Pathways of HERG inactivation

1999 ◽  
Vol 277 (1) ◽  
pp. H199-H210 ◽  
Author(s):  
Johann Kiehn ◽  
Antonio E. Lacerda ◽  
Arthur M. Brown
Keyword(s):  
Xenopus Oocytes ◽  
Single Channel ◽  
Inward Rectification ◽  
Single Channels ◽  
Channel Current ◽  
Cardiac Action ◽  
Current Voltage ◽  
Inwardly Rectifying ◽  
Channel Properties ◽  
Current Voltage Relation

The rapid, repolarizing K+ current in cardiomyocytes ( I Kr) has unique inwardly rectifying properties that contribute importantly to the downstroke of the cardiac action potential. The human ether-à-go-go-related gene ( HERG) expresses a macroscopic current virtually identical to I Kr, but a description of the single-channel properties that cause rectification is lacking. For this reason we measured single-channel and macropatch currents heterologously expressed by HERG in Xenopus oocytes. Our experiments had two main findings. First, the single-channel current-voltage relation showed inward rectification, and conductance was 9.7 pS at −100 mV and 3.9 pS at 100 mV when measured in symmetrical 100 mM K+ solutions. Second, single channels frequently showed no openings during depolarization but nevertheless revealed bursts of openings during repolarization. This type of gating may explain the inward rectification of HERG currents. To test this hypothesis, we used a three-closed state kinetics model and obtained rate constants from fits to macropatch data. Results from the model are consistent with rapid inactivation from closed states as a significant source of HERG rectification.

scholarly journals Modeling Macroscopic Currents of Ion Channels

2021 ◽  
Author(s):  
Di Wu
Keyword(s):  
Open Channel ◽  
Single Channel ◽  
Ion Permeation ◽  
Model Studies ◽  
Proposed Model ◽  
Current Voltage ◽  
Improved Model ◽  
Boltzmann Model ◽  
Channel Properties ◽  
Current Voltage Relation

Ion-channel functions are often studied by the current-voltage relation, which is commonly fitted by the Boltzmann equation, a powerful model widely used nowadays. However, the Boltzmann model is restricted to a two-state ion-permeation process. Here we present an improved model that comprises a flexible number of states and incorporates both the single-channel conductance and the open-channel probability. Employing the channel properties derived from the single-channel recording experiments, the proposed model is able to describe various current-voltage relations, especially the reversal ion-permeation curves showing the inward- and outward-rectifications. We demonstrate the applicability of the proposed model using the published patch-clamp data of BK and MthK potassium channels, and discuss the similarity of the two channels based on the model studies.

scholarly journals Block of the cyclic GMP-gated channel of vertebrate rod and cone photoreceptors by l-cis-diltiazem.

1992 ◽  
Vol 100 (5) ◽  
pp. 783-801 ◽  
Author(s):  
L W Haynes
Keyword(s):  
Binding Site ◽  
Single Molecule ◽  
Single Channel ◽  
Inward Rectification ◽  
Pipette Solution ◽  
Current Voltage ◽  
Voltage Dependent ◽  
Extracellular Side ◽  
Current Voltage Relation ◽  
Cytoplasmic Side

Inside-out patches were excised from catfish rod or cone outer segments. Single channel and macroscopic currents were recorded from GMP-gated channels activated by 1 mM cGMP in low divalent buffered saline. Currents were blocked by the application of micromolar concentrations of l-cis-diltiazem to the cytoplasmic side of the patch. The concentration dependence of block indicated that a single molecule was sufficient to block a channel and that all channels were susceptible to block. The dissociation constant for the rod channel was an order of magnitude smaller than for the cone channel, but the voltage dependence of block was nearly identical. The macroscopic current-voltage relation in the presence of blocker was inwardly rectifying and superficially resembled voltage-dependent block by an impermeant blocker occluding the ion-conducting pore of the channel. Block by diltiazem acting from the extracellular side of the channel was investigated by including 5 microM diltiazem in the recording pipette solution. The macroscopic current-voltage relation again showed inward rectification, inconsistent with the idea that diltiazem acts by occluding the pore at the external side. The kinetics of block by diltiazem applied to the intra- and extracellular side were measured in cone patches containing only a single channel. The unbinding rates were similar in both cases, suggesting a single binding site. Differences in the binding rate were consistent with greater accessibility to the binding site from the cytoplasmic side. Block from the cytoplasmic side was independent of pH, suggesting that the state of ionization of diltiazem was not related to its ability to block the channel in a voltage-dependent fashion. These observations are inconsistent with a pore-occluding blocker, but could be explained if the hydrophobic portion of diltiazem partitioned into the hydrophobic core of the channel protein, perhaps altering the gating of the channel.

scholarly journals Single Channel Properties of P2X2 Purinoceptors

1999 ◽  
Vol 113 (5) ◽  
pp. 695-720 ◽  
Author(s):  
Shinghua Ding ◽  
Frederick Sachs
Keyword(s):  
Conformational Changes ◽  
Single Channel ◽  
Excess Noise ◽  
Hill Coefficient ◽  
Inward Rectification ◽  
Channel Current ◽  
Single Channel Current ◽  
The Mean ◽  
Single Channel Currents ◽  
Channel Properties

The single channel properties of cloned P2X2 purinoceptors expressed in human embryonic kidney (HEK) 293 cells and Xenopus oocytes were studied in outside-out patches. The mean single channel current–voltage relationship exhibited inward rectification in symmetric solutions with a chord conductance of ∼30 pS at −100 mV in 145 mM NaCl. The channel open state exhibited fast flickering with significant power beyond 10 kHz. Conformational changes, not ionic blockade, appeared responsible for the flickering. The equilibrium constant of Na+ binding in the pore was ∼150 mM at 0 mV and voltage dependent. The binding site appeared to be ∼0.2 of the electrical distance from the extracellular surface. The mean channel current and the excess noise had the selectivity: K+ > Rb+ > Cs+ > Na+ > Li+. ATP increased the probability of being open (Po) to a maximum of 0.6 with an EC50 of 11.2 μM and a Hill coefficient of 2.3. Lowering extracellular pH enhanced the apparent affinity of the channel for ATP with a pKa of ∼7.9, but did not cause a proton block of the open channel. High pH slowed the rise time to steps of ATP without affecting the fall time. The mean single channel amplitude was independent of pH, but the excess noise increased with decreasing pH. Kinetic analysis showed that ATP shortened the mean closed time but did not affect the mean open time. Maximum likelihood kinetic fitting of idealized single channel currents at different ATP concentrations produced a model with four sequential closed states (three binding steps) branching to two open states that converged on a final closed state. The ATP association rates increased with the sequential binding of ATP showing that the binding sites are not independent, but positively cooperative. Partially liganded channels do not appear to open. The predicted Po vs. ATP concentration closely matches the single channel current dose–response curve.

Effects of divalent cations on single-channel conduction properties of XenopusIP3 receptor

1998 ◽  
Vol 275 (1) ◽  
pp. C179-C188 ◽  
Author(s):  
Don-On Daniel Mak ◽  
J. Kevin Foskett
Keyword(s):  
Single Channel ◽  
Divalent Cations ◽  
Channel Current ◽  
Current Voltage ◽  
Conduction Pathway ◽  
Wide Range ◽  
Trisphosphate Receptor ◽  
Channel Properties ◽  
Current Blocking ◽  
Conduction Properties

The effects of Mg2+ and Ba2+ on single-channel properties of the inositol 1,4,5-trisphosphate receptor (IP3R) were studied by patch clamp of isolated nuclei from Xenopusoocytes. In 140 mM K+ the IP3R channel kinetics and presence of conductance substates were similar over a range (0–9.5 mM) of free Mg2+. In 0 mM Mg2+ the channel current-voltage ( I-V) relation was linear with conductance of ∼320 pS. Conductance varied slowly and continuously over a wide range (SD ≈ 60 pS) and sometimes fluctuated during single openings. The presence of Mg2+ on either or both sides of the channel reduced the current (blocking constant ∼0.6 mM in symmetrical Mg2+), as well as the range of conductances observed, and made the I-V relation nonlinear (slope conductance ∼120 pS near 0 mV and ∼360 pS at ±70 mV in symmetrical 2.5 mM Mg2+). Ba2+ exhibited similar effects on channel conductance. Mg2+ and Ba2+ permeated the channel with a ratio of permeability of Ba2+ to Mg2+ to K+ of 3.5:2.6:1. These results indicate that divalent cations induce nonlinearity in the I-V relation and reduce current by a mechanism involving permeation block of the IP3R due to strong binding to site(s) in the conduction pathway. Furthermore, stabilization of conductance by divalent cations reveals a novel interaction between the cations and the IP3R.

Na(+)-activated nonselective cation channels in primary olfactory neurons

1995 ◽  
Vol 73 (5) ◽  
pp. 1774-1781 ◽  
Author(s):  
A. B. Zhainazarov ◽  
B. W. Ache
Keyword(s):  
Neuronal Excitability ◽  
Single Channel ◽  
Cation Channel ◽  
Hill Coefficient ◽  
Inward Rectification ◽  
Dependent Manner ◽  
Open Probability ◽  
Concentration Dependent Manner ◽  
Channel Current ◽  
Current Voltage

1. Excised inside-out patch recordings were used to describe a novel cation channel from cultured lobster olfactory receptor neurons that is activated by [Na+]i. 2. The channel is reversibly activated by intracellular Na+ as low as 5 mM. The half-effect concentration of intracellular Na+ is approximately 60 mM at -60 mV. The dependence of the channel open probability on [Na+]i is sigmoidal with a Hill coefficient of 3.1, indicating that more than one Na+ must bind to activate the channel. 3. The channel is equally permeable to Na+, K+, and Li+. In symmetrical 210 mM Na+, the open channel current-voltage relationship shows slight inward rectification at positive potentials. The slope conductance of the channel is 107 pS between -90 and 0 mV. 4. Although the channel is not activated by voltage in the absence of intracellular Na+, the gating of the channel is dependent on voltage as well as [Na+]i and [Na+]o. 5. Both intracellular Ca2+ and Mg2+ reversibly affect channel activity in a concentration-dependent manner starting at 1 microM. Ca2+ decreases both the open probability and the single channel amplitude, whereas Mg2+ decreases the open probability but has no effect on the single channel amplitude. Ba2+ (5 mM), but not 20 mM Cs+ and 100 microM amiloride, reversibly block the channel. 6. We speculate that this novel cation channel regulates neuronal excitability by accentuating the rate and/or the magnitude of depolarization of the cell to odors.

scholarly journals Single-Channel Properties of Recombinant Acid-Sensitive Ion Channels Formed by the Subunits Asic2 and Asic3 from Dorsal Root Ganglion Neurons Expressed in Xenopus Oocytes

2001 ◽  
Vol 117 (6) ◽  
pp. 563-572 ◽  
Author(s):  
Ping Zhang ◽  
Cecilia M. Canessa
Keyword(s):  
Dorsal Root Ganglion ◽  
Dorsal Root ◽  
Xenopus Oocytes ◽  
Single Channel ◽  
Dorsal Root Ganglion Neurons ◽  
Channel Current ◽  
Single Channel Current ◽  
The Mean ◽  
Ganglion Neurons ◽  
Channel Properties

The acid-sensitive ion channels known as ASIC are gated by external protons. A set of these channels is expressed in dorsal root ganglion neurons where they may participate in the transduction of mechanical and nociceptive stimuli. Here, we have examined the single-channel properties of channels formed by the subunits ASIC2 and ASIC3 expressed in Xenopus oocytes using outside-out patches. The mean single-channel current-voltage relationship is linear with a slope conductance of 18 pS between −80 and −40 mV in 150 mM Na+ outside and 150 mM K+ inside the patch pipet. The selectivity for monovalent cations has the sequence Na+ > Li+ > K+. Divalent cations such as Ca2+ do not permeate, but instead block the channel when applied to the extracellular side. External protons increase the probability of channels being open to a maximum of 0.8 with an EC50 of 16 ± 4 μM and a Hill coefficient of 2.7 ± 0.3, whereas the mean single-channel current amplitude is independent of external pH. Analysis of the kinetics of single channels indicates the presence of at least four modes of activity (Mod1 to Mod4) in addition to an inactivated state. Three of the modes exhibit distinct kinetics, and can be unambiguously identified on the basis of open probability (PoMod1 = 0.5 ± 0.05; PoMod2 > 0.9 ± 0.05; PoMod3 < 0.1). Mode 4, which has a Po in the range of 0.5–0.8, may constitute a distinct mode or alternatively, it represents transitions between the other three modes of activity. Increasing [H+]o increases the frequency of entering the modes with high Po (modes 1, 2, and 4) and the time the channel spends in the modes with high activity.

scholarly journals Effects of Strontium on the Permeation and Gating Phenotype of Calcium Channels in Hair Cells

2008 ◽  
Vol 100 (4) ◽  
pp. 2115-2124 ◽  
Author(s):  
Adrian Rodriguez-Contreras ◽  
Ping Lv ◽  
Jun Zhu ◽  
Hyo Jeong Kim ◽  
Ebenezer N. Yamoah
Keyword(s):  
Hair Cell ◽  
Single Channel ◽  
Single Channels ◽  
Physiological Concentration ◽  
Closed State ◽  
Dependent Inactivation ◽  
Intracellular Ca ◽  
High Concentrations ◽  
Latency Distributions ◽  
Channel Properties

To minimize the effects of Ca2+ buffering and signaling, this study sought to examine single Ca2+ channel properties using Sr2+ ions, which substitute well for Ca2+ but bind weakly to intracellular Ca2+ buffers. Two single-channel fluctuations were distinguished by their sensitivity to dihydropyridine agonist (L-type) and insensitivity toward dihydropyridine antagonist (non-L-type). The L- and non-L-type single channels were observed with single-channel conductances of 16 and 19 pS at 70 mM Sr2+ and 11 and 13 pS at 5 mM Sr2+, respectively. We obtained KD estimates of 5.2 and 1.9 mM for Sr2+ for L- and non-L-type channels, respectively. At Ca2+ concentration of ∼2 mM, the single-channel conductances of Sr2+ for the L-type channel was ∼1.5 and 4.0 pS for the non-L-type channels. Thus the limits of single-channel microdomain at the membrane potential of a hair cell (e.g., −65 mV) for Sr2+ ranges from 800 to 2,000 ion/ms, assuming an ECa of 100 mV. The channels are ≥4-fold more sensitive at the physiological concentration ranges than at concentrations >10 mM. Additionally, the channels have the propensity to dwell in the closed state at high concentrations of Sr2+, which is reflected in the time constant of the first latency distributions. It is concluded that the concentration of the permeant ion modulates the gating of hair cell Ca2+ channels. Finally, the closed state/s that is/are altered by high concentrations of Sr2+ may represent divalent ion-dependent inactivation of the L-type channel.

scholarly journals Single-channel properties of glycine receptors expressed in Xenopus oocytes injected with synthetic RNAs

1992 ◽  
Vol 58 ◽  
pp. 231
Author(s):  
H. Akagi ◽  
A. Momiyama ◽  
K. Hirai ◽  
R. Kohama ◽  
F. Hishinuma ◽  
...  
Keyword(s):  
Xenopus Oocytes ◽  
Single Channel ◽  
Glycine Receptors ◽  
Channel Properties

Single-channel basis for conductance increase induced by isoflurane in Shaker H4 IR K+ channels

2001 ◽  
Vol 280 (5) ◽  
pp. C1130-C1139 ◽  
Author(s):  
Jichang Li ◽  
Ana M. Correa
Keyword(s):  
Time Course ◽  
Xenopus Oocytes ◽  
Single Channel ◽  
Noise Analysis ◽  
Single Channels ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Open Probability ◽  
Slowing Down ◽  
Conductance Increase

Volatile anesthetics modulate the function of various K+ channels. We previously reported that isoflurane induces an increase in macroscopic currents and a slowing down of current deactivation of Shaker H4 IR K+ channels. To understand the single-channel basis of these effects, we performed nonstationary noise analysis of macroscopic currents and analysis of single channels in patches from Xenopus oocytes expressing Shaker H4 IR. Isoflurane (1.2% and 2.5%) induced concentration-dependent, partially reversible increases in macroscopic currents and in the time course of tail currents. Noise analysis of currents (70 mV) revealed an increase in unitary current (∼17%) and maximum open probability (∼20%). Single-channel conductance was larger (∼20%), and opening events were more stable, in isoflurane. Tail-current slow time constants increased by 41% and 136% in 1.2% and 2.5% isoflurane, respectively. Our results show that, in a manner consistent with stabilization of the open state, isoflurane increased the macroscopic conductance of Shaker H4 IR K+ channels by increasing the single-channel conductance and the open probability.

scholarly journals Endogenous Protease Activation of ENaC

2005 ◽  
Vol 126 (4) ◽  
pp. 339-352 ◽  
Author(s):  
Adedotun Adebamiro ◽  
Yi Cheng ◽  
John P. Johnson ◽  
Robert J. Bridges
Keyword(s):  
Single Channel ◽  
Apical Membrane ◽  
Open Channels ◽  
Na Channel ◽  
Fluctuation Analysis ◽  
Protease Inhibition ◽  
Open Probability ◽  
Channel Current ◽  
Single Channel Current ◽  
Channel Properties

Endogenous serine proteases have been reported to control the reabsorption of Na+ by kidney- and lung-derived epithelial cells via stimulation of electrogenic Na+ transport mediated by the epithelial Na+ channel (ENaC). In this study we investigated the effects of aprotinin on ENaC single channel properties using transepithelial fluctuation analysis in the amphibian kidney epithelium, A6. Aprotinin caused a time- and concentration-dependent inhibition (84 ± 10.5%) in the amiloride-sensitive sodium transport (INa) with a time constant of 18 min and half maximal inhibition constant of 1 μM. Analysis of amiloride analogue blocker–induced fluctuations in INa showed linear rate–concentration plots with identical blocker on and off rates in control and aprotinin-inhibited conditions. Verification of open-block kinetics allowed for the use of a pulse protocol method (Helman, S.I., X. Liu, K. Baldwin, B.L. Blazer-Yost, and W.J. Els. 1998. Am. J. Physiol. 274:C947–C957) to study the same cells under different conditions as well as the reversibility of the aprotinin effect on single channel properties. Aprotinin caused reversible changes in all three single channel properties but only the change in the number of open channels was consistent with the inhibition of INa. A 50% decrease in INa was accompanied by 50% increases in the single channel current and open probability but an 80% decrease in the number of open channels. Washout of aprotinin led to a time-dependent restoration of INa as well as the single channel properties to the control, pre-aprotinin, values. We conclude that protease regulation of INa is mediated by changes in the number of open channels in the apical membrane. The increase in the single channel current caused by protease inhibition can be explained by a hyperpolarization of the apical membrane potential as active Na+ channels are retrieved. The paradoxical increase in channel open probability caused by protease inhibition will require further investigation but does suggest a potential compensatory regulatory mechanism to maintain INa at some minimal threshold value.

Sign in / Sign up

Export Citation Format

Share Document