The effects of the antiarrhythmic drug propafenone at c-type kv1.4 channels in Xenopus laevis oocytes were studied with the two-electrode voltage-clamp techinique. Defolliculated oocytes (stage V-VI) were injected with transcribed cRNAs of ferret Kv1.4 delta N channels. During recording, oocytes were continuously perfused with control solution or propafenone. Propafenone decreased the currents during voltage steps. The block was voltage-, use-, and concentration- dependent manners. The block was increased with positive going potentials. The voltage dependence of block could be fitted with the sum of monoexponential and a linear function. Propafenone accelerated the inactivate of current during the voltage step. The concentration of half-maximal block (IC(50)) was 121 micrometer/L. With high, normal, and low extracellular potassium concentrations, the changes of IC(50) value had no significant statistical differences. The block of propafenone was PH- dependent in high-, normal- and low- extracellular potassium concentrations. Acidification of the extracellular solution to PH 6.0 increased the IC50 values to 463 micrometer/L, alkalization to PH 8.0 reduced it to 58 micrometer/L. The results suggest that propafenone blocks the kv1.4 delta N channel in the open state and give some hints for an intracellular site of action.
Animals ; Anti-Arrhythmia Agents/*pharmacology ; Hydrogen-Ion Concentration ; Inhibitory Concentration 50 ; Kv1.4 Potassium Channel/*antagonists & inhibitors/metabolism ; Oocytes/drug effects/metabolism ; Patch-Clamp Techniques ; Potassium/*metabolism ; Potassium Channel Blockers/*pharmacology ; Propafenone/*pharmacology ; Xenopus laevis

It has been shown that cell volume regulation mechanisms play important roles in various cell functions. We demonstrated previously that volume-activated chloride channels were involved in cell volume regulation. The present study aimed to clarify the roles of various types of potassium channels in regulatory volume decrease (RVD) induced by hypotonic challenges in human nasopharyngeal carcinoma cells (CNE-2Z cells). The whole-cell patch clamp technique was used to record hypotonic challenge-induced potassium currents. During current recordings, cells were held at 0 mV and stepped to +/-46 and +/-92 mV, repeatedly. The cell volume was computed from cell diameters. The changes of cell volume were monitored and analyzed by the time-lapse imaging technique. The results showed that the exposure to 160 mOsm/L hypotonic solution caused the cells to swell by (144.5+/-4.2)%, activated a potassium current (59.2 pA/pF+/-13.3 pA/pF at 92 mV), and induced RVD. Cell volume was recovered from hypotonic challenge-induced swelling by (48.9+/-4.6)% after 20 min. The potassium current (at 92 mV) and RVD were inhibited by the calcium-dependent potassium channel blocker, clotrimazole (100 mumol/L), by (98.5+/-2.8)% and (89.3+/-4.9)%, respectively. Depletion of extracellular calcium prevented the activation of the hypotonic challenge-induced potassium current and inhibited the process of RVD. The voltage-gated potassium channel blocker, 4-AP (5 mmol/L), partially inhibited the hypotonic challenge-activated potassium currents by (66.6+/-5.3)% (at 92 mV). These results suggest that the Ca(2+)-dependent potassium channel is the main component of volume-activated potassium channels and plays an important role in volume regulation of CNE-2Z cells. The voltage-gated potassium channels may also contribute in part to the formation of the volume-activated potassium current.
Carcinoma ; Cell Line, Tumor ; Cell Size ; Clotrimazole ; pharmacology ; Humans ; Hypotonic Solutions ; pharmacology ; Nasopharyngeal Neoplasms ; pathology ; Patch-Clamp Techniques ; Potassium Channel Blockers ; pharmacology ; Potassium Channels, Calcium-Activated ; metabolism

Rapidly activating component of delayed rectifier potassium current (I(Kr)) plays a key role in the repolarization phase of cardiac action potential. Human ether-a-go-go-related gene (HERG) encodes the alpha subunit of this potassium channel. Mutations of HERG gene induce genetic long QT syndrome (LQTS). Furthermore, I(Kr)/HERG is the target of some drugs which may cause cardiac QT interval prolongation. Some other drugs with different chemical structures also may block the channel and prolong QT interval, which even developed into acquired arrhythmias. This review summarized the recent progress of structure, gating mechanisms and functions of I(Kr)/HERG channel, I(Kr)/HERG related arrhythmias, interaction between K+ channel and drugs, and strategies of grading-up the I(Kr)/HERG target.
Anti-Arrhythmia Agents ; adverse effects ; pharmacology ; therapeutic use ; Arrhythmias, Cardiac ; drug therapy ; metabolism ; Ether-A-Go-Go Potassium Channels ; antagonists & inhibitors ; chemistry ; genetics ; metabolism ; Humans ; Ion Channel Gating ; Long QT Syndrome ; drug therapy ; etiology ; genetics ; metabolism ; Mutation ; Potassium Channel Blockers ; pharmacology

The present study was carried out to determine the functional properties of Kv4.2 expressed in mammalian cells in comparison with native transient potassium outward current (I(A)) in the hippocampal neurons. Transient transfection, cell culture and whole cell voltage clamp techniques were used. The results showed that I(A) in cultured rat hippocampal neurons and Kv4.2 expressed in HEK293 cells both displayed "A"-type current properties. The activation curves of I(A) and Kv4.2 were better fitted by simple Boltzmann function with V(1/2) 10.0+/-3.3 mV, k 13.9+/-2.6 mV for I(A) and V1/2 -9.7+/-4.1 mV, k 15.8+/-5.7 mV for Kv4.2, respectively. The steady-state inactivation curves of I(A) had a midpoint of -93.0+/-11.4 mV and a slope of 9.0+/-1.5 mV. The voltage-dependence of inactivation for Kv4.2 exhibited midpoint and slope values of -59.4+/-12.2 mV and 8.0+/-3.1 mV, respectively. The time constants (tau) of recovery from inactivation of I(A) and Kv4.2 were 27.9+/-14.1 ms and 172.8+/-10.0 ms, respectively. These results suggest that Kv4.2 is probably a major isoform contributing to I(A) in hippocampus neurons.
Animals ; Animals, Newborn ; Cells, Cultured ; Female ; Gene Transfer Techniques ; Hippocampus ; metabolism ; physiology ; Ion Transport ; Male ; Neurons ; metabolism ; physiology ; Patch-Clamp Techniques ; Potassium Channel Blockers ; Potassium Channels ; genetics ; physiology ; Potassium Channels, Voltage-Gated ; Rats ; Rats, Wistar ; Shal Potassium Channels

To study the functional expression of KCNQ gene in outer hair cells (OHCs) and Deiters' cells, the effects of linopirdine on the whole cell K(+) current were investigated by using the whole cell variant of patch clamp technique in the present study. The outward tetraethylammonium (TEA)-sensitive K(+) current and the inward K(+) current (I(Kn)) in OHCs were recorded and measured before and after the administration of linopirdine. Simultaneously, the whole cell currents in Deiters?cells were also observed in normal solution and in the presence of linopirdine. After the application of 100 micromol/L linopirdine to OHCs, the peak K(+) current was reversibly blocked and the late K(+) current was partly reduced. In addition, the decay time constant of the TEA-sensitive K(+) current was prolonged in the presence of 100 micromol/L linopirdine. The inward current in OHCs was totally inhibited after the superfusion of 100 mmol/L and 200 micromol/L linopirdine respectively. The outward rectifier K(+) current (Ik) was the dominant K(+) current in the whole cell currents in Deiters' cells. In the presence of 200 micromol/L linopirdine, the I(K) current was not significantly affected. Our findings demonstrate that the KCNQ heteromeric or homomeric potassium channel is possibly the molecular basis for the peak outward K(+) current and that the inward I(Kn) current is mediated by KCNQ potassium channel. KCNQ potassium channel in OHCs can not only permit the K(+) efflux but also limit the depolarization. In the present study, no expression of KCNQ potassium channel is found in Deiters' cells.
Animals ; Cochlea ; cytology ; Electrophysiology ; Guinea Pigs ; Hair Cells, Auditory, Outer ; cytology ; metabolism ; Indoles ; pharmacology ; KCNQ Potassium Channels ; Patch-Clamp Techniques ; Potassium Channel Blockers ; pharmacology ; Potassium Channels ; physiology ; Potassium Channels, Voltage-Gated ; genetics ; Pyridines ; pharmacology ; Vestibular Nucleus, Lateral ; cytology

AIMTo observe the regulatory volume decrease (RVD) process of human intestine cells and investigate its ion channel mechanism.

METHODSCultured human intestine cells were exposed to hypotonic solution and the cell volume was measured using Coulter Counter System. RT-PCR was explored to detect the mRNA expression of Ca(2+) -activated K+ channel.

RESULTSHuman intestine cells showed a RVD process and this process could be blocked by Cl- channel blocker NPPB and K+ channel blocker TEA. Further results demonstrated the subtype of K+ channel involved in RVD was an intermediate-conductance, Ca(2+) -activated K+ channel (IK) because of its high sensitivity to clotrimazole. RT-PCR results also showed the expression of IK in this cell line.

CONCLUSIONThe RVD process of intestine cell was dependent on the parallel activation of Cl- channel and K+ channel. The subtype of K+ channel in volved in the RVD process was IK channel.

Cell Line ; Cell Size ; drug effects ; Chloride Channels ; antagonists & inhibitors ; metabolism ; Epithelial Cells ; cytology ; Humans ; Hypotonic Solutions ; Intestine, Small ; cytology ; Potassium Channel Blockers ; pharmacology ; Potassium Channels ; metabolism ; Potassium Channels, Calcium-Activated ; metabolism

Kv2.1 channel currents in pancreatic beta-cells are thought to contribute to action potential repolarization and thereby modulate insulin secretion. Because of its central role in this important physiological process, Kv2.1 channel is a promising target for the treatment of type 2 diabetes. Jingzhaotoxin-XI (JZTX-XI) is a novel peptide neurotoxin isolated from the venom of the spider Chilobrachys jingzhao. Two-microelectrode voltage clamp experiments had showed that the toxin inhibited Kv2.1 potassium currents expressed in Xenopus Laevis oocytes. In order to investigate the structure-function relationship of JZTX-XI, the natural toxin and a mutant of JZTX-XI in which Arg3 was replaced by Ala, were synthesized by solid-phase chemistry method with Fmoc-protected amino acids on the PS3 automated peptide synthesizer. Reverse-phase high performance liquid chromatography (RP-HPLC) and matrix assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) were used to monitor the oxidative refolding process of synthetic linear peptides to find the optimal renaturation conditions of these toxins. The experiments also proved that the relative molecular masses of refolded peptides were in accordance with their theoretical molecular masses. RP-HPLC chromatogram of co-injected native and refolded JZTX-XI was a single peak. Under the whole-cell patch-clamp mode, JZTX-XI could completely inhibit hKv2.1 and hNav1.5 channels currents expressed in HEK293T cells with IC50 values of 95.8 nmol/L and 437.1 nmol/L respectively. The mutant R3A-JZTX-XI could also inhibit hKv2.1 and hNav1.5 channel currents expressed in HEK293T cells with IC50 values of 1.22 micromol/L and 1.96 micromol/L respectively. However, the prohibitive levels of R3A-JZTX-XI on hKv2.1 and hNav1.5 channels were reduced by about 12.7 times and 4.5 times respectively, indicating that Arg3 was a key amino acid residue relative to the hKv2.1 channel activity of JZTX-XI, but it is also an amino acid residue correlated with the binding activity of JZTX-XI to hNav1.5 channel. Our findings should be helpful to develop JZTX-XI into a molecular probe and drug candidate targeting to Kv2.1 potassium channel in the pancreas.
Animals ; HEK293 Cells ; Humans ; Insulin-Secreting Cells ; metabolism ; Mutant Proteins ; genetics ; pharmacology ; NAV1.5 Voltage-Gated Sodium Channel ; metabolism ; Neurotoxins ; chemical synthesis ; genetics ; pharmacology ; Protein Refolding ; Shab Potassium Channels ; antagonists & inhibitors ; metabolism ; Sodium Channel Blockers ; pharmacology ; Spider Venoms ; genetics ; pharmacology ; Transfection

Hepatic stellate cells (HSCs) are known to play a role in the pathogenesis of the increased intrahepatic vascular resistance found in chronic liver diseases. The aim of this study was to evaluate the K+ and Ca2+ currents in cultured HSCs from rat liver, through the patch-clamp technique. Most cells were positive for desmin immunostain after isolation and in alpha-smooth muscle actin immunostain after 10 - 14 days of culturing. Outward and inward rectifying K+ currents were confirmed. Two different types of K+ currents were distinguished: one with the inward rectifying current and the other without. The outward K+ currents consisted of at least four components: tetraethylammonium (TEA) -sensitive current, 4-aminopyridine (4-AP) -sensitive current, pimozide-sensitive current and three blocker-resistant current. The peaks of the outward K+ currents evoked by a depolarizing pulse were decreased to 32.0 +/- 3.0, 62.8 +/- 3.7 and 32.8 +/- 3.5% by 5 mM TEA, 2 mM 4-AP and 15microM pimozide, respectively. Moreover, the combined application of three blockers caused 86.6 +/- 4.8% suppression. The inward currents evoked hyperpolarizing pulses were inwardly rectifying and almost blocked by Ba2+. Elevation of external K+ increased the inward current amplitude and positively shifted its reversal potential. Voltage- dependent Ca2+ currents which were completely abolished by Cd2+ and nimodipine were detected in 14 day cultured HSCs. In this study, the cultured HSCs were found to express outward K+ currents composed of multiple pharmacological components, Ba2+-sensitive inward rectifying K+ current and L-type Ca2+ current.
Animals ; Calcium/*metabolism ; Calcium Channel Blockers/pharmacology ; Calcium Channels, L-Type/*physiology ; Cells, Cultured ; Hepatocytes/cytology/*physiology ; Immunohistochemistry ; Male ; Membrane Potentials/drug effects/physiology ; Patch-Clamp Techniques ; Potassium/*metabolism ; Potassium Channel Blockers/pharmacology ; Potassium Channels, Voltage-Gated/*physiology ; Rats ; Rats, Sprague-Dawley

Lindera erythrocarpa Makino (Lauraceae) is used as a traditional medicine for analgesic, antidote, and antibacterial purposes and shows anti-tumor activity. We studied the effects of Lindera erythrocarpa on the human ether-a-go-go-related gene (HERG) channel, which appears of importance in favoring cancer progression in vivo and determining cardiac action potential duration. Application of MeOH extract of Lindera erythrocarpa showed a dose-dependent decrease in the amplitudes of the outward currents measured at the end of the pulse (I(HERG)) and the tail currents of HERG (I(tail)). When the BuOH fraction and H2O fraction of Lindera erythrocarpa were added to the perfusate, both I(HERG) and I(tail) were suppressed, while the hexane fraction, CHCl3 fraction, and EtOAc fraction did not inhibit either I(HERG) or I(tail). The potential required for half-maximal activation caused by EtOAc fraction, BuOH fraction, and H2O fraction shifted significantly. The BuOH fraction and H2O fraction (100 microgram/mL) decreased gmax by 59.6% and 52.9%, respectively. The H2O fraction- and BuOH fraction-induced blockades of I(tail) progressively decreased with increasing depolarization, showing the voltage-dependent block. Our findings suggest that Lindera erythrocarpa, a traditional medicine, blocks HERG channel, which could contribute to its anticancer and cardiac arrhythmogenic effect.
Animals ; Butanols/chemistry/metabolism ; Ether-A-Go-Go Potassium Channels/*antagonists & inhibitors/metabolism ; Female ; Humans ; Lindera/*chemistry ; Oocytes/cytology/physiology ; Patch-Clamp Techniques ; Plant Extracts/*metabolism ; Potassium Channel Blockers/*metabolism ; Xenopus laevis

The purpose of this study was to investigate the effect of a mitochondrial ATP-sensitive potassium channel (mitoK(ATP)) opener, diazoxide (DE), on Fas/FasL protein expressions in rat heart suffered from long-term hypothermic preservation. The Langendorff isolated rat heart model was used. The hearts were stored in 4 °C Celsior solution with or without (control) DE for 8 h followed by 60 min of reperfusion. The recovery of rate-pressure product (RPP) was observed. Apoptotic cardiomyocytes were detected by TdT-mediated dUTP nick end labeling (TUNEL) technique. The expressions of Fas/FasL proteins were also analyzed by immunohistochemical method. The results showed that compared with the control group, DE (30 mmol/L) increased the recovery of RPP during reperfusion, reduced the percentage of apoptotic cells and the expressions of Fas and FasL proteins in rat hearts suffered from 8 h of hypothermic preservation. The above effects of DE were attenuated by a mitoK(ATP) channel inhibitor 5-hydroxydecanoate (5-HD). These results indicate that DE could alleviate rat myocardial injury induced by ischemia-reperfusion through reducing the expressions of Fas and FasL proteins via opening of mitoK(ATP)channel.
Animals ; Apoptosis ; Cryopreservation ; Decanoic Acids ; pharmacology ; Diazoxide ; pharmacology ; Fas Ligand Protein ; metabolism ; Heart ; drug effects ; Hydroxy Acids ; pharmacology ; Myocardium ; metabolism ; Myocytes, Cardiac ; cytology ; drug effects ; Potassium Channel Blockers ; pharmacology ; Potassium Channels ; Rats ; fas Receptor ; metabolism