of 4-HNE in regulating AIFm2 translocation and cardiomyocyte death during an ischemia-reperfusion injury in the heart. Following ischemia both RNA and protein expression of AIFm2 significantly increased in the ischemic heart compared to sham. Also, 4-HNE adducted AIFm2 translocated from mitochondria to the nucleus shown by western blot analysis in ischemic heart. The mass spectrometric analysis was done to see the modification site on AIFm2 by 4-HNE and revealed that His 174 and Cys 187 are two sites on AIFm2 where 4-HNE adduction occurred. To identify the modification site responsible for AIFm2 translocation we performed site-direct mutagenesis in H9C2 cardiomyocyte, where Histidine 174 was replaced by arginine and Cys 187 was replaced by threonine. When the ischemia-reperfusion injury was induced, only Histidine 174 mutant failed to translocate to the nucleus indicating His 174 modification by 4-HNE was responsible for AIFm2 translocation. To further support the transport mechanism, protein expression of Importin; an adaptor protein responsible for the transfer of proteins to the nucleus was increased in the ischemic heart compared to sham. Collectively, those results for the first time identify the unique role of 4-HNE modification on AIFm2 protein during an ischemia-reperfusion injury in the heart.

Oxytocin protects rat heart against ischemia–reperfusion injury via pathway involving mitochondrial ATP-dependent potassium channel

Peptides ◽  
2010 ◽  
Vol 31 (7) ◽  
pp. 1341-1345 ◽  
Author(s):  
Ali Mohammad Alizadeh ◽  
Mahdieh Faghihi ◽  
Hamid Reza Sadeghipour ◽  
Fahimeh MohammadGhasemi ◽  
Alireza Imani ◽  
...  
Keyword(s):  
Potassium Channel ◽  
Reperfusion Injury ◽  
Rat Heart ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion
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