Association between Hsp90 and the ClC-2 chloride channel upregulates channel function

2006 ◽  
Vol 290 (1) ◽  
pp. C45-C56 ◽  
Author(s):  
Alexandre Hinzpeter ◽  
Joanna Lipecka ◽  
Franck Brouillard ◽  
Maryvonne Baudoin-Legros ◽  
Michal Dadlez ◽  
...  
Keyword(s):  
Chloride Channel ◽  
Fluid Transport ◽  
Cell Swelling ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
Mass Spectrometry Identification ◽  
Voltage Dependent ◽  
Channel Expression ◽  
Associated Proteins ◽  
Cellular Stresses

The voltage-dependent ClC-2 chloride channel has been implicated in a variety of physiological functions, including fluid transport across specific epithelia. ClC-2 is activated by hyperpolarization, weakly acidic external pH, intracellular Cl−, and cell swelling. To add more insight into the mechanisms involved in ClC-2 regulation, we searched for associated proteins that may influence ClC-2 activity. With the use of immunoprecipitation of ClC-2 from human embryonic kidney-293 cells stably expressing the channel, followed by electrophoretic separation of coimmunoprecipitated proteins and mass spectrometry identification, Hsp70 and Hsp90 were unmasked as possible ClC-2 interacting partners. Association of Hsp90 with ClC-2 was confirmed in mouse brain. Inhibition of Hsp90 by two specific inhibitors, geldanamycin or radicicol, did not affect total amounts of ClC-2 but did reduce plasma membrane channel abundance. Functional experiments using the whole cell configuration of the patch-clamp technique showed that inhibition of Hsp90 reduced ClC-2 current amplitude and impaired the intracellular Cl− concentration [Cl−]-dependent rightward shift of the fractional conductance. Geldanamycin and radicicol increased both the slow and fast activation time constants in a chloride-dependent manner. Heat shock treatment had the opposite effect. These results indicate that association of Hsp90 with ClC-2 results in greater channel activity due to increased cell surface channel expression, facilitation of channel opening, and enhanced channel sensitivity to intracellular [Cl−]. This association may have important pathophysiological consequences, enabling increased ClC-2 activity in response to cellular stresses such as elevated temperature, ischemia, or oxidative reagents.

Direct block of cloned hKv1.5 channel by cytochalasins, actin-disrupting agents

2005 ◽  
Vol 289 (2) ◽  
pp. C425-C436 ◽  
Author(s):  
Bok Hee Choi ◽  
Jung-Ah Park ◽  
Kyung-Ryoul Kim ◽  
Ggot-Im Lee ◽  
Yong-Tae Lee ◽  
...  
Keyword(s):  
Actin Filament ◽  
Cytochalasin B ◽  
Delayed Rectifier ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
Independent Manner ◽  
Concentration Dependent Manner ◽  
Voltage Range ◽  
Whole Cell Patch Clamp ◽  
Voltage Dependent

The action of cytochalasins, actin-disrupting agents on human Kv1.5 channel (hKv1.5) stably expressed in Ltk− cells was investigated using the whole cell patch-clamp technique. Cytochalasin B inhibited hKv1.5 currents rapidly and reversibly at +60 mV in a concentration-dependent manner with an IC50 of 4.2 μM. Cytochalasin A, which has a structure very similar to cytochalasin B, inhibited hKv1.5 (IC50 of 1.4 μM at +60 mV). Pretreatment with other actin filament disruptors cytochalasin D and cytochalasin J, and an actin filament stabilizing agent phalloidin had no effect on the cytochalasin B-induced inhibition of hKv1.5 currents. Cytochalasin B accelerated the decay rate of inactivation for the hKv1.5 currents. Cytochalasin B-induced inhibition of the hKv1.5 channels was voltage dependent with a steep increase over the voltage range of the channel's opening. However, the inhibition exhibited voltage independence over the voltage range in which channels are fully activated. Cytochalasin B produced no significant effect on the steady-state activation or inactivation curves. The rate constants for association and dissociation of cytochalasin B were 3.7 μM/s and 7.5 s−1, respectively. Cytochalasin B produced a use-dependent inhibition of hKv1.5 current that was consistent with the slow recovery from inactivation in the presence of the drug. Cytochalasin B (10 μM) also inhibited an ultrarapid delayed rectifier K+ current ( IK,ur) in human atrial myocytes. These results indicate that cytochalasin B primarily blocks activated hKv1.5 channels and endogenous IK,ur in a cytoskeleton-independent manner as an open-channel blocker.

Inhibition of CLC-2 chloride channel expression interrupts expansion of fetal lung cysts

2004 ◽  
Vol 286 (2) ◽  
pp. L420-L426 ◽  
Author(s):  
Carol J. Blaisdell ◽  
Marcelo M. Morales ◽  
Ana Carolina Oliveira Andrade ◽  
Penelope Bamford ◽  
Michael Wasicko ◽  
...  
Keyword(s):  
Chloride Channel ◽  
Fluid Transport ◽  
Fetal Lung ◽  
Chloride Secretion ◽  
Normal Lung ◽  
Apical Surface ◽  
Lung Cysts ◽  
Lung Fluid ◽  
Lung Morphogenesis ◽  
Channel Expression

Normal lung morphogenesis is dependent on chloride-driven fluid transport. The molecular identity of essential fetal lung chloride channel(s) has not been elucidated. CLC-2 is a chloride channel, which is expressed on the apical surface of the developing respiratory epithelium. CLC-2-like pH-dependent chloride secretion exists in fetal airway cells. We used a 14-day fetal rat lung submersion culture model to examine the role of CLC-2 in lung development. In this model, the excised fetal lung continues to grow, secrete fluid, and become progressively cystic in morphology ( 26 ). We inhibited CLC-2 expression in these explants, using antisense oligonucleotides, and found that lung cyst morphology was disrupted. In addition, transepithelial voltage ( Vt) of lung explants transfected with antisense CLC-2 was inhibited with Vt = -1.5 ± 0.2 mV (means + SE) compared with -3.7 ± 0.3 mV (means + SE) for mock-transfected controls and -3.3 ± 0.3 mV (means + SE) for nonsense oligodeoxynucleotide-transfected controls. This suggests that CLC-2 is important for fetal lung fluid production and that it may play a role in normal lung morphogenesis.

Calcium Channel Currents in Acutely Dissociated Intracardiac Neurons From Adult Rats

1997 ◽  
Vol 77 (4) ◽  
pp. 1769-1778 ◽  
Author(s):  
Seong-Woo Jeong ◽  
Robert D. Wurster
Keyword(s):  
Calcium Channel ◽  
Fold Change ◽  
Dependent Manner ◽  
High Concentration ◽  
Patch Clamp Technique ◽  
Adult Rats ◽  
Voltage Dependent ◽  
Steady State Inactivation ◽  
Channel Currents ◽  
Intracardiac Neurons

Jeong, Seong-Woo and Robert D. Wurster. Calcium channel currents in acutely dissociated intracardiac neurons from adult rats. J. Neurophysiol. 77: 1769–1778, 1997. With the use of the whole cell patch-clamp technique, multiple subtypes of voltage-activated calcium channels, as indicated by measuring Ba2+ currents, were pharmacologically identified in acutely dissociated intracardiac neurons from adult rats. All tested neurons that were held at −80 mV displayed only high-voltage-activated (HVA) Ca2+ channel currents that were completely blocked by 100 μM CdCl2. The current density of HVA Ca2+ currents was dependent on the external Ca2+ concentration. The Ba2+ (5 mM) currents were half-activated at −16.3 mV with a slope of 5.6 mV per e-fold change. The steady-state inactivation was also voltage dependent with half-inactivation at −33.7 mV and a slope of −12.1 mV per e-fold change. The most effective L-type channel activator, FPL 64176 (2 μM), enhanced the Ba2+ current in a voltage-dependent manner. When cells were held at −80 mV, the saturating concentration (10 μM) of nifedipine blocked ∼11% of the control Ba2+ current. The major component of the Ca2+ channels was N type (63%), which was blocked by a saturating concentration (1 μM) of ω-conotoxin GVIA. Approximately 19% of the control Ba2+ current was sensitive to ω-conotoxin MVIIC (5 μM) but insensitive to low concentrations (30 and 100 nM) of ω-agatoxin IVA (ω-Aga IVA). In addition, a high concentration (1 μM) of ω-Aga IVA occluded the effect of ω-conotoxin MVIIC. Taken together, these results indicate that the ω-conotoxin MVIIC-sensitive current represents only the Q type of Ca2+ channels. The current that was insensitive to nifedipine and various toxins represents the R-type current (7%), which was sensitive to 100 μM NiCl2. In conclusion, the intracardiac neurons from adult rats express at least four different subtypes (L, N, Q, and R) of HVA Ca2+ channels. This information is essential for understanding the regulation of synaptic transmission and excitability of intracardiac neurons by different neurotransmitters and neural regulation of cardiac functions.

Preferential potentiation by hypotonic cell swelling of muscarinic cation current in guinea pig ileum

1997 ◽  
Vol 272 (1) ◽  
pp. C240-C253 ◽  
Author(s):  
Y. Waniishi ◽  
R. Inoue ◽  
Y. Ito
Keyword(s):  
Smooth Muscle ◽  
Guinea Pig ◽  
Muscarinic Receptor ◽  
Time Course ◽  
Cell Swelling ◽  
Patch Clamp Technique ◽  
Cationic Current ◽  
Whole Cell Patch Clamp ◽  
Voltage Dependent ◽  
Hypotonic Cell Swelling

The effects of hypotonic cell swelling (HCS) on muscarinic receptor-activated cationic current in guinea pig ileal smooth muscle were investigated by the whole cell patch-clamp technique. With nystatin-perforated recording, reduced external tonicity from 312 to 262 mosM caused cell swelling but hardly affected the membrane currents activated by depolarization, such as outward-rectifying K and voltage-dependent Ca currents. In contrast, the inward current evoked by carbachol at -60 mV was greatly increased (approximately 50%) by the same extent of hypotonicity. This effect is likely to occur through potentiation of nonselective cation channels coupled to the muscarinic receptor (mNSCCs) and probably does not involve elevated intracellular Ca2+ concentration ([Ca2+]i), since neither removal of external Ca2+ nor [Ca2+]i buffering with 10 mM 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid significantly affected the results. Furthermore, the time course and degree of this potentiation closely matched those of video-microscopically monitored HCS. These results support the view that mechanosensitive modulation may be a powerful mechanism to regulate mNSCCs activity in gut smooth muscle, together with membrane potential and [Ca2+]i.

Charybdotoxin block of Ca2+-activated K+ channels in colonic muscle depends on membrane potential dynamics

1998 ◽  
Vol 274 (3) ◽  
pp. C673-C680 ◽  
Author(s):  
Bret W. Frey ◽  
Andreas Carl ◽  
Nelson G. Publicover
Keyword(s):  
Membrane Potential ◽  
Longitudinal Muscle ◽  
Circular Muscle ◽  
Resting Membrane Potential ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
Whole Cell Patch Clamp ◽  
Rate Of Increase ◽  
Voltage Dependent ◽  
Simple Kinetic Model

Charybdotoxin (ChTX) is a specific blocker of Ca2+-activated K+ channels. The voltage- and time-dependent dynamics of ChTX block were investigated using canine colonic myocytes and the whole cell patch-clamp technique with step and ramp depolarization protocols. During prolonged step depolarizations, K+ current slowly increased in the continued presence of ChTX (100 nM). The rate of increase depended on membrane potential with an e-fold change for every 60 mV. During ramp depolarizations, the effectiveness of ChTX block depended significantly on the rate of the ramp (50% at 0.01 V/s to 80% at 0.5 V/s). Results are consistent with a mechanism in which ChTX slowly “unbinds” in a voltage-dependent manner. A simple kinetic model was developed in which ChTX binds to both open and closed states. Slow unbinding is consistent with ChTX having little effect on electrical slow waves recorded from circular muscle while causing depolarization and contraction of longitudinal muscle, which displays more rapid “spikes.” Resting membrane potential and membrane potential dynamics are important determinants of ChTX action.

scholarly journals Ascorbic acid modulation of calcium channels in pancreatic beta cells.

1993 ◽  
Vol 102 (3) ◽  
pp. 503-523 ◽  
Author(s):  
R V Parsey ◽  
D R Matteson
Keyword(s):  
Ascorbic Acid ◽  
Calcium Channels ◽  
Beta Cells ◽  
Pancreatic Beta Cells ◽  
Inactivation Kinetics ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
Voltage Dependent ◽  
Voltage Dependent Calcium Channels ◽  
Metal Catalyzed

We have studied the effect of ascorbic acid on voltage-dependent calcium channels in pancreatic beta cells. Using the whole-cell and perforated-patch variants of the patch clamp technique to record calcium tail currents, we have shown that the slowly deactivating (SD) calcium channel, which is similar to the T-type channel in other cells, is inhibited in a voltage-dependent manner by ascorbic acid (AA). The other channels that carry inward current in beta cells, FD calcium channels and sodium channels, are unaffected by AA. Ascorbic acid causes a voltage-dependent decrease in the magnitude of the SD channel conductance which can be explained by the hypothesis that approximately 50-60% of the channels have their voltage dependence shifted by approximately 62 mV in the depolarizing direction. Thus, ascorbate appears to modify only a fraction of the SD channels. The activation kinetics of the ascorbate-modified channels are slower than control channels in a manner that is consistent with this hypothesis. Deactivation and inactivation kinetics are unaffected by ascorbate. These effects of ascorbate require metal ions, and it appears that some of the activity of ascorbate is due to a product of its metal catalyzed oxidation, perhaps dehydroascorbate.

scholarly journals Effects of suplatast tosilate on acutely dissociated sensory and paratracheal ganglia neurons

2016 ◽  
Vol 311 (4) ◽  
pp. L770-L778 ◽  
Author(s):  
Jian-Rong Zhou ◽  
Tetsuya Shirasaki ◽  
Fumio Soeda ◽  
Kazuo Takahama
Keyword(s):  
Acetylcholine Release ◽  
Inhibitory Concentration ◽  
Lower Airway ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
Concentration Dependent Manner ◽  
Pathological Conditions ◽  
Voltage Dependent ◽  
Pretreatment Time ◽  
Postganglionic Nerve

In this study, we investigated the effects of suplatast on acutely dissociated single neurons of sensory and paratracheal ganglia using a patch-clamp technique. Suplatast had little effect on various responses caused by capsaicin, acid, bradykinin, serotonin, and adenosine 5′-triphosphate in rat sensory neurons. Suplatast, even at 10−3 M, also did not induce any current at various membrane potentials in rat and guinea pig paratracheal ganglia neurons. Furthermore, acetylcholine- and bradykinin-induced depolarizations were not affected by suplatast. On the other hand, in rat paratracheal ganglia neurons, 10−5 M nicotine-induced current were inhibited by suplatast in a concentration-dependent manner with a 50% inhibitory concentration of 9.86 × 10−5 M. The effect was noncompetitive and voltage dependent. Furthermore, the effect was use independent and not affected by the pretreatment time of suplatast. The results suggested that suplatast may inhibit neurotransmission at the paratracheal ganglia via the inhibition of nicotinic current. Thus suplatast may attenuate cough production through the improvement of pathological conditions of the lower airway via suppressed acetylcholine release from the postganglionic nerve terminal.

A novel approach allows identification of K channels in the lateral membrane of rat CCD

1994 ◽  
Vol 266 (5) ◽  
pp. F813-F822 ◽  
Author(s):  
W. H. Wang ◽  
C. M. McNicholas ◽  
A. S. Segal ◽  
G. Giebisch
Keyword(s):  
Membrane Potential ◽  
Cell Membrane ◽  
Cell Membrane Potential ◽  
K Channel ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
K Channels ◽  
Lateral Membrane ◽  
Novel Approach ◽  
Voltage Dependent

We have developed a novel approach to study K channels in the lateral membrane of principal cells (PC) in rat cortical collecting ducts (CCD). The technique consists of 1) exposing the CCD apical membrane, 2) removing the intercalated cells adjoining a PC by gentle suction through a pipette, and 3) applying patch-clamp technique to the lateral membrane of PC. Functional viability of the PC was confirmed by three indexes: 1) maintenance of physiological cell membrane potentials (-85 +/- 3 mV); 2) depolarization of the cell membrane potential with 1 mM Ba2+; and 3) hyperpolarization of the cell potential with 0.1 mM amiloride. Two types of K channels were identified: a low-conductance K channel and an intermediate-conductance K channel. In cell-attached patches the slope conductance of the low-conductance K channel was 27 pS and that of the intermediate-conductance K channel was 45 pS. The open probability (Po) of the 27-pS K channel was 0.81 +/- 0.02 and was not voltage dependent. In contrast, the Po of the 45-pS K channel was 0.23 +/- 0.01 at the spontaneous cell membrane potential and was increased by hyperpolarization. In addition, decrease of the bath pH from 7.4 to 6.7 reduced the 27-pS K channel current amplitude in a voltage-dependent manner, but the Po was not affected. Finally, two time constants were required to fit open- and closed-time histograms of both populations of K channels. Application of 1 mM Ba2+ completely blocked these K channels. We conclude that two types of K channel are present in the basolateral membrane of PC.

Mechanism and role of high-potassium-induced reduction of intracellular Ca2+ concentration in rat osteoclasts

2003 ◽  
Vol 285 (2) ◽  
pp. C457-C466 ◽  
Author(s):  
Hiroshi Kajiya ◽  
Fujio Okamoto ◽  
Hidefumi Fukushima ◽  
Keisuke Takada ◽  
Koji Okabe
Keyword(s):  
Dependent Manner ◽  
Patch Clamp Technique ◽  
High Potassium ◽  
Entry Pathway ◽  
Pit Formation ◽  
Motile Cells ◽  
High K ◽  
Voltage Dependent ◽  
Intracellular Ca ◽  
Actin Rings

Osteoclasts are multinucleated, bone-resorbing cells that show structural and functional differences between the resorbing and nonresorbing (motile) states during the bone resorption cycle. In the present study, we measured intracellular Ca2+ concentration ([Ca2+]i) in nonresorbing vs. resorbing rat osteoclasts. Basal [Ca2+]i in osteoclasts possessing pseudopodia (nonresorbing/motile state) was around 110 nM and significantly higher than that in actin ring-forming osteoclasts (resorbing state, around 50 nM). In nonresorbing/motile osteoclasts, exposure to high K+ reduced [Ca2+]i, whereas high K+ increased [Ca2+]i in resorbing state osteoclasts. In nonresorbing/motile cells, membrane depolarization and hyperpolarization applied by the patch-clamp technique decreased and increased [Ca2+]i, respectively. Removal of extracellular Ca2+ or application of 300 μM La3+ reduced [Ca2+]i to ∼50 nM in nonresorbing/motile osteoclasts, and high-K+-induced reduction of [Ca2+]i could not be observed under these conditions. Neither inhibition of intracellular Ca2+ stores or plasma membrane Ca2+ pumps nor blocking of L- and N-type Ca2+ channels significantly reduced [Ca2+]i. Exposure to high K+ inhibited the motility of nonresorbing osteoclasts and reduced the number of actin rings and pit formation in resorbing osteoclasts. These results indicate that in nonresorbing/motile osteoclasts, a La3+-sensitive Ca2+ entry pathway is continuously active under resting conditions, keeping [Ca2+]i high. Changes in membrane potential regulate osteoclastic motility by controlling the net amount of Ca2+ entry in a “reversed” voltage-dependent manner, i.e., depolarization decreases and hyperpolarization increases [Ca2+]i.

A potassium channel in the apical membrane of rabbit thick ascending limb of Henle's loop

1990 ◽  
Vol 258 (2) ◽  
pp. F244-F253 ◽  
Author(s):  
W. H. Wang ◽  
S. White ◽  
J. Geibel ◽  
G. Giebisch
Keyword(s):  
Apical Membrane ◽  
Rabbit Kidney ◽  
K Channel ◽  
Thick Ascending Limb ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
Bath Solution ◽  
Henle's Loop ◽  
Voltage Dependent ◽  
Inside Out

We used the patch-clamp technique to study the activity of single potassium channels in the apical membrane of isolated thick ascending limbs of Henle's loop (TAL) of rabbit kidneys. In cell-attached patches with NaCl Ringer or high-K+ solution in the bath and 140 mM K+ in the pipette, an inwardly rectifying K+ channel was observed with an inward slope conductance of 22.0 +/- 0.5 pS and outward slope conductance of 10.2 +/- 0.3 pS at 22 degrees C (n = 15). The channel was highly selective for K+, with a calculated permeability ratio for K(+)-to-Na+ of 20:1 (n = 4). The open probability (Po) of the channel was 0.89 +/- 0.03 (n = 15) and was not voltage dependent. In inside-out patches with 140 mM K+ in both the bath and the pipette solutions, both Po and conductance of the channel were similar to that in cell-attached patches. Addition of 0.1 mM Ba2+ to the pipette solution reduced Po of the channel in a voltage-dependent manner. Lowering the pH of the bath solution from 7.4 to 6.9 or increasing Ca2+ concentration from 0 to 0.5 mM in inside-out patches did not alter either Po or conductance of the channel. Addition of 2 mM ATP to the bath solution completely inhibited channel activity. This ATP-induced inhibition was fully reversible and was found to be dependent on the ratio of ATP to ADP, since adding 1 mM ADP to the bath solution relieved the ATP-induced blockade. The property of this small-conductance K+ channel make it a likely candidate for recycling of K+ across the apical membrane of TAL of the rabbit kidney. ATP and ADP are possible intracellular regulators of the channel's activity.

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