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.

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.

A maxi Cl(−) channel in cultured pavement cells from the gills of the freshwater rainbow trout Oncorhynchus mykiss

2001 ◽  
Vol 204 (10) ◽  
pp. 1783-1794 ◽  
Author(s):  
M.J. O'Donnell ◽  
S.P. Kelly ◽  
C.A. Nurse ◽  
C.M. Wood
Keyword(s):  
Rainbow Trout ◽  
Oncorhynchus Mykiss ◽  
Single Channel ◽  
Primary Cultures ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Pavement Cells ◽  
Cl Channel ◽  
Rainbow Trout Oncorhynchus Mykiss ◽  
Inside Out

Primary cultures of pavement cells from the gills of a freshwater fish, the rainbow trout Oncorhynchus mykiss, have been studied for the first time using the patch-clamp technique. Gigaohm seals were obtained in approximately 95 % of cells studied, and channel activity was evident in a high proportion (>90 %). A large-conductance Cl(−) channel was evident in 6 % of cell-attached and in 31 % of inside-out patches. Single-channel conductance in inside-out patches was 372 pS, and current/voltage relationships were linear over the range −60 to +60 mV. The channel was activated by patch excision, and activation was often associated with polarization of the patch. The mean number of channels per patch was 1.9, and there were several subconductance states. The relationship between channel activity (NP(o)) and voltage was in the form of an inverted U, and channel activity was highest between 0 and +20 mV. Large-conductance Cl(−) channels showed a progressive time-dependent reduction in current in response to sustained polarization to voltages outside the range −20 mV to +20 mV. Permeability ratios (P) of Cl(−) to other anions were P(HCO3)/P(Cl)=0.81, P(SO4)/P(Cl)=0.31 and P(isethionate)/P(Cl)=0.53. The channel was blocked by Zn(2+), SITS, DIDS and diphenylamine carboxylate. This is the first description of a large-conductance Cl(−) channel in gill cells from freshwater or marine species. Possible functions of the channel are discussed.

TRPM7 Is Involved in Volume Regulation in Salivary Glands

2017 ◽  
Vol 96 (9) ◽  
pp. 1044-1050 ◽  
Author(s):  
J.M. Kim ◽  
S. Choi ◽  
K. Park
Keyword(s):  
Critical Role ◽  
Regulatory Volume Decrease ◽  
Specific Inhibitor ◽  
Acinar Cells ◽  
Cell Swelling ◽  
Hypotonic Solution ◽  
Current Profile ◽  
Patch Clamp Technique ◽  
Extracellular Medium ◽  
Intracellular Ca

Under hypotonic conditions, the regulatory volume decrease (RVD) is essential to maintain physiological homeostasis and functions in diverse biological systems. Intracellular Ca2+ has been reported as an important mediator of this response, but the underlying Ca2+ mechanism responsible for RVD is still controversial. Here we investigate the role of Ca2+ in the RVD response using live-cell imaging, microspectrofluorimetry, and a patch-clamp technique. A typical RVD was observed in submandibular gland acinar cells after swelling in a hypotonic solution, whereas intracellular Ca2+ chelation completely inhibited the RVD response. The incidence and magnitude of the Ca2+ transient were proportional to the degree of hypotonicity of the extracellular medium, and there was a close relationship between intracellular Ca2+ concentration and the volumetric changes of the cells. Notably, this response was mediated by Ca2+-induced Ca2+ release, which is triggered by Ca2+ influx via stretch-activated TRPM7 channels. Furthermore, we detected the generation of Cl− currents in the swelling acinar cells upon hypotonic stress, and the current profile matched that of the Ca2+-activated Cl− currents. A specific inhibitor of Cl− currents also inhibited the RVD response. In conclusion, an intracellular Ca2+ increase in response to osmotically induced cell swelling plays a critical role in RVD in salivary gland acinar cells.

scholarly journals Cl− and K+ conductances activated by cell swelling in primary cultures of rabbit distal bright convoluted tubules

1997 ◽  
Vol 273 (5) ◽  
pp. F680-F697 ◽  
Author(s):  
I. Rubera ◽  
M. Tauc ◽  
C. Poujeol ◽  
M. T. Bohn ◽  
M. Bidet ◽  
...  
Keyword(s):  
Osmotic Shock ◽  
Ion Selectivity ◽  
Primary Cultures ◽  
Regulatory Volume Decrease ◽  
Ionic Currents ◽  
Cell Swelling ◽  
Disulfonic Acid ◽  
Patch Clamp Technique ◽  
Pipette Solution ◽  
Whole Cell

Ionic currents induced by cell swelling were characterized in primary cultures of rabbit distal bright convoluted tubule (DCTb) by the whole cell patch-clamp technique. Cl− currents were produced spontaneously by whole cell recording with an isotonic pipette solution or by exposure to a hypotonic stress. Initial Cl− currents exhibited outwardly rectifying current-voltage relationship, whereas steady-state currents showed strong decay with depolarizing pulses. The ion selectivity sequence was I−= Br− > Cl− ≫ glutamate. Currents were inhibited by 0.1 mM 5-nitro-2-(3-phenylpropylamino)benzoic acid and 1 mM 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid and strongly blocked by 1 mM diphenylamine-2-carboxylate. Currents were insensitive to intracellular Ca2+but required the presence of extracellular Ca2+. They were not activated in cells pretreated with 200 nM staurosporine, 50 μM LaCl3, 10 μM nifedipine, 100 μM verapamil, 5 μM tamoxifen, and 50 μM dideoxyforskolin. Staurosporine, tamoxifen, verapamil, or the absence of external Ca2+ was without effect on the fully developed Cl−currents. Osmotic shock also activated K+ currents in Cl−-free conditions. These currents were time independent, activated at depolarized potentials, and inhibited by 5 mM BaCl2. The activation of Cl− and K+ currents by an osmotic shock may be implicated in regulatory volume decrease in DCTb cells.

Hypotonic swelling stimulates L-type Ca2+ channel activity in vascular smooth muscle cells through PKC

2004 ◽  
Vol 287 (2) ◽  
pp. C413-C421 ◽  
Author(s):  
Yanfeng Ding ◽  
Dean Schwartz ◽  
Philip Posner ◽  
Juming Zhong
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
Portal Vein ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Cell Swelling ◽  
Hypotonic Solution ◽  
Channel Activity ◽  
Rabbit Portal Vein ◽  
Hypotonic Swelling

It has been suggested that L-type Ca2+ channels play an important role in cell swelling-induced vasoconstriction. However, there is no direct evidence that Ca2+ channels in vascular smooth muscle are modulated by cell swelling. We tested the hypothesis that L-type Ca2+ channels in rabbit portal vein myocytes are modulated by hypotonic cell swelling via protein kinase activation. Ba2+ currents ( IBa) through L-type Ca2+ channels were recorded in smooth muscle cells freshly isolated from rabbit portal vein with the conventional whole cell patch-clamp technique. Superfusion of cells with hypotonic solution reversibly enhanced Ca2+ channel activity but did not alter the voltage-dependent characteristics of Ca2+ channels. Bath application of selective inhibitors of protein kinase C (PKC), Ro-31–8425 or Go-6983, prevented IBa enhancement by hypotonic swelling, whereas the specific protein kinase A (PKA) inhibitor KT-5720 had no effect. Bath application of phorbol 12,13-dibutyrate (PDBu) significantly increased IBa under isotonic conditions and prevented current stimulation by hypotonic swelling. However, PDBu did not have any effect on IBa when cells were first exposed to hypotonic solution. Furthermore, downregulation of endogenous PKC by overnight treatment of cells with PDBu prevented current enhancement by hypotonic swelling. These data suggest that hypotonic cell swelling can enhance Ca2+ channel activity in rabbit portal vein smooth muscle cells through activation of PKC.

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.

Potassium channels in primary cultures of seawater fish gill cells. II. Channel activation by hypotonic shock

2000 ◽  
Vol 279 (5) ◽  
pp. R1659-R1670 ◽  
Author(s):  
C. Duranton ◽  
E. Mikulovic ◽  
M. Tauc ◽  
M. Avella ◽  
P. Poujeol
Keyword(s):  
Cellular Localization ◽  
Present Report ◽  
Basolateral Membrane ◽  
Primary Cultures ◽  
Scorpion Venom ◽  
Convincing Evidence ◽  
Cell Swelling ◽  
Hypotonic Solution ◽  
Hypotonic Shock ◽  
Apical Side

Previous studies performed on apical membranes of seawater fish gills in primary culture have demonstrated the existence of stretch-activated K+channels with a conductance of 122 pS. The present report examines the involvement of K+ channels in ion transport mechanisms and cell swelling. In the whole cell patch-clamp configuration, K+ currents were produced by exposing cells to a hypotonic solution or to 1 μM ionomycin. These K+ currents were inhibited by the addition of quinidine and charybdotoxin to the bath solution. Isotopic efflux measurements were performed on cells grown on permeable supports using 86Rb+ as a tracer to indicate potassium movements. Apical and basolateral membrane86Rb effluxes were stimulated by the exposure of cells to a hypotonic medium. During the hypotonic shock, the stimulation of86Rb efflux on the apical side of the monolayer was inhibited by 500 μM quinidine or 100 μM gadolinium but was insensitive to scorpion venom [ Leirus quinquestriatus hebraeus (LQH)]. An increased 86Rb efflux across the basolateral membrane was also reduced by the addition of quinidine and LQH venom but was not modified by gadolinium. Moreover, basolateral and apical membrane 86Rb effluxes were not modified by bumetanide or thapsigargin. There is convincing evidence for two different populations of K+ channels activated by hypotonic shock. These populations can be separated according to their cellular localization (apical or basolateral membrane) and as a function of their kinetic behavior and pharmacology.

Volume-sensitive outwardly rectifying chloride channel in white adipocytes from normal and diabetic mice

2010 ◽  
Vol 298 (4) ◽  
pp. C900-C909 ◽  
Author(s):  
Hana Inoue ◽  
Nobuyuki Takahashi ◽  
Yasunobu Okada ◽  
Masato Konishi
Keyword(s):  
Current Density ◽  
Chloride Channel ◽  
Volume Regulation ◽  
Cell Types ◽  
Cell Swelling ◽  
Hypotonic Solution ◽  
Diabetic Mice ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
White Adipocytes

The volume-sensitive outwardly rectifying (VSOR) chloride channel is ubiquitously expressed and involved in cell volume regulation after osmotic swelling, called regulatory volume decrease (RVD), in various cell types. In adipocytes, the expression of the VSOR channel has not been explored to date. Here, by employing the whole-cell patch-clamp technique, we examined whether or not the VSOR channel is expressed in white adipocytes freshly isolated from epididymal fat pads of normal (C57BL/6 or KK) and diabetic (KKAy) mice. Whole cell voltage-clamp recordings revealed that Cl− currents were gradually activated upon cell swelling induced by application of a hypotonic solution, both in normal and diabetic adipocytes. Although both the mean cell size (or cell capacitance) and the current magnitude in KKAy adipocytes were larger than those in C57BL/6 cells, the current density was significantly lower in KKAy adipocytes (23.32 ± 1.94 pA in C57BL/6 adipocytes vs. 13.04 ± 2.41 pA in KKAy adipocytes at +100 mV). Similarly, the current density in diabetic KKAy adipocytes was lower than that in adipocytes from KK mice (a parental strain of KKAy mice), which do not present diabetes until an older age. The current was inhibited by Cl− channel blockers, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and glibenclamide, or hypertonic solution, and showed outward rectification and inactivation kinetics at large positive potentials. These electrophysiological and pharmacological properties are consistent with those of the VSOR channel in other cell types. Moreover, adipocytes showed RVD, which was inhibited by NPPB. In KKAy adipocytes, RVD was significantly slower (τ; 8.42 min in C57BL/6 adipocytes vs. 11.97 min in KKAy adipocytes) and incomplete during the recording period (25 min). It is concluded that the VSOR channel is functionally expressed and involved in volume regulation in white adipocytes. RVD is largely impaired in adipocytes from diabetic mice, presumably as a consequence of the lower density of the functional VSOR channel in the plasma membrane.

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.

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