scholarly journals CFTR Gating I

2005 ◽  
Vol 125 (4) ◽  
pp. 361-375 ◽  
Author(s):  
Silvia G. Bompadre ◽  
Tomohiko Ai ◽  
Jeong Han Cho ◽  
Xiaohui Wang ◽  
Yoshiro Sohma ◽  
...  
Keyword(s):  
Single Channel ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Kinetic Mechanism ◽  
Inhibitory Effect ◽  
Unexpected Result ◽  
Ovary Cells ◽  
Gating Mechanisms ◽  
Inside Out ◽  
R Domain

The CFTR chloride channel is activated by phosphorylation of serine residues in the regulatory (R) domain and then gated by ATP binding and hydrolysis at the nucleotide binding domains (NBDs). Studies of the ATP-dependent gating process in excised inside-out patches are very often hampered by channel rundown partly caused by membrane-associated phosphatases. Since the severed ΔR-CFTR, whose R domain is completely removed, can bypass the phosphorylation-dependent regulation, this mutant channel might be a useful tool to explore the gating mechanisms of CFTR. To this end, we investigated the regulation and gating of the ΔR-CFTR expressed in Chinese hamster ovary cells. In the cell-attached mode, basal ΔR-CFTR currents were always obtained in the absence of cAMP agonists. Application of cAMP agonists or PMA, a PKC activator, failed to affect the activity, indicating that the activity of ΔR-CFTR channels is indeed phosphorylation independent. Consistent with this conclusion, in excised inside-out patches, application of the catalytic subunit of PKA did not affect ATP-induced currents. Similarities of ATP-dependent gating between wild type and ΔR-CFTR make this phosphorylation-independent mutant a useful system to explore more extensively the gating mechanisms of CFTR. Using the ΔR-CFTR construct, we studied the inhibitory effect of ADP on CFTR gating. The Ki for ADP increases as the [ATP] is increased, suggesting a competitive mechanism of inhibition. Single channel kinetic analysis reveals a new closed state in the presence of ADP, consistent with a kinetic mechanism by which ADP binds at the same site as ATP for channel opening. Moreover, we found that the open time of the channel is shortened by as much as 54% in the presence of ADP. This unexpected result suggests another ADP binding site that modulates channel closing.

scholarly journals Oligomycin inhibits store-operated channels by a mechanism independent of its effects on mitochondrial ATP

1997 ◽  
Vol 324 (3) ◽  
pp. 971-980 ◽  
Author(s):  
Jwa Hwa CHO ◽  
M. BALASUBRAMANYAM ◽  
Galina CHERNAYA ◽  
Jeffrey P. GARDNER ◽  
Abraham AVIV ◽  
...  
Keyword(s):  
T Cells ◽  
Rank Order ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Inhibitory Effect ◽  
Jurkat T Cells ◽  
Ovary Cells ◽  
Atp Production ◽  
Patch Pipette ◽  
Pump Activity

Inhibitors of mitochondrial oxidative metabolism have been proposed to interfere with Ca2+ influx mediated by store-operated channels (SOC), secondary to their effects on ATP production. We assessed SOC activity by 45Ca2+ influx and fluorimetric measurements of free Ca2+ or Mn2+ quench in thapsigargin-treated Chinese hamster ovary cells and Jurkat T-cells, and additionally by electrophysiological measurements of the Ca2+-release-activated Ca2+ current (Icrac) in Jurkat T-cells. Various mitochondrial antagonists were confirmed to inhibit SOC. However, the following evidence supported the proposal that oligomycin, in particular, exerts an inhibitory effect on SOC in addition to its known actions on mitochondria and Na+-pump activity: (i) the concentrations of oligomycin required to inhibit SOC-mediated Ca2+ influx or Icrac (half-inhibitory concentration ∼2 μM) were nearly 50-fold higher than the concentrations that blocked mitochondrial ATP production; (ii) the rank order of potency of oligomycins A, B and C for decreasing SOC-mediated Ca2+ influx or Icrac differed from that known for inhibition of mitochondrial function; (iii) oligomycin blocked Icrac under voltage clamp and with intracellular Na+ and K+ concentrations fixed by dialysis from the patch pipette, arguing that the effect was not secondary to membrane polarization or pump activity; and (iv) fixing the cytosolic ATP concentration by dialysis from the patch pipette attenuated rotenone- but not oligomycin-mediated inhibition of Icrac. Oligomycin also blocked volume-activated Cl- currents, a profile common to some other known blockers of SOC that are not known mitochondrial inhibitors. These findings raise the possibility that oligomycin interacts directly with SOC, and thus may extend the known pharmacological profile for this type of Ca2+-influx pathway.

Dibasic phosphorylation sites in the R domain of CFTR have stimulatory and inhibitory effects on channel activation

2004 ◽  
Vol 287 (3) ◽  
pp. C737-C745 ◽  
Author(s):  
Horia Vais ◽  
Rugang Zhang ◽  
William W. Reenstra
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Phosphorylation Sites ◽  
Dependent Manner ◽  
Channel Activity ◽  
Ovary Cells ◽  
Channel Opening ◽  
Excised Patches ◽  
A Site ◽  
R Domain

To better understand the mechanisms by which PKA-dependent phosphorylation regulates CFTR channel activity, we have assayed open probabilities ( Po), mean open time, and mean closed time for a series of CFTR constructs with mutations at PKA phosphorylation sites in the regulatory (R) domain. Forskolin-stimulated channel activity was recorded in cell-attached and inside-out excised patches from transiently transfected Chinese hamster ovary cells. Wild-type CFTR and constructs with a single Ser-to-Ala mutation as well as octa (Ser-to-Ala mutations at 8 sites) and constructs with one or two Ala-to-Ser mutations were studied. In cell-attached patches, Ser-to-Ala mutations at amino acids 700, 795, and 813 decreased Po, whereas Ser-to-Ala mutations at 737 and 768 increased Po. In general, differences in Po were due to differences in mean closed time. For selected constructs with either high or low values of Po, channel activity was measured in excised patches. With 1 mM ATP, Po was similar to that observed in cell-attached patches, but with 10 mM ATP, all constructs tested showed elevated Po values. ATP-dependent increases in Po were due to reductions in mean closed time. These results indicate that R-domain phosphorylation affects ATP binding and not the subsequent steps of hydrolysis and channel opening. A model was developed whereby R-domain phosphorylation, in a site-dependent manner, alters equilibrium between forms of CFTR with low and high affinities for ATP.

Retigabine Stimulates Human KCNQ2/Q3 Channels in the Presence of Bupivacaine

2004 ◽  
Vol 101 (2) ◽  
pp. 430-438 ◽  
Author(s):  
Mark A. Punke ◽  
Patrick Friederich
Keyword(s):  
Membrane Potential ◽  
Local Anesthetic ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Inhibitory Effect ◽  
Hill Coefficient ◽  
Patch Clamp Technique ◽  
Ovary Cells ◽  
Novel Approach ◽  
Concentration Response Curve

Background Inhibition of KCNQ2/Q3 channels may cause convulsion in humans. The interaction of bupivacaine with these channels is unknown. The anticonvulsant retigabine activates KCNQ2/Q3 channels and may reverse inhibitory actions of bupivacaine. Potassium channel stimulation may thus constitute a novel approach to treat local anesthetic-induced seizures. The aim of this study was to characterize bupivacaine effects on KCNQ2/Q3 channels and to investigate whether retigabine reverses the effects of the local anesthetic. Methods KCNQ2/Q3 channels were transiently expressed in Chinese hamster ovary cells. The effects of bupivacaine and retigabine were studied with the patch-clamp technique. Results Bupivacaine inhibited KCNQ2/Q3 channels in a concentration-dependent and reversible manner. The concentration-response curve was described by a Hill equation (IC50 = 173 +/- 7 microm, Hill coefficient = 1.4 +/- 0.1, mean +/- SEM, n = 37). The inhibitory effect did not differ between bupivacaine and levobupivacaine (42 +/- 4%, n = 7, versus 42 +/- 5%, n = 10; P > 0.05). Ropivacaine was four times less potent than bupivacaine. The inhibition of KCNQ2/Q3 channels by bupivacaine resulted in a significant and reversible depolarization of the membrane potential. Retigabine (300 nm-10 microm) reversed the inhibitory action of bupivacaine on KCNQ2/Q3 channels as well as the depolarization of the membrane potential. Conclusions The anticonvulsant retigabine at nanomolar concentrations reverses the inhibitory effect of micromolar concentrations of bupivacaine. Our results allow the hypothesis that activation of KCNQ2/Q3 channels by retigabine may offer a novel therapeutic approach for the treatment of bupivacaine-induced seizures.

scholarly journals Antiarrhythmic Mechanisms of Chinese Herbal Medicine Dingji Fumai Decoction

2020 ◽  
Vol 2020 ◽  
pp. 1-9
Author(s):  
Bo Liang ◽  
Yan Zhou ◽  
Ling Fu ◽  
Hui-Ling Liao
Keyword(s):  
Ventricular Arrhythmia ◽  
Connexin 43 ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Barium Chloride ◽  
Inhibitory Effect ◽  
Ovary Cells ◽  
Whole Cell Patch Clamp ◽  
And Oxidative Stress ◽  
Electrophysiological Mechanism

Background. Dingji Fumai decoction (DFD) is used to treat ventricular arrhythmia, and it has provided a very good curative effect. However, its cellular electrophysiological mechanism is unknown. Methods. Electrocardiogram was recorded, and oxidative stress response and ion-channel-related molecules were detected in rats with barium chloride- and aconitine-induced ventricular arrhythmia. Moreover, whole-cell patch-clamp assay was used to investigate the inhibitory effect of DFD on Nav1.5 in Chinese hamster ovary cells. Results. DFD prolonged the occurrence time and shortened the duration of ventricular arrhythmia, decreased the malondialdehyde and increased the superoxide dismutase, and alleviated the activation of Na+-K+-ATPase and connexin-43. DFD suppressed Nav1.5dose-dependently with an IC50 of 24.0 ± 2.4 mg/mL. Conclusions. The clinical antiarrhythmic mechanisms of DFD are based on its antioxidant potential, alleviation of Na+-K+-ATPase and connexin-43, and class I antiarrhythmic properties by suppressing Nav1.5dose-dependently with an IC50 of 24.0 ± 2.4 mg/mL.

Interaction of Ropivacaine with Cloned Cardiac Kv4.3/KChIP2.2 Complexes

2004 ◽  
Vol 101 (6) ◽  
pp. 1347-1356 ◽  
Author(s):  
Patrick Friederich ◽  
Anna Solth
Keyword(s):  
Local Anesthetic ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Outward Current ◽  
Ventricular Myocardium ◽  
Inhibitory Effect ◽  
Transient Outward Current ◽  
Qtc Interval ◽  
Ovary Cells ◽  
Ic50 Value

Background Inhibition of cardiac K channels by local anesthetic may contribute to QTc interval prolongation of the electrocardiogram and induction of ventricular arrhythmia. The transient outward current Ito has been identified as a toxicologically relevant target of bupivacaine. S(-)-ropivacaine has been developed as a safer alternative to bupivacaine. The effects of S(-)-ropivacaine on Ito have not been investigated. In human ventricular myocardium, Ito is formed by Kv4.3 and KChIP2.2 subunits. Therefore, the aim of this study was to establish the effects of S(-)-ropivacaine on human Kv4.3/KChIP2.2 channels. Methods Kv4.3/KChIP2.2 complementary DNA cloned from human heart was transiently transfected in Chinese hamster ovary cells. The pharmacologic effects of S(-)-ropivacaine were investigated with the patch clamp method. Results Ropivacaine inhibited Kv4.3/KChIP2.2 channels in a concentration-dependent, stereospecific, and reversible manner. The IC50 value of S(-)-ropivacaine for inhibition of the charge conducted by Kv4.3/KChIP2.2 channel was 117 +/- 21 microm (n = 30). The local anesthetic accelerated macroscopic current decline with an IC50 value of 77 +/- 11 microm (n = 30). It shifted the midpoint of channel activation into the depolarizing direction, and it slowed recovery from inactivation without altering steady state inactivation. Kv4.3 channels are more sensitive to the inhibitory effect than Kv4.3/KChIP2.2 channels. Conclusions : The results are consistent with the idea that ropivacaine, by blocking Kv4.3/KChIP2.2 from the open state, interferes with the gating modifying effects of KChIP2.2 on Kv4.3 channels. Inhibition of Kv4.3/KChIP2.2 channels by the local anesthetic may contribute to the deterioration of cardiac function during events of intoxication.

scholarly journals Expression of TRPC3 in Chinese Hamster Ovary Cells Results in Calcium-activated Cation Currents Not Related to Store Depletion

1997 ◽  
Vol 138 (6) ◽  
pp. 1333-1341 ◽  
Author(s):  
Christof Zitt ◽  
Alexander G. Obukhov ◽  
Carsten Strübing ◽  
Andrea Zobel ◽  
Frank Kalkbrenner ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Membrane Receptors ◽  
Calcium Stores ◽  
Channel Activity ◽  
Ovary Cells ◽  
Cytosolic Side ◽  
Green Fluorescent ◽  
Inside Out

TRPC3 (or Htrp3) is a human member of the trp family of Ca2+-permeable cation channels. Since expression of TRPC3 cDNA results in markedly enhanced Ca2+ influx in response to stimulation of membrane receptors linked to phospholipase C (Zhu, X., J. Meisheng, M. Peyton, G. Bouley, R. Hurst, E. Stefani, and L. Birnbaumer. 1996. Cell. 85:661–671), we tested whether TRPC3 might represent a Ca2+ entry pathway activated as a consequence of depletion of intracellular calcium stores. CHO cells expressing TRPC3 after intranuclear injection of cDNA coding for TRPC3 were identified by fluorescence from green fluorescent protein. Expression of TRPC3 produced cation currents with little selectivity for Ca2+ over Na+. These currents were constitutively active, not enhanced by depletion of calcium stores with inositol-1,4,5-trisphosphate or thapsigargin, and attenuated by strong intracellular Ca2+ buffering. Ionomycin led to profound increases of currents, but this effect was strictly dependent on the presence of extracellular Ca2+. Likewise, infusion of Ca2+ into cell through the patch pipette increased TRPC3 currents. Therefore, TRPC3 is stimulated by a Ca2+-dependent mechanism. Studies on TRPC3 in inside-out patches showed cation-selective channels with 60-pS conductance and short (<2 ms) mean open times. Application of ionomycin to cells increased channel activity in cell-attached patches. Increasing the Ca2+ concentration on the cytosolic side of inside-out patches (from 0 to 1 and 30 μM), however, failed to stimulate channel activity, even in the presence of calmodulin (0.2 μM). We conclude that TRPC3 codes for a Ca2+-permeable channel that supports Ca2+-induced Ca2+-entry but should not be considered store operated.

scholarly journals Effect of Colcemid on the Centriole Cycle in Chinese Hamster Ovary Cells

1982 ◽  
Vol 53 (1) ◽  
pp. 155-171 ◽  
Author(s):  
RYOKO KURIYAMA
Keyword(s):  
Chinese Hamster Ovary ◽  
Structural Changes ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Inhibitory Effect ◽  
Ultrastructural Changes ◽  
Prolonged Treatment ◽  
Ovary Cells ◽  
Almost All ◽  
Daughter Centriole

The structural changes in the centrioles in Chinese hamster ovary cells were monitored by electron microscopy of whole mount preparations to investigate the effects of colcemid on the events in the centriole cycle. The population of mitotic cells increased with time of incubation with colcemid, but the arrest at mitosis by this drug was soon overcome, resulting in the formation of nuclei and a change in the shape of the cells again spreading over the substrate. The maximal mitotic index was reached every 25 h in the presence of either 0.10 or 0.91 μg/ml of colcemid. During this time, cells became multinucleated, increased greatly in size, and accumulated 8 to 10-nm filamentous bundles in the cytoplasm instead of microtubules, almost all of which had been depolymerized after exposure to colcemid. In the cells that were continuously treated with colcemid, a pair of centrioles became disoriented and each subsequently produced a daughter centriole. However, these daughter centrioles elongated to only half their full length; many unusual figures in the centriolar pairs resulted from their proceeding normally to the phases for disorientation and nucleation for centrioles in the next cycle. Although the rate of centriole elongation and the frequency of formation of the daughter centrioles were decreased by increasing the concentration of colcemid, the disorientation of the centrioles was not disturbed by this drug. The inhibitory effect of colcemid on centriolar nucleation and elongation was found to be totally reveisible; the formation and elongation of new daughter centrioles occurred again just after removal of the drug. Prolonged treatment of cells with colcemid caused ultrastructural changes in the centrioles, such as the outgrowth of microtubules from the wall of centriolar triplets or the formation of unusual bundles of microtubules around the centrioles.

Small linear chloride channels are endogenous to nonepithelial cells

1992 ◽  
Vol 263 (3) ◽  
pp. C708-C713 ◽  
Author(s):  
S. E. Gabriel ◽  
E. M. Price ◽  
R. C. Boucher ◽  
M. J. Stutts
Keyword(s):  
Cell Lines ◽  
Chloride Channels ◽  
Single Channel ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cell Types ◽  
Ovary Cells ◽  
3T3 Fibroblasts ◽  
Whole Cell Patch Clamp ◽  
Cl Channels

We used both single-channel and whole cell patch-clamp techniques to characterize chloride channels and currents endogenous to Sf9 cells, 3T3 fibroblasts, and Chinese hamster ovary cells. In cell-attached patches from these cell types, anion channels were observed with low ohmic conductance (4-11 ps), linear current-voltage relationships, and little time- or voltage-dependent behavior. These channels are very similar to the Cl- channels reported to appear concomitant with the expression of cystic fibrosis transmembrane conductance regulator (CFTR) in these cell lines. The presence of such endogenous channels suggests either that low levels of CFTR are present in all of these cell lines prior to transfection or that an endogenous non-CFTR channel is present in these cell types. Our results suggest that at least some of the channel behaviors attributed to expressed, recombinant CFTR in previous studies may have been due to these endogenous Cl- channels.

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