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.

scholarly journals A vacuolar phosphate transporter essential for phosphate homeostasis in Arabidopsis

2015 ◽  
Vol 112 (47) ◽  
pp. E6571-E6578 ◽  
Author(s):  
Jinlong Liu ◽  
Lei Yang ◽  
Mingda Luan ◽  
Yuan Wang ◽  
Chi Zhang ◽  
...  
Keyword(s):  
Single Channel ◽  
Functional Characterization ◽  
Phosphate Transporter ◽  
Wild Type ◽  
Phosphate Homeostasis ◽  
Open Probability ◽  
Mesophyll Cells ◽  
Single Channel Analysis ◽  
Channel Analysis

Inorganic phosphate (Pi) is stored in the vacuole, allowing plants to adapt to variable Pi availability in the soil. The transporters that mediate Pi sequestration into vacuole remain unknown, however. Here we report the functional characterization of Vacuolar Phosphate Transporter 1 (VPT1), an SPX domain protein that transports Pi into the vacuole in Arabidopsis. The vpt1 mutant plants were stunted and consistently retained less Pi than wild type plants, especially when grown in medium containing high levels of Pi. In seedlings, VPT1 was expressed primarily in younger tissues under normal conditions, but was strongly induced by high-Pi conditions in older tissues, suggesting that VPT1 functions in Pi storage in young tissues and in detoxification of high Pi in older tissues. As a result, disruption of VPT1 rendered plants hypersensitive to both low-Pi and high-Pi conditions, reducing the adaptability of plants to changing Pi availability. Patch-clamp analysis of isolated vacuoles showed that the Pi influx current was severely reduced in vpt1 compared with wild type plants. When ectopically expressed in Nicotiana benthamiana mesophyll cells, VPT1 mediates vacuolar influx of anions, including Pi, SO42−, NO3−, Cl−, and malate with Pi as that preferred anion. The VPT1-mediated Pi current amplitude was dependent on cytosolic phosphate concentration. Single-channel analysis showed that the open probability of VPT1 was increased with the increase in transtonoplast potential. We conclude that VPT1 is a transporter responsible for vacuolar Pi storage and is essential for Pi adaptation in Arabidopsis.

Characterization of a newly found stretch-activated KCa,ATP channel in cultured chick ventricular myocytes

1999 ◽  
Vol 276 (6) ◽  
pp. H1827-H1838 ◽  
Author(s):  
Takashi Kawakubo ◽  
Keiji Naruse ◽  
Tatsuaki Matsubara ◽  
Nigishi Hotta ◽  
Masahiro Sokabe
Keyword(s):  
Single Channel ◽  
Ventricular Myocytes ◽  
Heart Cells ◽  
Patch Clamp Technique ◽  
Selective Channel ◽  
New Type ◽  
Inside Out ◽  
Ion Selectivities ◽  
Channel Type

With the use of the patch-clamp technique, five kinds of stretch-activated (SA) ion channels were identified on the basis of their single-channel conductances and ion selectivities in cultured chick ventricular myocytes. Because a high-conductance K+-selective channel predominated among these channels, we concentrated on characterizing its properties mostly using excised inside-out patches. With 145 mM KCl solution in the pipette and the bath, the channel had a conductance of 199.8 ± 8.2 pS ( n = 22). The ion selectivities among K+, Na+, Ca2+, and Cl− as estimated from their permeability ratios were P Na/ P K= 0.03, P Ca/ P K= 0.025, and P Cl/ P K= 0.026. The probability of the channel being open (Po) increased with the Ca2+concentration in the bath ([Ca2+]b; dissociation constant K d = 0.51 μM at +30 mV) and membrane potential (voltage at half-maximal Po= 39.4 mV at 0.35 μM [Ca2+]b). The channel was blocked by gadolinium, tetraethylammonium, and charybdotoxin from the extracellular surface and, consequently, was identified as a Ca2+-activated K+(KCa) channel type. The channel was also reversibly activated by ATP applied to the intracellular surface ( K d = 0.74 mM at 0.10 μM [Ca2+]bat +30 mV). From these data taken together, we concluded that the channel is a new type of KCachannel that could be designated as an “SA KCa,ATP channel.” To our knowledge, this is the first report of KCa channel in heart cells.

IP3-activated Ca2+ channels in the plasma membrane of cultured vascular endothelial cells

1995 ◽  
Vol 269 (3) ◽  
pp. C733-C738 ◽  
Author(s):  
L. Vaca ◽  
D. L. Kunze
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
Single Channel ◽  
Vascular Endothelial Cells ◽  
Channel Activity ◽  
Open Probability ◽  
Aortic Endothelial Cells ◽  
Control Value ◽  
Selective Channel ◽  
Inside Out

Although it is clear that D-myo-inositol 1,4,5-trisphosphate (IP3) plays an important role in the activation of Ca2+ influx, the mechanisms by which this occurs remain controversial. In an attempt to determine the role of IP3 in the activation of Ca2+ influx, patch-clamp single-channel experiments in the cell-attached, inside-out, and outside-out configurations were performed on cultured bovine aortic endothelial cells (BAEC). The results presented indicate that both IP3 and intracellular Ca2+ can modulate the activity of a Ca(2+)-selective channel found in the plasma membrane of these cells. Addition of 10 microM IP3 increased channel open probability (P(o)) from a control value of 0.12 +/- 0.05 to 0.7 +/- 0.13 at a constant intracellular Ca2+ of 1 nM in excised inside-out patches. D-Myo-inositol 1,3,4,5-tetrakisphosphate at 50 microM was ineffective in altering channel P(o). Channel activity declined after approximately 2 min in the continuous presence of IP3. Three to four minutes after addition of IP3, channel P(o) was reduced from 0.7 +/- 0.2 to 0.2 +/- 0.1, indicating that an additional regulator might be required to maintain channel activity in excised patches. The channel was reversibly blocked by application of 1 microgram/ml heparin to the intracellular side of inside-out patches. This Ca(2+)-selective channel is indistinguishable from the depletion-activated Ca2+ channel we have previously described in BAEC.

Characterization of Mechanisms Mediating Drug Resistance in Human Leukemic Cells Selected with High Concentration Methotrexate

Pteridines ◽  
1991 ◽  
Vol 3 (1-2) ◽  
pp. 119-121
Author(s):  
M. Kavallaris ◽  
M. Haber ◽  
M. D. Norris ◽  
L. H. Matherly ◽  
B. W. Stewart
Keyword(s):  
Drug Resistance ◽  
Leukemic Cells ◽  
High Concentration ◽  
Human Leukemic Cells

scholarly journals Comparison of Effects of the Ethanolic Extracts of Brazilian Propolis on Human Leukemic Cells As Assessed with the MTT Assay

2012 ◽  
Vol 2012 ◽  
pp. 1-6 ◽  
Author(s):  
Gilberto C. Franchi ◽  
Cleber S. Moraes ◽  
Viviane C. Toreti ◽  
Andreas Daugsch ◽  
Alexandre E. Nowill ◽  
...  
Keyword(s):  
K562 Cells ◽  
Leukemic Cells ◽  
Cytotoxic Activities ◽  
Human Leukemia ◽  
Cancer Cell Growth ◽  
Brazilian Propolis ◽  
Human Leukemic Cells ◽  
Dioxin Toxicity ◽  
Resinous Product

Propolis is a resinous product collected by honey bees. It was also reported that propolis has a wide variety of biological actions, including antimicrobial activity and antioxidant, anti-inflammatory, and suppressive effects of dioxin toxicity activities. The aim of this study was to compare the in vitro cytotoxic activities of green propolis (G12) and red propolis (G13) in human leukemia cells. These cells were incubated with different concentrations of propolis and 48 hours after the IC50was calculated for each cell. The results showed that the red propolis has cytotoxic effect in vitro higher than green propolis. Red propolis was showed to be cytostatic in K562 cells and caused the same amount of apoptosis as its control Gleevec. In conclusion, these results showed that red propolis is more cytotoxic than the green propolis in a variety of human cell lines of leukemia. Red propolis may contain drugs capable of inhibiting cancer cell growth. Therefore, further isolation of respective chemical ingredients from the red propolis (G13) for identification of the activities is necessary.

scholarly journals Functional characterization of a voltage-gated anion channel from teleost fish intestinal epithelium

1988 ◽  
Vol 136 (1) ◽  
pp. 383-403 ◽  
Author(s):  
C. A. Loretz ◽  
C. R. Fourtner
Keyword(s):  
Single Channel ◽  
Basolateral Membrane ◽  
Functional Characterization ◽  
Anion Channel ◽  
Intestinal Epithelial ◽  
Channel Conductance ◽  
Patch Clamp Technique ◽  
Voltage Gated

An anion channel was isolated, using patch-clamp technique, from the basolateral membrane of goby intestinal epithelial cells. Single-channel conductance varied over a range from 20 to 90 pS. The channel was voltage-gated over the physiological range of cell membrane potential with depolarization increasing the proportion of time in the open state. There was no Ca2+ sensitivity. The selectivity sequence was SO4(2-) greater than Cl- greater than Mes-. The channel may function in vivo as one of several avenues of basolateral membrane Cl- exit with the voltage-gating property serving to match basolateral Cl- exit to apical entry.

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.

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