Abstract MP220: Identification And Functional Characterization Of Exosomal Bk Channels

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Shridhar Sanghvi ◽  
Julie A Dougherty ◽  
Parker Evans ◽  
Divya Sridharan ◽  
Devasena Ponnalagu ◽  
...  
Keyword(s):  
Ion Channel ◽  
Reperfusion Injury ◽  
Single Channel ◽  
Ischemia Reperfusion ◽  
Functional Characterization ◽  
Physiological Role ◽  
Bk Channels ◽  
Target Cells ◽  
Open Probability ◽  
Planar Bilayers

Extracellular vesicles (EVs) specifically exosomes are important in mediating intracellular communications, and are capable of transferring genetic information between cells. Exosomes are used as a drug delivery vehicle to carry cargo for targeted therapy and have emerged as one of the most promising candidate for treating cardiovascular diseases. Exosomes get packaged inside the cell, excise out to the extracellular environment, and deliver the cargo to the target cells. However, the precise mechanism of how exosomes handle the differential ionic environment and the physiological role of their ion channels is not determined. Given that potassium (K + ) ions has the largest gradient, we focused on identifying the presence and physiological relevance of K+ channels in exosomes. Using the in silico approach, several ion channel candidates were identified, the most prominent ion channel being large conductance Ca 2+ and voltage-activated potassium channel (BK). To record BK in exosomes, we incorporated planar bilayers and a novel electrophysiology approach called near field electrophysiology (NFE), as the canonical patch-clamp methods are not feasible due to the size of EVs. Our NFE indicates a presence of K + channels in intact exosomes and 45% of them are sensitive to IbTX. Since IbTX specifically blocks, BK channels, we estimated 2 functional channels (single-channel conductance of 300 pS with 50% open probability) in a single exosome. Plasma-derived exosomes from BK +/+ and BK -/- mice subjected to differential K + gradient indicated that functional BK channels exist in exosomes, and help in maintaining their structural integrity. Furthermore, plasma derived exosomes from BK +/+ mice showed cardioprotection from ischemia-reperfusion injury whereas exosomes from BK -/- mice did not. Thus, the presence of BK determines the packaging as well as cardioprotective function of exosomes. Overall, the study for the first time indicate a presence of functional ion channel (BK) in exosomes which plays a role in protecting cells and heart against ischemia and reperfusion injury.

scholarly journals TOK Homologue in Neurospora crassa: First Cloning and Functional Characterization of an Ion Channel in a Filamentous Fungus

2003 ◽  
Vol 2 (1) ◽  
pp. 181-190 ◽  
Author(s):  
Stephen K. Roberts
Keyword(s):  
Ion Channels ◽  
Ion Channel ◽  
Neurospora Crassa ◽  
Filamentous Fungus ◽  
Single Channel ◽  
Functional Characterization ◽  
Reversal Potential ◽  
Channel Activity ◽  
Biophysical Properties ◽  
Patch Clamp Technique

ABSTRACT In contrast to animal and plant cells, very little is known of ion channel function in fungal physiology. The life cycle of most fungi depends on the “filamentous” polarized growth of hyphal cells; however, no ion channels have been cloned from filamentous fungi and comparatively few preliminary recordings of ion channel activity have been made. In an attempt to gain an insight into the role of ion channels in fungal hyphal physiology, a homolog of the yeast K+ channel (ScTOK1) was cloned from the filamentous fungus, Neurospora crassa. The patch clamp technique was used to investigate the biophysical properties of the N. crassa K+ channel (NcTOKA) after heterologous expression of NcTOKA in yeast. NcTOKA mediated mainly time-dependent outward whole-cell currents, and the reversal potential of these currents indicated that it conducted K+ efflux. NcTOKA channel gating was sensitive to extracellular K+ such that channel activation was dependent on the reversal potential for K+. However, expression of NcTOKA was able to overcome the K+ auxotrophy of a yeast mutant missing the K+ uptake transporters TRK1 and TRK2, suggesting that NcTOKA also mediated K+ influx. Consistent with this, close inspection of NcTOKA-mediated currents revealed small inward K+ currents at potentials negative of EK. NcTOKA single-channel activity was characterized by rapid flickering between the open and closed states with a unitary conductance of 16 pS. NcTOKA was effectively blocked by extracellular Ca2+, verapamil, quinine, and TEA+ but was insensitive to Cs+, 4-aminopyridine, and glibenclamide. The physiological significance of NcTOKA is discussed in the context of its biophysical properties.

scholarly journals Therapeutic Inhibition of Acid Sensing Ion Channel 1a Recovers Heart Function After Ischemia-Reperfusion Injury

Circulation ◽  
2021 ◽  
Author(s):  
Meredith A. Redd ◽  
Sarah E. Scheuer ◽  
Natalie J. Saez ◽  
Yusuke Yoshikawa ◽  
Han Sheng Chiu ◽  
...  
Keyword(s):  
Ion Channel ◽  
Reperfusion Injury ◽  
Electromechanical Coupling ◽  
Ischemia Reperfusion Injury ◽  
Ex Vivo ◽  
Heart Function ◽  
Ischemia Reperfusion ◽  
Genetic Ablation ◽  
Organ Level

Background: Ischemia-reperfusion injury (IRI) is one of the major risk factors implicated in morbidity and mortality associated with cardiovascular disease. During cardiac ischemia, the build-up of acidic metabolites results in decreased intracellular and extracellular pH that can reach as low as 6.0-6.5. The resulting tissue acidosis exacerbates ischemic injury and significantly impacts cardiac function. Methods: We used genetic and pharmacological methods to investigate the role of acid sensing ion channel 1a (ASIC1a) in cardiac IRI at the cellular and whole organ level. Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) as well as ex vivo and in vivo models of IRI were used to test the efficacy of ASIC1a inhibitors as pre- and post-conditioning therapeutic agents. Results: Analysis of human complex trait genetics indicate that variants in the ASIC1 genetic locus are significantly associated with cardiac and cerebrovascular ischemic injuries. Using hiPSC-CMs in vitro and murine ex vivo heart models, we demonstrate that genetic ablation of ASIC1a improves cardiomyocyte viability after acute IRI. Therapeutic blockade of ASIC1a using specific and potent pharmacological inhibitors recapitulates this cardioprotective effect. We used an in vivo model of myocardial infarction (MI) and two models of ex vivo donor heart procurement and storage as clinical models to show that ASIC1a inhibition improves post-IRI cardiac viability. Use of ASIC1a inhibitors as pre- or post-conditioning agents provided equivalent cardioprotection to benchmark drugs, including the sodium-hydrogen exchange inhibitor zoniporide. At the cellular and whole organ level, we show that acute exposure to ASIC1a inhibitors has no impact on cardiac ion channels regulating baseline electromechanical coupling and physiological performance. Conclusions: Collectively, our data provide compelling evidence for a novel pharmacological strategy involving ASIC1a blockade as a cardioprotective therapy to improve the viability of hearts subjected to IRI.

CFTR in K562 human leukemic cells

2003 ◽  
Vol 285 (2) ◽  
pp. C480-C488 ◽  
Author(s):  
Yanina A. Assef ◽  
Alicia E. Damiano ◽  
Elsa Zotta ◽  
Cristina Ibarra ◽  
Basilio A. Kotsias
Keyword(s):  
Single Channel ◽  
Functional Characterization ◽  
Leukemic Cells ◽  
Cftr Gene ◽  
Human Leukemia ◽  
Selectivity Ratio ◽  
Open Probability ◽  
Selective Channel ◽  
Human Leukemic Cells

In this study, the expression and functional characterization of CFTR (cystic fibrosis transmembrane regulator) was determined in K562 chronic human leukemia cells. Expression of the CFTR gene product was determined by RT-PCR and confirmed by immunohistochemistry and Western blot analysis. Functional characterization of CFTR Cl- channel activity was conducted with patch-clamp techniques. Forskolin, an adenylyl cyclase activator, induced an anion-selective channel with a linear current-voltage relationship and a single-channel conductance of 11 pS. This cAMP-activated channel had a Pgluconate/PCl or PF/PCl perm-selectivity ratio of 0.35 and 0.30, respectively, and was inhibited by the CFTR blocker glibenclamide and the anti-CFTR antibody MAb 13-1, when added to the cytoplasmatic side of the patch. Glibenclamide decreased the open probability increasing the frequency of open-to-closed transitions. Addition of 200 μM DIDS caused an irreversible block of the channels when added to the cytosolic side of inside-out patches. These and other observations indicate a widespread distribution of CFTR gene expression and suggest that this channel protein may function in most human cells to help maintain cellular homeostasis.

Fast BK-Type Channel Mediates the Ca2+-Activated K+ Current in Crayfish Muscle

1999 ◽  
Vol 82 (4) ◽  
pp. 1655-1661 ◽  
Author(s):  
Alfonso Araque ◽  
Washington Buño
Keyword(s):  
Electrical Activity ◽  
Single Channel ◽  
Muscle Fibers ◽  
Ionic Channels ◽  
Bk Channels ◽  
Intrinsic Properties ◽  
Open Probability ◽  
Crayfish Muscle ◽  
Fast Activation ◽  
Inside Out

The role of the Ca2+-activated K+ current ( I K(Ca)) in crayfish opener muscle fibers is functionally important because it regulates the graded electrical activity that is characteristic of these fibers. Using the cell-attached and inside-out configurations of the patch-clamp technique, we found three different classes of channels with properties that matched those expected of the three different ionic channels mediating the depolarization-activated macroscopic currents previously described (Ca2+, K+, and Ca2+-dependent K+ currents). We investigated the properties of the ionic channels mediating the extremely fast activating and persistent I K(Ca). These voltage- and Ca2+-activated channels had a mean single-channel conductance of ∼ 70 pS and showed a very fast activation. Both the single-channel open probability and the speed of activation increased with depolarization. Both parameters also increased in inside-out patches, i.e., in high Ca2+concentration. Intracellular loading with the Ca2+ chelator bis(2-aminophenoxy) ethane- N, N,N′,N′-tetraacetic acid gradually reduced and eventually prevented channel openings. The channels opened at very brief delays after the pulse depolarization onset (<5 ms), and the time-dependent open probability was constant during sustained depolarization (≤560 ms), matching both the extremely fast activation kinetics and the persistent nature of the macroscopic I K(Ca). However, the intrinsic properties of these single channels do not account for the partial apparent inactivation of the macroscopic I K(Ca), which probably reflects temporal Ca2+ variations in the whole muscle fiber. We conclude that the channels mediating I K(Ca) in crayfish muscle are voltage- and Ca2+-gated BK channels with relatively small conductance. The intrinsic properties of these channels allow them to act as precise Ca2+ sensors that supply the exact feedback current needed to control the graded electrical activity and therefore the contraction of opener muscle fibers.

scholarly journals KCNMA1 Encoded Cardiac BK Channels Afford Protection against Ischemia-Reperfusion Injury

PLoS ONE ◽  
2014 ◽  
Vol 9 (7) ◽  
pp. e103402 ◽  
Author(s):  
Ewa Soltysinska ◽  
Bo Hjorth Bentzen ◽  
Maria Barthmes ◽  
Helle Hattel ◽  
A. Brianne Thrush ◽  
...  
Keyword(s):  
Reperfusion Injury ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Bk Channels

Ba(2+)-sensitive K+ channels in basal membrane of confluent Madin-Darby canine kidney cells

1994 ◽  
Vol 267 (6) ◽  
pp. F1007-F1014
Author(s):  
X. Y. Wang ◽  
P. J. Harris ◽  
R. E. Kemm
Keyword(s):  
Single Channel ◽  
Mdck Cells ◽  
Physiological Role ◽  
K Channel ◽  
Madin Darby Canine Kidney ◽  
Open Probability ◽  
Cytosolic Ph ◽  
K Channels ◽  
Darby Canine Kidney ◽  
Canine Kidney

A method is described for gaining access to the basolateral membranes of confluent Madin-Darby canine kidney (MDCK) cells by surgical reflection of the cell layer overlying fluid-filled domes. Single-channel recordings from cell-attached inside-out and outside-out configurations revealed two K+ channels located in the basal membranes of the highly differentiated monolayers. With 140 mmol/l KCl in pipette, the intermediate-conductance K+ channel displayed outward rectification in cell-attached configuration with channel conductances of 65 pS for outward part and 17 pS for inward part. In excised-patch recording, this channel had a conductance of 92 pS with 140 mmol/l KCl on the extracellular side of the patch and 5 mmol/l KCl on the cytosolic side. The maximum conductance obtained in symmetrical KCl (140 mmol/l) solution was 140 pS. Ba2+ (1 mmol/l) and tetraethylammonium (5 mmol/l) blocked this channel reversibly. Channel open probability (Po) was reduced from 0.41 at cytosolic pH 7.4 to 0.14 at pH 6.8 and increased to 0.64 at pH 8.0. The channel activity was significantly inhibited by elevation of intracellular Ca2+. A small-conductance K+ channel was also observed mainly in excised patches with single-channel conductance of 48 pS in symmetrical KCl solutions. However, the activity of this channel was partially obscured by the intermediate-conductance K+ channel and further analysis was not possible. A physiological role of these channels in mediating K+ recycling through the monolayer is suggested.

Large conducting potassium channel reconstituted from airway smooth muscle

1992 ◽  
Vol 262 (3) ◽  
pp. L327-L336 ◽  
Author(s):  
D. Savaria ◽  
C. Lanoue ◽  
A. Cadieux ◽  
E. Rousseau
Keyword(s):  
Smooth Muscle ◽  
Lipid Bilayers ◽  
Airway Smooth Muscle ◽  
Single Channel ◽  
Specific Inhibitor ◽  
Physiological Role ◽  
Membrane Receptors ◽  
K Channel ◽  
Open Probability ◽  
K Channels

Microsomal fractions were prepared from canine and bovine airway smooth muscle (ASM) by differential and gradient centrifugations. Surface membrane vesicles were characterized by binding assays and incorporated into planar lipid bilayers. Single-channel activities were recorded in symmetric or asymmetric K+ buffer systems and studied under voltage and Ca2+ clamp conditions. A large-conductance K(+)-selective channel (greater than 220 pS in 150 mM K+) displaying a high Ca2+, low Ba2+, and charybdotoxin (CTX) sensitivity was identified. Time analysis of single-channel recordings revealed a complex kinetic behavior compatible with the previous schemes proposed for Ca(2+)-activated K+ channels in a variety of biological surface membranes. We now report that the open probability of the channel at low Ca2+ concentration is enhanced on in vitro phosphorylation, which is mediated via an adenosine 3',5'-cyclic monophosphate-dependent protein kinase. In addition to this characterization at the molecular level, a second series of pharmacological experiments were designed to assess the putative role of this channel in ASM strips. Our results show that 50 nM CTX, a specific inhibitor of the large conducting Ca(2+)-dependent K+ channel, prevents norepinephrine transient relaxation on carbamylcholine-precontracted ASM strips. It was also shown that CTX reversed the steady-state relaxation induced by vasoactive intestinal peptide and partially antagonized further relaxation induced by cumulative doses of this potent bronchodilatator. Thus it is proposed that the Ca(2+)-activated K+ channels have a physiological role because they are indirectly activated on stimulation of various membrane receptors via intracellular mechanisms.

Ischemia/Reperfusion Injury and Ion Channel Blockade

1998 ◽  
pp. 195-214
Author(s):  
Satoshi Takeo ◽  
Kouichi Tanonaka ◽  
Jian-Xun Liu ◽  
Toru Kamiyama ◽  
Itaru Ohoi ◽  
...  
Keyword(s):  
Ion Channel ◽  
Reperfusion Injury ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Channel Blockade

Antiarrhythmic efficacy of CPUY102122, a multiple ion channel blocker, on rabbits with ischemia/reperfusion injury

2014 ◽  
Vol 66 (6) ◽  
pp. 1022-1030
Author(s):  
Minhui Wang ◽  
Jiaojiao Shan ◽  
Qian Yang ◽  
Xianglei Ma ◽  
Sisi Jin ◽  
...  
Keyword(s):  
Ion Channel ◽  
Reperfusion Injury ◽  
Channel Blocker ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Antiarrhythmic Efficacy

Peroxynitrite reversibly inhibits Ca2+-activated K+ channels in rat cerebral artery smooth muscle cells

2000 ◽  
Vol 278 (6) ◽  
pp. H1883-H1890 ◽  
Author(s):  
Anna K. Brzezinska ◽  
Debebe Gebremedhin ◽  
William M. Chilian ◽  
Balaraman Kalyanaraman ◽  
Stephen J. Elliott
Keyword(s):  
Single Channel ◽  
Cerebral Arteries ◽  
Ionic Current ◽  
Inhibitory Effect ◽  
Bk Channels ◽  
Bk Channel ◽  
Channel Activity ◽  
Open Probability ◽  
Whole Cell ◽  
Cell Current

Peroxynitrite (ONOO−) is a contractile agonist of rat middle cerebral arteries. To determine the mechanism responsible for this component of ONOO−bioactivity, the present study examined the effect of ONOO− on ionic current and channel activity in rat cerebral arteries. Whole cell recordings of voltage-clamped cells were made under conditions designed to optimize K+ current. The effects of iberiotoxin, a selective inhibitor of large-conductance Ca2+-activated K+ (BK) channels, and ONOO− (10–100 μM) were determined. At a pipette potential of +50 mV, ONOO− inhibited 39% of iberiotoxin-sensitive current. ONOO− was selective for iberiotoxin-sensitive current, whereas decomposed ONOO− had no effect. In excised, inside-out membrane patches, channel activity was recorded using symmetrical K+solutions. Unitary currents were sensitive to increases in internal Ca2+ concentration, consistent with activity due to BK channels. Internal ONOO− dose dependently inhibited channel activity by decreasing open probability and mean open times. The inhibitory effect of ONOO− could be overcome by reduced glutathione. Glutathione, added after ONOO−, restored whole cell current amplitude to control levels and reverted single-channel gating to control behavior. The inhibitory effect of ONOO− on membrane K+ current is consistent with its contractile effects in isolated cerebral arteries and single myocytes. Taken together, our data suggest that ONOO− has the potential to alter cerebral vascular tone by inhibiting BK channel activity.

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