scholarly journals Electromagnetic energy (670 nm) stimulates vasodilation through activation of the large conductance potassium channel (BKCa)

PLoS ONE ◽  
2021 ◽  
Vol 16 (10) ◽  
pp. e0257896
Author(s):  
Debebe Gebremendhin ◽  
Brian Lindemer ◽  
Dorothee Weihrauch ◽  
David R. Harder ◽  
Nicole L. Lohr
Keyword(s):  
Potassium Channels ◽  
Electromagnetic Energy ◽  
State Probability ◽  
Tissue Loss ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Potassium Efflux ◽  
Α Subunit ◽  
Membrane Hyperpolarization ◽  
Channel Opening

Introduction Peripheral artery disease (PAD) is a highly morbid condition in which impaired blood flow to the limbs leads to pain and tissue loss. Previously we identified 670 nm electromagnetic energy (R/NIR) to increase nitric oxide levels in cells and tissue. NO elicits relaxation of smooth muscle (SMC) by stimulating potassium efflux and membrane hyperpolarization. The actions of energy on ion channel activity have yet to be explored. Here we hypothesized R/NIR stimulates vasodilation through activation of potassium channels in SMC. Methods Femoral arteries or facial arteries from C57Bl/6 and Slo1-/- mice were isolated, pressurized to 60 mmHg, pre-constricted with U46619, and irradiated twice with energy R/NIR (10 mW/cm2 for 5 min) with a 10 min dark period between irradiations. Single-channel K+ currents were recorded at room temperature from cell-attached and excised inside-out membrane patches of freshly isolated mouse femoral arterial muscle cells using the patch-clamp technique. Results R/NIR stimulated vasodilation requires functional activation of the large conductance potassium channels. There is a voltage dependent outward current in SMC with light stimulation, which is due to increases in the open state probability of channel opening. R/NIR modulation of channel opening is eliminated pharmacologically (paxilline) and genetically (BKca α subunit knockout). There is no direct action of light to modulate channel activity as excised patches did not increase the open state probability of channel opening. Conclusion R/NIR vasodilation requires indirect activation of the BKca channel.

PACAP38 Modulates Activity of NMDA Receptors in Cultured Chick Cortical Neurons

1997 ◽  
Vol 78 (4) ◽  
pp. 2231-2234 ◽  
Author(s):  
Guo Jun Liu ◽  
Barry W. Madsen
Keyword(s):  
Nmda Receptor ◽  
Nmda Receptors ◽  
Cortical Neurons ◽  
Second Messengers ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Low Concentrations ◽  
Channel Opening ◽  
Pituitary Adenylate Cyclase ◽  
Intracellular Second Messengers

Liu, Guo Jun and Barry W. Madsen. PACAP38 modulates activity of NMDA receptors in cultured chick cortical neurons. J. Neurophysiol. 78: 2231–2234, 1997. The outside-out recording mode of the patch-clamp technique was used to study modulatory effects of pituitary adenylate cyclase-activating polypeptide (PACAP38) on N-methyl-d-aspartate (NMDA) receptor activity in cultured chick cortical neurons. Biphasic concentration-dependent effects of PACAP38 on channel opening frequency induced by NMDA (20 μM) and glycine (1 μM) were found, with low concentrations (0.5–2 nM) of PACAP38 increasing activity and higher concentrations (10–1,000 nM) causing inhibition. These effects were reversible, reduced with higher concentrations of glycine (2–10 μM) but not by 200 μM NMDA, and inhibited by 10 μM 7-chlorokynurenic acid. In addition, 1 μM PACAP6–38 (a PACAP antagonist) inhibited channel activity due to 20 μM NMDA and 1 μM glycine by 66%, and this inhibition was reduced to 13% in the additional presence of 2 nM PACAP38. These observations suggest thatPACAP38 has a direct modulatory effect on the NMDA receptor that is independent of intracellular second messengers and probably mediated through the glycine coagonist site(s).

Apical membrane potassium channels in frog diluting segment: stimulation by furosemide

1992 ◽  
Vol 262 (4) ◽  
pp. F606-F614 ◽  
Author(s):  
A. M. Hurst ◽  
M. Hunter
Keyword(s):  
Potassium Channels ◽  
Apical Membrane ◽  
Inward Rectification ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
High Potassium ◽  
Diluting Segment ◽  
Pipette Solution ◽  
Significant Change

The patch-clamp technique was used to study the activity of apical membrane potassium channels in frog isolated everted diluting segments, and the effect of transport inhibitors on channel activity was assessed. In cell-attached patches with a high-potassium pipette solution and Ringer in the bath the channels show inward rectification (inward conductance, 25.1 pS; outward conductance, 10.5 pS). The channel is selective for potassium over sodium and is voltage dependent with depolarization increasing channel open probability (Po). Furosemide increased channel activity, which resulted exclusively from a significant increase in the number (N) of channels in the patch (control, 2.3 +/- 0.3, n = 8; furosemide, 4.0 +/- 0.4, n = 14) without any significant change in Po. Amiloride blocked the stimulatory effect of furosemide by reducing N to 1.4 +/- 0.6 (n = 6), and amiloride alone also reduced N with no significant change in Po. This suggests that the increase in N in response to furosemide may be secondary to a rise in intracellular pH mediated by activation of the apical Na-H exchanger.

Potassium channels in primary cultures of seawater fish gill cells. I. Stretch-activated K+ channels

2000 ◽  
Vol 279 (5) ◽  
pp. R1647-R1658 ◽  
Author(s):  
C. Duranton ◽  
E. Mikulovic ◽  
M. Tauc ◽  
M. Avella ◽  
P. Poujeol
Keyword(s):  
Potassium Channels ◽  
Primary Cultures ◽  
Dicentrarchus Labrax ◽  
Sea Bass ◽  
Cell Swelling ◽  
Hypotonic Solution ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Ionic Substitution ◽  
Gill Cells

Previous studies using the patch-clamp technique demonstrated the presence of a small conductance Cl− channel in the apical membrane of respiratory gill cells in primary culture originating from sea bass Dicentrarchus labrax. We used the same technique here to characterize potassium channels in this model. A K+ channel of 123 ± 3 pS was identified in the cell-attached configuration with 140 mM KCl in the bath and in the pipette. The activity of the channel declined rapidly with time and could be restored by the application of a negative pressure to the pipette (suction) or by substitution of the bath solution with a hypotonic solution (cell swelling). In the excised patch inside-out configuration, ionic substitution demonstrated a high selectivity of this channel for K+ over Na+ and Ca2+. The mechanosensitivity of this channel to membrane stretching via suction was also observed in this configuration. Pharmacological studies demonstrated that this channel was inhibited by barium (5 mM), quinidine (500 μM), and gadolinium (500 μM). Channel activity decreased when cytoplasmic pH was decreased from 7.7 to 6.8. The effect of membrane distension by suction and exposure to hypotonic solutions on K+ channel activity is consistent with the hypothesis that stretch-activated K+ channels could mediate an increase in K+ conductance during cell swelling.

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.

The LQT-associated calmodulin mutant E141G induces disturbed Ca2+-dependent binding and a flickering gating mode of the CaV1.2 channel

2020 ◽  
Vol 318 (5) ◽  
pp. C991-C1004
Author(s):  
Jingyang Su ◽  
Qinghua Gao ◽  
Lifeng Yu ◽  
Xuanxuan Sun ◽  
Rui Feng ◽  
...  
Keyword(s):  
Single Channel ◽  
Missense Variant ◽  
Inhibitory Effect ◽  
Channel Activity ◽  
Peptide Fragments ◽  
Patch Clamp Technique ◽  
Open Time ◽  
Channel Opening ◽  
Lqt Syndrome ◽  
Calmodulin Mutant

Calmodulin (CaM) mutations are associated with congenital long QT (LQT) syndrome (LQTS), which may be related to the dysregulation of the cardiac-predominant Ca2+ channel isoform CaV1.2. Among various mutants, CaM-E141G was identified as a critical missense variant. However, the interaction of this CaM mutant with the CaV1.2 channel has not been determined. In this study, by utilizing a semiquantitative pull-down assay, we explored the interaction of CaM-E141G with CaM-binding peptide fragments of the CaV1.2 channel. Using the patch-clamp technique, we also investigated the electrophysiological effects of the mutant on CaV1.2 channel activity. We found that the maximum binding (Bmax) of CaM-E141G to the proximal COOH-terminal region, PreIQ-IQ, PreIQ, IQ, and NT (an NH2-terminal peptide) was decreased (by 17.71–59.26%) compared with that of wild-type CaM (CaM-WT). In particular, the Ca2+-dependent increase in Bmax became slower with the combination of CaM-E141G + PreIQ and IQ but faster in the case of NT. Functionally, CaM-WT and CaM-E141G at 500 nM Ca2+ decreased CaV1.2 channel activity to 24.88% and 55.99%, respectively, compared with 100 nM Ca2+, showing that the inhibitory effect was attenuated in CaM-E141G. The mean open time of the CaV1.2 channel was increased, and the number of blank traces with no channel opening was significantly decreased. Overall, CaM-E141G exhibits disrupted binding with the CaV1.2 channel and induces a flickering gating mode, which may result in the dysfunction of the CaV1.2 channel and, thus, the development of LQTS. The present study is the first to investigate the detailed binding properties and single-channel gating mode induced by the interaction of CaM-E141G with the CaV1.2 channel.

scholarly journals Inhibition of phosphatidylinositol 3-kinase stimulates activity of the small-conductance K channel in the CCD

2006 ◽  
Vol 290 (4) ◽  
pp. F806-F812 ◽  
Author(s):  
Dimin Li ◽  
Yuan Wei ◽  
Elisa Babilonia ◽  
Zhijian Wang ◽  
Wen-Hui Wang
Keyword(s):  
Collecting Duct ◽  
Sk Channels ◽  
Channel Activity ◽  
Phosphatidylinositol 3 Kinase ◽  
Patch Clamp Technique ◽  
Α Subunit ◽  
Sk Channel ◽  
Low K ◽  
Small Conductance ◽  
Phosphatidylinositol 3

We used Western blotting to examine the expression of phosphatidylinositol 3-kinase (PI3K) in the renal cortex and outer medulla and employed the patch-clamp technique to study the effect of PI3K on the ROMK-like small-conductance K (SK) channels in the cortical collecting duct (CCD). Low K intake increased the expression of the 110-kDa α-subunit (p110α) of PI3K compared with rats on a normal-K diet. Because low K intake increases superoxide levels ( 2 ), the possibility that increases in superoxide anions may be responsible for the effect of low K intake on the expression of PI3K is supported by finding that addition of H2O2 stimulates the expression of p110α in M1 cells. Inhibition of PI3K with either wortmannin or LY-294002 significantly increased channel activity in the CCD from rats on a K-deficient (KD) diet or on a normal-K diet. The stimulatory effect of wortmannin on ROMK channel activity cannot be mimicked by inhibition of phospholipase C with U-73122. This suggests that the effect of inhibiting PI3K was not the result of increasing the phosphatidylinositol 4,5-bisphosphate level. Moreover, application of the exogenous phosphatidylinositol 3,4,5-trisphosphate analog had no effect on channel activity in excised patches. Because low K intake has been shown to increase the activity of protein tyrosine kinase (PTK), we explored the role of the interaction between PTK and PI3K in the regulation of the SK channel activity. Inhibition of PTK increased SK channel activity in the CCD from rats on a KD diet. However, addition of wortmannin did not further increase ROMK channel activity. Also, the effect of wortmannin was abolished by treatment of CCD with phalloidin. We conclude that PI3K is involved in mediating the effect of low K intake on ROMK channel activity in the CCD and that the effect of PI3K on SK channels requires the involvement of PTK and the cytoskeleton.

scholarly journals Inhibition of phosphodiesterases relaxes detrusor smooth muscle via activation of the large-conductance voltage- and Ca2+-activated K+ channel

2012 ◽  
Vol 302 (9) ◽  
pp. C1361-C1370 ◽  
Author(s):  
Wenkuan Xin ◽  
Qiuping Cheng ◽  
Rupal P. Soder ◽  
Georgi V. Petkov
Keyword(s):  
Smooth Muscle ◽  
Guinea Pig ◽  
Bk Channels ◽  
Bk Channel ◽  
Dependent Manner ◽  
Detrusor Smooth Muscle ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Membrane Hyperpolarization ◽  
Pde Inhibition

Detrusor smooth muscle (DSM) exhibits increased spontaneous phasic contractions under pathophysiological conditions such as detrusor overactivity (DO). Our previous studies showed that activation of cAMP signaling pathways reduces DSM contractility by increasing the large-conductance voltage- and Ca2+-activated K+ (BK) channel activity. Here, we tested the hypothesis whether inhibition of phosphodiesterases (PDEs) can reduce guinea pig DSM excitability and contractility by increasing BK channel activity. Utilizing isometric tension recordings of DSM isolated strips and the perforated patch-clamp technique on freshly isolated DSM cells, we examined the mechanism of DSM relaxation induced by PDE inhibition. Inhibition of PDEs by 3-isobutyl-1-methylxanthine (IBMX), a nonselective PDE inhibitor, significantly reduced DSM spontaneous and carbachol-induced contraction amplitude, frequency, duration, muscle force integral, and tone in a concentration-dependent manner. IBMX significantly reduced electrical field stimulation-induced contractions of DSM strips. Blocking BK channels with paxilline diminished the inhibitory effects of IBMX on DSM contractility, indicating a role for BK channels in DSM relaxation mediated by PDE inhibition. IBMX increased the transient BK currents (TBKCs) frequency by ∼3-fold without affecting the TBKCs amplitude. IBMX increased the frequency of the spontaneous transient hyperpolarizations by ∼2-fold and hyperpolarized the DSM cell resting membrane potential by ∼6 mV. Blocking the BK channels with paxilline abolished the IBMX hyperpolarizing effects. Under conditions of blocked Ca2+ sources for BK channel activation, IBMX did not affect the depolarization-induced steady-state whole cell BK currents. Our data reveal that PDE inhibition with IBMX relaxes guinea pig DSM via TBKCs activation and subsequent DSM cell membrane hyperpolarization.

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

Molecules ◽  
2021 ◽  
Vol 26 (11) ◽  
pp. 3233
Author(s):  
Aleksandra Sek ◽  
Rafal P. Kampa ◽  
Bogusz Kulawiak ◽  
Adam Szewczyk ◽  
Piotr Bednarczyk
Keyword(s):  
Potassium Channels ◽  
Potassium Channel ◽  
Epithelial Cell Line ◽  
Bkca Channel ◽  
Channel Activity ◽  
Potassium Channel Opener ◽  
Patch Clamp Technique ◽  
Bkca Channels ◽  
Bronchial Epithelial ◽  
Channel Opener

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.

Acute effects of repetitive depolarization on sodium current in chick myocytes

1991 ◽  
Vol 260 (6) ◽  
pp. H1810-H1818
Author(s):  
M. R. Gold ◽  
G. R. Strichartz
Keyword(s):  
Time Course ◽  
Sodium Current ◽  
Complete Recovery ◽  
Acute Effects ◽  
Na Current ◽  
Patch Clamp Technique ◽  
Membrane Hyperpolarization ◽  
Atrial Cells ◽  
Whole Cell Patch Clamp ◽  
Recovery From Inactivation

Acute effects of repetitive depolarization on the inward Na+ current (INa) of cultured embryonic chick atrial cells were studied using the whole cell patch-clamp technique. Stimulation rates of 1 Hz or greater produced a progressive decrement of peak INa. With depolarizations to 0 mV of 150-ms duration, applied at 2 Hz from a holding potential of -100 mV, the steady-state decrement was approximately 20%. The magnitude of this effect increased with stimulation frequency and with test potential depolarization and decreased with membrane hyperpolarization. Analysis of INa kinetics revealed that reactivation was sufficiently slow to preclude complete recovery from inactivation with interpulse intervals less than 1,000 ms. Moreover, reactivation accelerated markedly with membrane hyperpolarization, in parallel with the response to repetitive stimulation. The multiexponential time course of recovery of peak INa from repetitive depolarization was similar to that observed after single stimuli; however, there was a shift toward a greater proportion of current recovering with the slower of two time constants. It is concluded that incomplete recovery from inactivation is responsible for the decrement in INa observed with short interpulse intervals.

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