Functional and pharmacological properties of canine ERG potassium channels

2003 ◽  
Vol 284 (1) ◽  
pp. H256-H267 ◽  
Author(s):  
Jixin Wang ◽  
Kimberly Della Penna ◽  
Hao Wang ◽  
Jerzy Karczewski ◽  
Thomas M. Connolly ◽  
...  
Keyword(s):  
Inward Rectification ◽  
Delayed Rectifier ◽  
Pharmacological Properties ◽  
Hek 293 ◽  
Whole Cell Patch Clamp ◽  
Channel Opening ◽  
Maximum Activation ◽  
Voltage Dependent ◽  
Cardiac Delayed Rectifier ◽  
Action Potential Repolarization

We established HEK-293 cell lines that stably express functional canine ether-à-go-go-related gene (cERG) K+ channels and examined their biophysical and pharmacological properties with whole cell patch clamp and35S-labeled MK-499 ([35S]MK-499) binding displacement. Functionally, cERG current had the hallmarks of cardiac delayed rectifier K+ current ( I Kr). Channel opening was time- and voltage dependent with threshold near −40 mV. The half-maximum activation voltage was −7.8 ± 2.4 mV at 23°C, shifting to −31.9 ± 1.2 mV at 36°C. Channels activated with a time constant of 13 ± 1 ms at +20 mV, showed prominent inward rectification at depolarized potentials, were highly K+ selective (Na+-to-K+permeability ratio = 0.007), and were potently inhibited by I Kr blockers. Astemizole, terfenadine, cisapride, and MK-499 inhibited cERG and human ERG (hERG) currents with IC50 values of 1.3, 13, 19, and 15 nM and 1.2, 9, 14, and 21 nM, respectively, and competitively displaced [35S]MK-499 binding from cERG and hERG with IC50 values of 0.4, 12, 35, and 0.6 nM and 0.8, 5, 47, and 0.7 nM, respectively. cERG channels had biophysical properties appropriate for canine action potential repolarization and were pharmacologically sensitive to agents known to prolong QT. A novel MK-499 binding assay provides a new tool to detect agents affecting ERG channels.

scholarly journals Quantitative Analysis of the Voltage-dependent Gating of Mouse Parotid ClC-2 Chloride Channel

2005 ◽  
Vol 126 (6) ◽  
pp. 591-603 ◽  
Author(s):  
Jose Antonio de Santiago ◽  
Keith Nehrke ◽  
Jorge Arreola
Keyword(s):  
Chloride Channel ◽  
Time Course ◽  
Inward Rectification ◽  
Chloride Currents ◽  
Hek 293 ◽  
Whole Cell Patch Clamp ◽  
Voltage Dependent ◽  
State Models ◽  
New Perspective ◽  
Opening And Closing

Various ClC-type voltage-gated chloride channel isoforms display a double barrel topology, and their gating mechanisms are thought to be similar. However, we demonstrate in this work that the nearly ubiquitous ClC-2 shows significant differences in gating when compared with ClC-0 and ClC-1. To delineate the gating of ClC-2 in quantitative terms, we have determined the voltage (Vm) and time dependence of the protopore (Pf) and common (Ps) gates that control the opening and closing of the double barrel. mClC-2 was cloned from mouse salivary glands, expressed in HEK 293 cells, and the resulting chloride currents (ICl) were measured using whole cell patch clamp. WT channels had ICl that showed inward rectification and biexponential time course. Time constants of fast and slow components were ∼10-fold different at negative Vm and corresponded to Pf and Ps, respectively. Pf and Ps were ∼1 at −200 mV, while at Vm ≥ 0 mV, Pf ∼ 0 and Ps ∼ 0.6. Hence, Pf dominated open kinetics at moderately negative Vm, while at very negative Vm both gates contributed to gating. At Vm ≥ 0 mV, mClC-2 closes by shutting off Pf. Three- and two-state models described the open-to-closed transitions of Pf and Ps, respectively. To test these models, we mutated conserved residues that had been previously shown to eliminate or alter Pf or Ps in other ClC channels. Based on the time and Vm dependence of the two gates in WT and mutant channels, we constructed a model to explain the gating of mClC-2. In this model the E213 residue contributes to Pf, the dominant regulator of gating, while the C258 residue alters the Vm dependence of Pf, probably by interacting with residue E213. These data provide a new perspective on ClC-2 gating, suggesting that the protopore gate contributes to both fast and slow gating and that gating relies strongly on the E213 residue.

scholarly journals Zn2+ Slows Down CaV3.3 Gating Kinetics: Implications for Thalamocortical Activity

2007 ◽  
Vol 98 (4) ◽  
pp. 2274-2284 ◽  
Author(s):  
M. Cataldi ◽  
V. Lariccia ◽  
V. Marzaioli ◽  
A. Cavaccini ◽  
G. Curia ◽  
...  
Keyword(s):  
Neuronal Excitability ◽  
Whole Cell ◽  
Hek 293 ◽  
Gating Kinetics ◽  
Epileptiform Discharges ◽  
Whole Cell Patch Clamp ◽  
Recovery From Inactivation ◽  
293 Cells ◽  
Channel Opening ◽  
Current Activation

We employed whole cell patch-clamp recordings to establish the effect of Zn2+ on the gating the brain specific, T-type channel isoform CaV3.3 expressed in HEK-293 cells. Zn2+ (300 μM) modified the gating kinetics of this channel without influencing its steady-state properties. When inward Ca2+ currents were elicited by step depolarizations at voltages above the threshold for channel opening, current inactivation was significantly slowed down while current activation was moderately affected. In addition, Zn2+ slowed down channel deactivation but channel recovery from inactivation was only modestly changed. Zn2+ also decreased whole cell Ca2+ permeability to 45% of control values. In the presence of Zn2+, Ca2+ currents evoked by mock action potentials were more persistent than in its absence. Furthermore, computer simulation of action potential generation in thalamic reticular cells performed to model the gating effect of Zn2+ on T-type channels (while leaving the kinetic parameters of voltage-gated Na+ and K+ unchanged) revealed that Zn2+ increased the frequency and the duration of burst firing, which is known to depend on T-type channel activity. In line with this finding, we discovered that chelation of endogenous Zn2+ decreased the frequency of occurrence of ictal-like epileptiform discharges in rat thalamocortical slices perfused with medium containing the convulsant 4-aminopyridine (50 μM). These data demonstrate that Zn2+ modulates CaV3.3 channel gating thus leading to increased neuronal excitability. We also propose that endogenous Zn2+ may have a role in controlling thalamocortical oscillations.

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.

scholarly journals Characterization of α3 Glycine Receptors with Ginkgolide B and Picrotoxin

2018 ◽  
Author(s):  
Sampurna Chakrabarti ◽  
Anil Neelakantan ◽  
Malcolm M. Slaughter
Keyword(s):  
Beta Subunits ◽  
Ginkgolide B ◽  
Glycine Receptors ◽  
Hek 293 ◽  
Whole Cell Patch Clamp ◽  
293 Cells ◽  
Voltage Dependent ◽  
The Central Nervous System ◽  
Subunit Isoforms

AbstractGinkgolide B (GB) and picrotoxin (PTX) are antagonists of the major inhibitory receptors of the central nervous system: GABA and glycine receptors (GlyRs). GlyRs contain one or more of the four alpha subunit isoforms of which α1 and α2 have been extensively studied. This report compares GB and PTX block of α3 GlyRs expressed in HEK 293 cells, using whole-cell patch clamp techniques. In CNS, α3 exists as a heteropentamer in conjunction with beta subunits in a 2α:3β ratio. Thus, the nature of block was also tested in α3β heteromeric glycine receptors. GB and PTX blocked α3 GlyRs both in the presence (liganded state) and absence of glycine (unliganded state). This property is unique to α3 subunits; α1 and α2 subunits are only blocked in the liganded state. The GB block of α3 GlyRs is voltage-dependent (more effective when the cell is depolarized) and non-competitive, while the PTX block is competitive and not voltage-dependent. The heteromeric and homomeric α3 GlyRs recovered significantly faster from unliganded GB block compared to liganded GB block, but no such distinction was found for PTX block suggesting more than one binding site for GB. This study sheds light on features of the α3 GlyR that distinguish it from the more widely studied α1 and α2 subunits. Understanding these properties can help decipher the physiological functioning of GlyRs in the CNS and may permit development of subunit specific drugs.

scholarly journals Fast inactivation causes rectification of the IKr channel.

1996 ◽  
Vol 107 (5) ◽  
pp. 611-619 ◽  
Author(s):  
P S Spector ◽  
M E Curran ◽  
A Zou ◽  
M T Keating ◽  
M C Sanguinetti
Keyword(s):  
Outward Current ◽  
K Channel ◽  
Delayed Rectifier ◽  
Fast Inactivation ◽  
Time Constants ◽  
Recovery From Inactivation ◽  
Current Voltage ◽  
Voltage Dependent ◽  
Cardiac Delayed Rectifier ◽  
Cytoplasmic Side

The mechanism of rectification of HERG, the human cardiac delayed rectifier K+ channel, was studied after heterologous expression in Xenopus oocytes. Currents were measured using two-microelectrode and macropatch voltage clamp techniques. The fully activated current-voltage (I-V) relationship for HERG inwardly rectified. Rectification was not altered by exposing the cytoplasmic side of a macropatch to a divalent-free solution, indicating this property was not caused by voltage-dependent block of outward current by Mg2+ or other soluble cytosolic molecules. The instantaneous I-V relationship for HERG was linear after removal of fast inactivation by a brief hyperpolarization. The time constants for the onset of and recovery from inactivation were a bell-shaped function of membrane potential. The time constants of inactivation varied from 1.8 ms at +50 mV to 16 ms at -20 mV; recovery from inactivation varied from 4.7 ms at -120 mV to 15 ms at -50 mV. Truncation of the NH2-terminal region of HERG shifted the voltage dependence of activation and inactivation by +20 to +30 mV. In addition, the rate of deactivation of the truncated channel was much faster than wild-type HERG. The mechanism of HERG rectification is voltage-gated fast inactivation. Inactivation of channels proceeds at a much faster rate than activation, such that no outward current is observed upon depolarization to very high membrane potentials. Fast inactivation of HERG and the resulting rectification are partly responsible for the prolonged plateau phase typical of ventricular action potentials.

Isolation and characterization of IKr in cardiac myocytes by Cs+ permeation

2006 ◽  
Vol 290 (3) ◽  
pp. H1038-H1049 ◽  
Author(s):  
Shetuan Zhang
Keyword(s):  
Cardiac Myocytes ◽  
Potassium Current ◽  
Ventricular Myocytes ◽  
Delayed Rectifier ◽  
Pharmacological Properties ◽  
Membrane Expression ◽  
Isolation And Characterization ◽  
Recovery From Inactivation ◽  
Voltage Dependent ◽  
Herg Current

Isolation of the rapidly activating delayed rectifier potassium current ( IKr) from other cardiac currents has been a difficult task for quantitative study of this current. The present study was designed to separate IKr using Cs+ in cardiac myocytes. Cs+ have been known to block a variety of K+ channels, including many of those involved in the cardiac action potential such as inward rectifier potassium current IK1 and the transient outward potassium current Ito. However, under isotonic Cs+ conditions (135 mM Cs+), a significant membrane current was recorded in isolated rabbit ventricular myocytes. This current displayed the voltage-dependent onset of and recovery from inactivation that are characteristic to IKr. Consistently, the current was selectively inhibited by the specific IKr blockers. The biophysical and pharmacological properties of the Cs+-carried human ether-a-go-go-related gene (hERG) current were very similar to those of the Cs+-carried IKr in ventricular myocytes. The primary sequence of the selectivity filter in hERG was in part responsible for the Cs+ permeability, which was lost when the sequence was changed from GFG to GYG, characteristic of other, Cs+-impermeable K+ channels. Thus the unique high Cs+ permeability in IKr channels provides an effective way to isolate IKr current. Although the biophysical and pharmacological properties of the Cs+-carried IKr are different from those of the K+-carried IKr, such an assay enables IKr current to be recorded at a level that is large enough and sufficiently robust to evaluate any IKr alterations in native tissues in response to physiological or pathological changes. It is particularly useful for exploring the role of reduction of IKr in arrhythmias associated with heart failure and long QT syndrome due to the reduced hERG channel membrane expression.

Magnesium shifts voltage dependence of activation of delayed rectifier I(K) in guinea pig ventricular myocytes

1997 ◽  
Vol 272 (3) ◽  
pp. H1292-H1301 ◽  
Author(s):  
B. A. Williams ◽  
G. N. Beatch
Keyword(s):  
Guinea Pig ◽  
Ventricular Myocytes ◽  
Inward Rectification ◽  
Delayed Rectifier ◽  
Maximal Effect ◽  
Patch Clamp Technique ◽  
Whole Cell Patch Clamp ◽  
K Current ◽  
Dose Dependent Decrease ◽  
20 Nm

The sensitivity of the delayed rectifier K+ current (I(K)) to intracellular Mg2+ was investigated in guinea pig ventricular myocytes using the whole cell patch-clamp technique. An increase in free intracellular Mg2+ concentration ([Mg2+]i) led to a dose-dependent decrease in I(K) with a half-maximal effect of approximately 20 nM. Activation of I(K) was shifted toward more positive voltages on increasing [Mg2+]i, but little effect was observed on activation and deactivation kinetics. Isoproterenol increased I(K) and was partially reversible in both control and 100 nM [Mg2+]i. The antiarrhythmic drug dofetilide was used to separate I(K) into its two components, rapidly activating (I(Kr)) and slowly activating (I(Ks)). The magnitude of both components decreased to a similar extent with an increase in [Mg2+]i. As [Mg2+]i was reduced, however, the number of experiments in which the dofetilide-sensitive current I(Kr) displayed inward rectification was reduced. In contrast to results previously reported for frog myocytes, it is unlikely that Mg2+ effects on guinea pig I(K) are mediated by a protein phosphatase.

Actions of diazoxide on CA1 neurons in hippoeampal slices from rats

1995 ◽  
Vol 73 (5) ◽  
pp. 608-618 ◽  
Author(s):  
G. Erdemli ◽  
K. Krnjević
Keyword(s):  
High Voltage ◽  
Outward Current ◽  
Inward Rectification ◽  
Delayed Rectifier ◽  
Sprague Dawley ◽  
Na Current ◽  
Ca1 Neurons ◽  
Voltage Dependent ◽  
Inward Currents ◽  
Ca Currents

Membrane effects of diazoxide (DZX) were examined in CA1 pyramidal neurons, mainly by whole-cell recording in slices kept at 33 °C (from Sprague–Dawley rats). Bath applications of DZX (0.65 mM) did not significantly change the resting input conductance; but instantaneous inward rectification was reduced by 47 ± 14% (near –110 mV). There was a similar depression of a large, sustained voltage-dependent outward current (by 44 ± 11% near 0 mV). A nearly identical reduction of the outward current recorded in a Ca current suppressing medium (but not in 30 mM tetraethylammonium) indicated that the DZX-sensitive current includes the delayed rectifier. In Mn, low-Ca medium containing tetraethylammonium and carbachol, DZX potentiated (by 43 ± 12%) the D-type slowly decaying outward current seen after hyperpolarizing pulses at a holding potential of ≈ −50 mV. DZX abolished or depressed slow inward currents, such as the tetrodotoxin-sensitive persistent Na current, high voltage activated Ca currents (IC50 = 0.47 mM), and the Q current. In 6 of 13 cells recorded with electrodes containing either guanosine or adenosine diphosphate, DZX potentiated the voitage-dependent outward current, but input conductances were reduced. In conclusion, although there was little indication that it activates classical KATP channels in CA1 neurons, DZX strongly depresses several voltage-dependent, slowly inactivating outward and inward currents, which are important modulators of cell excitability.Key words: KATP channels, persistent Na current, high voltage activated Ca currents, delayed rectifier, D current, sulphonylureas, nucleotide diphosphates.

Potassium currents in the inner segment of single retinal cone photoreceptors

1990 ◽  
Vol 64 (6) ◽  
pp. 1929-1940 ◽  
Author(s):  
A. V. Maricq ◽  
J. I. Korenbrot
Keyword(s):  
Voltage Dependence ◽  
Delayed Rectifier ◽  
Half Maximum ◽  
Cone Photoreceptors ◽  
Delayed Rectifier Current ◽  
Maximum Activation ◽  
Voltage Dependent ◽  
Kinetics Of ◽  
K Currents ◽  
Steepness Factor

1. The K+ currents of cone inner segments isolated from the retina of a lizard were studied with the use of tight-seal electrodes in the whole cell configuration. To conduct these studies other identified currents in the cell were blocked. Co2+ blocked a voltage-dependent Ca2+ current and a Ca2(+)-dependent Cl- current, and Cs+ blocked an inward-rectifying current partially carried by K+. 2. The cells sustained a voltage-dependent K+ current that was blocked by tetraethylammonium (TEA)+ and had characteristics typical of the delayed rectifier. However, we found no evidence for the existence of “A”-type K+ currents or Ca2(+)-dependent K+ currents. 3. The delayed-rectifier current was nearly ideally selective for K+. Increasing external K+ concentration 10-fold shifted the reversal potential by 55 mV. 4. Analysis of the voltage dependence of the activation of the delayed-rectifier current revealed the existence of two distinct subclasses of this current. We referred to them as IdrL and IdrH for low and high threshold of voltage activation. 5. IdrL activated at voltages above -70 mV. Its dependence on voltage was described by Boltzmann's function with average half-maximum activation at -51 mV and steepness factor k = 7.5 mV. IdrH activated at voltages above -50 mV. Its dependence on voltage was described by Boltzmann's function with average half-maximum activation at -4.6 mV and steepness factor k = 17.1 mV. 6. Of nine cells analyzed in detail, one demonstrated IdrH alone, whereas the remaining had a variable mixture of the two current subtypes. At maximum activation the current through IdrL ranged between 0.3 and 0.5 of the total delayed-rectifier current. 7. The kinetics of activation of the total delayed-rectifier current were described by the sum of two exponentials the amplitudes and time constants of which were voltage dependent. However, the kinetics of the current subtypes were not resolved individually. The current inactivated slowly with a single-exponential time course that was voltage dependent. 8. The voltage dependence of the delayed-rectifier current indicates the current is active in a cone photoreceptor in the dark. The current is 20-30 pA in amplitude at the dark-membrane potential and outwardly directed. 9. IdrL may generate a rapid relaxation of photovoltages activated by dim lights--those that hyperpolarize the membrane by only a few millivolts. The delayed-rectifier currents help shape the action potentials that can be generated in isolated cone photoreceptors

Layer I neurons of the rat neocortex. II. Voltage-dependent outward currents

1996 ◽  
Vol 76 (2) ◽  
pp. 668-682 ◽  
Author(s):  
F. M. Zhou ◽  
J. J. Hablitz
Keyword(s):  
Time Constant ◽  
Time Course ◽  
Outward Current ◽  
Pyramidal Cells ◽  
Delayed Rectifier ◽  
Transient Outward Current ◽  
Pharmacological Properties ◽  
Voltage Dependent ◽  
Steady State Inactivation ◽  
Layer I

1. Whole cell patch-clamp techniques, combined with direct visualization of neurons, were used to study voltage-dependent potassium currents in layer 1 neurons and layer II/III pyramidal cells. 2. In the presence of tetrodotoxin, step depolarizations evoked an outward current. This current had a complex waveform and appeared to be a composite of early and late components. The early peak of the composite K+ outward current was larger in layer I neurons. 3. In both layer I and pyramidal cells, the composite outward K+ current could be separated into two components based on kinetic and pharmacological properties. The early component was termed I(A) because it was a transient outward current activating rapidly and then decaying. I(A) was more sensitive to blocking by 4-aminopyridine (4-AP) than tetraethylammonium (TEA). The second component, termed the delayed rectifier or I(DR), activated relatively slowly and did not decay significantly during a 200-ms test pulse. I(DR) was insensitive to blocking by 4-AP at concentrations up to 4 mM and blocked by > 60% by 40-60 mM TEA. 4. I(A) kinetics were examined in the presence of 40-60 mM TEA. Under these conditions, I(A) began to activate between -40 and -30 mV. Half-maximal activation occurred around 0 mV. In both layer I and pyramidal cells, the half-inactivation potential (Vh-inact) was around or more positive than -50 mV. At -60 mV, > 70% of I(A) conductance was available. I(A) decayed along a single exponential time course with a time constant of approximately 15 ms. This decay showed little voltage dependence. 5. In both layer I and pyramidal cells, I(DR) was studied in the presence of 4 mM 4-AP to block I(A) and in saline containing 0.2 mM Ca2+ and 3.6 mM Mg2+ to reduce contributions from Ca2+-dependent K+ currents. Under these conditions, I(DR) began to activate at -35 to -25 mV with Vh-act of 3.6 +/- 4.5 mV (mean +/- SD). The 10-90% rise time of I(DR) was 15 ms at 30 mV. At 2.2 ms after the onset of the command potential to +30 mV, I(DR) could reach a significant amplitude (approximately 1.5 nA in layer I neurons and 2.2 nA in pyramidal cells depending on the cell size). When long test pulses (> or = 1,000 ms) were used, a decay time constant approximately 800 ms at +40 mV was observed. In both layer I and pyramidal cells, steady state inactivation of I(DR) was minimal. 6. These results indicate that I(A) and I(DR) are the two major hyperpolarizing currents in layer I and pyramidal cells. The kinetics and pharmacological properties of I(A) and I(DR) were not significantly different in fast-spiking layer I neurons and regular-spiking layer II/III pyramidal cells. The relatively positive activation threshold (more than or equal to -40 mV) of both I(A) and I(DR) suggest that they do not play a role in neuronal behavior below action potential (AP) threshold and that their properties are more suitable to repolarize AP. The greater density of I(A) in layer I neurons appears responsible for fast spike generation.

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