scholarly journals Slow Inactivation in Shaker K Channels Is Delayed by Intracellular Tetraethylammonium

2008 ◽  
Vol 132 (6) ◽  
pp. 633-650 ◽  
Author(s):  
Vivian González-Pérez ◽  
Alan Neely ◽  
Christian Tapia ◽  
Giovanni González-Gutiérrez ◽  
Gustavo Contreras ◽  
...  
Keyword(s):  
Time Course ◽  
Inactivation Kinetics ◽  
Type I ◽  
Slow Inactivation ◽  
External Application ◽  
K Channels ◽  
Gating Charge ◽  
Channel Opening ◽  
Activation Gate ◽  
Free Membrane

After removal of the fast N-type inactivation gate, voltage-sensitive Shaker (Shaker IR) K channels are still able to inactivate, albeit slowly, upon sustained depolarization. The classical mechanism proposed for the slow inactivation observed in cell-free membrane patches—the so called C inactivation—is a constriction of the external mouth of the channel pore that prevents K+ ion conduction. This constriction is antagonized by the external application of the pore blocker tetraethylammonium (TEA). In contrast to C inactivation, here we show that, when recorded in whole Xenopus oocytes, slow inactivation kinetics in Shaker IR K channels is poorly dependent on external TEA but severely delayed by internal TEA. Based on the antagonism with internally or externally added TEA, we used a two-pulse protocol to show that half of the channels inactivate by way of a gate sensitive to internal TEA. Such gate had a recovery time course in the tens of milliseconds range when the interpulse voltage was −90 mV, whereas C-inactivated channels took several seconds to recover. Internal TEA also reduced gating charge conversion associated to slow inactivation, suggesting that the closing of the internal TEA-sensitive inactivation gate could be associated with a significant amount of charge exchange of this type. We interpreted our data assuming that binding of internal TEA antagonized with U-type inactivation (Klemic, K.G., G.E. Kirsch, and S.W. Jones. 2001. Biophys. J. 81:814–826). Our results are consistent with a direct steric interference of internal TEA with an internally located slow inactivation gate as a “foot in the door” mechanism, implying a significant functional overlap between the gate of the internal TEA-sensitive slow inactivation and the primary activation gate. But, because U-type inactivation is reduced by channel opening, trapping the channel in the open conformation by TEA would also yield to an allosteric delay of slow inactivation. These results provide a framework to explain why constitutively C-inactivated channels exhibit gating charge conversion, and why mutations at the internal exit of the pore, such as those associated to episodic ataxia type I in hKv1.1, cause severe changes in inactivation kinetics.

scholarly journals Correlation between Charge Movement and Ionic Current during Slow Inactivation in Shaker K+ Channels

1997 ◽  
Vol 110 (5) ◽  
pp. 579-589 ◽  
Author(s):  
Riccardo Olcese ◽  
Ramón Latorre ◽  
Ligia Toro ◽  
Francisco Bezanilla ◽  
Enrico Stefani
Keyword(s):  
Time Course ◽  
Voltage Dependence ◽  
Ionic Current ◽  
K Channel ◽  
Charge Movement ◽  
Slow Inactivation ◽  
Gating Currents ◽  
Similar Time ◽  
K Channels ◽  
Recovery From Inactivation

Prolonged depolarization induces a slow inactivation process in some K+ channels. We have studied ionic and gating currents during long depolarizations in the mutant Shaker H4-Δ(6–46) K+ channel and in the nonconducting mutant (Shaker H4-Δ(6–46)-W434F). These channels lack the amino terminus that confers the fast (N-type) inactivation (Hoshi, T., W.N. Zagotta, and R.W. Aldrich. 1991. Neuron. 7:547–556). Channels were expressed in oocytes and currents were measured with the cut-open-oocyte and patch-clamp techniques. In both clones, the curves describing the voltage dependence of the charge movement were shifted toward more negative potentials when the holding potential was maintained at depolarized potentials. The evidences that this new voltage dependence of the charge movement in the depolarized condition is associated with the process of slow inactivation are the following: (a) the installation of both the slow inactivation of the ionic current and the inactivation of the charge in response to a sustained 1-min depolarization to 0 mV followed the same time course; and (b) the recovery from inactivation of both ionic and gating currents (induced by repolarizations to −90 mV after a 1-min inactivating pulse at 0 mV) also followed a similar time course. Although prolonged depolarizations induce inactivation of the majority of the channels, a small fraction remains non–slow inactivated. The voltage dependence of this fraction of channels remained unaltered, suggesting that their activation pathway was unmodified by prolonged depolarization. The data could be fitted to a sequential model for Shaker K+ channels (Bezanilla, F., E. Perozo, and E. Stefani. 1994. Biophys. J. 66:1011–1021), with the addition of a series of parallel nonconducting (inactivated) states that become populated during prolonged depolarization. The data suggest that prolonged depolarization modifies the conformation of the voltage sensor and that this change can be associated with the process of slow inactivation.

scholarly journals The Effect of D2O on the Inactivation Kinetics and Recovery from Slow Inactivation of Shaker-IR K+ Channels

2015 ◽  
Vol 108 (2) ◽  
pp. 118a
Author(s):  
Tibor G. Szanto ◽  
Orsolya Szilagyi ◽  
Gyorgy Panyi
Keyword(s):  
Inactivation Kinetics ◽  
Slow Inactivation ◽  
K Channels

scholarly journals Inactivation of the sodium channel. II. Gating current experiments.

1977 ◽  
Vol 70 (5) ◽  
pp. 567-590 ◽  
Author(s):  
C M Armstrong ◽  
F Bezanilla
Keyword(s):  
Time Course ◽  
Voltage Dependence ◽  
Slow Component ◽  
Base Line ◽  
Charge Movement ◽  
Gating Current ◽  
Small Current ◽  
Gating Charge ◽  
Recovery From Inactivation ◽  
Activation Gate

Gating current (Ig) has been studied in relation to inactivation of Na channels. No component of Ig has the time course of inactivation; apparently little or no charge movement is associated with this step. Inactivation nonetheless affects Ig by immobilizing about two-thirds of gating charge. Immobilization can be followed by measuring ON charge movement during a pulse and comparing it to OFF charge after the pulse. The OFF:ON ratio is near 1 for a pulse so short that no inactivation occurs, and the ratio drops to about one-third with a time course that parallels inactivation. Other correlations between inactivation and immobilization are that: (a) they have the same voltage dependence; (b) charge movement recovers with the time coures of recovery from inactivation. We interpret this to mean that the immobilized charge returns slowly to "off" position with the time course of recovery from inactivation, and that the small current generated is lost in base-line noise. At -150 mV recover is very rapid, and the immobilized charge forms a distinct slow component of current as it returns to off position. After destruction of inactivation by pronase, there is no immobilization of charge. A model is presented in which inactivation gains its voltage dependence by coupling to the activation gate.

scholarly journals Stabilizing the Closed S6 Gate in the Shaker K v Channel Through Modification of a Hydrophobic Seal

2004 ◽  
Vol 124 (4) ◽  
pp. 319-332 ◽  
Author(s):  
Tetsuya Kitaguchi ◽  
Manana Sukhareva ◽  
Kenton J. Swartz
Keyword(s):  
Double Mutant ◽  
Single Channel ◽  
Ion Permeation ◽  
Ion Conduction ◽  
K Channels ◽  
Voltage Sensors ◽  
Gating Charge ◽  
Extracellular Region ◽  
Activation Gate ◽  
Primary Activation

The primary activation gate in K+ channels is thought to reside near the intracellular entrance to the ion conduction pore. In a previous study of the S6 activation gate in Shaker (Hackos et al., 2002), we found that mutation of V478 to W results in a channel that cannot conduct ions even though the voltage sensors are competent to translocate gating charge in response to membrane depolarization. In the present study we explore the mechanism underlying the nonconducting phenotype in V478W and compare it to that of W434F, a mutation located in an extracellular region of the pore that is nonconducting because the channel is predominantly found in an inactivated state. We began by examining whether the intracellular gate moves using probes that interact with the intracellular pore and by studying the inactivation properties of heterodimeric channels that are competent to conduct ions. The results of these experiments support distinct mechanisms underlying nonconduction in W434F and V478W, suggesting that the gate in V478W either remains closed, or that the mutation has created a large barrier to ion permeation in the open state. Single channel recordings for heterodimeric and double mutant constructs in which ion conduction is rescued suggest that the V478W mutation does not dramatically alter unitary conductance. Taken together, our results suggest that the V478W mutation causes a profound shift of the closed to open equilibrium toward the closed state. This mechanism is discussed in the context of the structure of this critical region in K+ channels.

scholarly journals Locked-Open Activation Gate Prevents the Recovery of Shaker K+ Channels from Slow Inactivation

2013 ◽  
Vol 104 (2) ◽  
pp. 123a
Author(s):  
Tibor Szanto G ◽  
Florina Zakany ◽  
Ferenc Papp ◽  
Zoltan Varga ◽  
Gyorgy Panyi
Keyword(s):  
Slow Inactivation ◽  
K Channels ◽  
Activation Gate

scholarly journals Extracellular Mg2+ Modulates Slow Gating Transitions and the Opening of Drosophila Ether-à-Go-Go Potassium Channels

2000 ◽  
Vol 115 (3) ◽  
pp. 319-338 ◽  
Author(s):  
Chih-Yung Tang ◽  
Francisco Bezanilla ◽  
Diane M. Papazian
Keyword(s):  
Time Course ◽  
Ionic Current ◽  
Ionic Currents ◽  
K Channel ◽  
Gating Currents ◽  
Gating Charge ◽  
Channel Opening ◽  
Voltage Dependent ◽  
Pore Opening ◽  
Sequential Activation

We have characterized the effects of prepulse hyperpolarization and extracellular Mg2+ on the ionic and gating currents of the Drosophila ether-à-go-go K+ channel (eag). Hyperpolarizing prepulses significantly slowed channel opening elicited by a subsequent depolarization, revealing rate-limiting transitions for activation of the ionic currents. Extracellular Mg2+ dramatically slowed activation of eag ionic currents evoked with or without prepulse hyperpolarization and regulated the kinetics of channel opening from a nearby closed state(s). These results suggest that Mg2+ modulates voltage-dependent gating and pore opening in eag channels. To investigate the mechanism of this modulation, eag gating currents were recorded using the cut-open oocyte voltage clamp. Prepulse hyperpolarization and extracellular Mg2+ slowed the time course of ON gating currents. These kinetic changes resembled the results at the ionic current level, but were much smaller in magnitude, suggesting that prepulse hyperpolarization and Mg2+ modulate gating transitions that occur slowly and/or move relatively little gating charge. To determine whether quantitatively different effects on ionic and gating currents could be obtained from a sequential activation pathway, computer simulations were performed. Simulations using a sequential model for activation reproduced the key features of eag ionic and gating currents and their modulation by prepulse hyperpolarization and extracellular Mg2+. We have also identified mutations in the S3–S4 loop that modify or eliminate the regulation of eag gating by prepulse hyperpolarization and Mg2+, indicating an important role for this region in the voltage-dependent activation of eag.

scholarly journals Voltage Gating of Shaker K+ Channels

1998 ◽  
Vol 112 (2) ◽  
pp. 223-242 ◽  
Author(s):  
Beatriz M. Rodríguez ◽  
Daniel Sigg ◽  
Francisco Bezanilla
Keyword(s):  
Temperature Dependence ◽  
Time Course ◽  
Recovery Period ◽  
Fluctuation Analysis ◽  
Kinetic Properties ◽  
Temperature Dependent ◽  
Open Probability ◽  
Voltage Range ◽  
K Channels ◽  
Gating Charge

Ionic (Ii) and gating currents (Ig) from noninactivating Shaker H4 K+ channels were recorded with the cut-open oocyte voltage clamp and macropatch techniques. Steady state and kinetic properties were studied in the temperature range 2–22°C. The time course of Ii elicited by large depolarizations consists of an initial delay followed by an exponential rise with two kinetic components. The main Ii component is highly temperature dependent (Q10 > 4) and mildly voltage dependent, having a valence times the fraction of electric field (z) of 0.2–0.3 eo. The Ig On response obtained between −60 and 20 mV consists of a rising phase followed by a decay with fast and slow kinetic components. The main Ig component of decay is highly temperature dependent (Q10 > 4) and has a z between 1.6 and 2.8 eo in the voltage range from −60 to −10 mV, and ∼0.45 eo at more depolarized potentials. After a pulse to 0 mV, a variable recovery period at −50 mV reactivates the gating charge with a high temperature dependence (Q10 > 4). In contrast, the reactivation occurring between −90 and −50 mV has a Q10 = 1.2. Fluctuation analysis of ionic currents reveals that the open probability decreases 20% between 18 and 8°C and the unitary conductance has a low temperature dependence with a Q10 of 1.44. Plots of conductance and gating charge displacement are displaced to the left along the voltage axis when the temperature is decreased. The temperature data suggests that activation consists of a series of early steps with low enthalpic and negative entropic changes, followed by at least one step with high enthalpic and positive entropic changes, leading to final transition to the open state, which has a negative entropic change.

scholarly journals Activation Gate Opening Precedes Slow Inactivation in Shaker K+ Channels

2012 ◽  
Vol 102 (3) ◽  
pp. 531a
Author(s):  
Ferenc Papp ◽  
Tibor G. Szanto ◽  
Zoltan Varga ◽  
Florina Zakany ◽  
Gyorgy Panyi
Keyword(s):  
Slow Inactivation ◽  
K Channels ◽  
Gate Opening ◽  
Activation Gate

scholarly journals The role of calcium ions in the closing of K channels.

1986 ◽  
Vol 87 (5) ◽  
pp. 817-832 ◽  
Author(s):  
C M Armstrong ◽  
D R Matteson
Keyword(s):  
Time Course ◽  
External Surface ◽  
Monovalent Cation ◽  
K Channel ◽  
Monovalent Cations ◽  
K Channels ◽  
Channel Opening ◽  
Voltage Curve ◽  
Channel Properties

The effects of external Ca ion on K channel properties were studied in squid giant axons. Increasing the Ca concentration from 20 to 100 mM slowed K channel opening, and was kinetically equivalent to decreasing the depolarizing step by approximately 25 mV. The same Ca increase had a much smaller effect on closing kinetics, equivalent to making the membrane potential more negative by approximately mV. With regard to the conductance-voltage curve, this Ca increase was about equivalent to decreasing the depolarizing step by approximately 10 mV. The presence of K or Rb in the bath slowed closing kinetics and made the time course more complex: there were pronounced slow components in Rb and, to a lesser extent, in K. Increasing the Ca concentration strongly antagonized the slowing caused by Rb or K. Thus, Ca has a strong effect on closing kinetics only in the presence of these monovalent cations. Rb and K do not significantly alter opening kinetics, nor do they alter Ca's ability to slow opening kinetics. High Ca slightly affects the instantaneous I-V curve by selectively depressing inward current at negative voltages. The results imply that Ca has two actions on K channels, and in only one, the action on closing, does it compete with monovalent cations. We propose (a) that opening kinetics are slowed by binding of Ca to negatively charged parts of the gating apparatus that are at the external surface of the channel protein when the channel is closed; monovalent cations do not compete effectively in this action; (b) Ca (or possibly Mg) normally occupies closed channels and has a latching effect. External K or Rb competes with Ca for channel occupancy. Channels close sluggishly when occupied by a monovalent cation and tend to reopen. Thus, slow closing results from occupancy by K or Rb instead of Ca. The data are well fit by a model based on these ideas.

scholarly journals A rapidly activating and slowly inactivating potassium channel cloned from human heart. Functional analysis after stable mammalian cell culture expression.

1993 ◽  
Vol 101 (4) ◽  
pp. 513-543 ◽  
Author(s):  
D J Snyders ◽  
M M Tamkun ◽  
P B Bennett
Keyword(s):  
Cell Lines ◽  
Time Course ◽  
Reversal Potential ◽  
Delayed Rectifier ◽  
Inactivation Kinetics ◽  
Constant Field ◽  
Slow Inactivation ◽  
Time Constants ◽  
K Concentration ◽  
External K

The electrophysiological properties of HK2 (Kv1.5), a K+ channel cloned from human ventricle, were investigated after stable expression in a mouse Ltk- cell line. Cell lines that expressed HK2 mRNA displayed a current with delayed rectifier properties at 23 degrees C, while sham transfected cell lines showed neither specific HK2 mRNA hybridization nor voltage-activated currents under whole cell conditions. The expression of the HK2 current has been stable for over two years. The dependence of the reversal potential of this current on the external K+ concentration (55 mV/decade) confirmed K+ selectivity, and the tail envelope test was satisfied, indicating expression of a single population of K+ channels. The activation time course was fast and sigmoidal (time constants declined from 10 ms to < 2 ms between 0 and +60 mV). The midpoint and slope factor of the activation curve were Eh = -14 +/- 5 mV and k = 5.9 +/- 0.9 (n = 31), respectively. Slow partial inactivation was observed especially at large depolarizations (20 +/- 2% after 250 ms at +60 mV, n = 32), and was incomplete in 5 s (69 +/- 3%, n = 14). This slow inactivation appeared to be a genuine gating process and not due to K+ accumulation, because it was present regardless of the size of the current and was observed even with 140 mM external K+ concentration. Slow inactivation had a biexponential time course with largely voltage-independent time constants of approximately 240 and 2,700 ms between -10 and +60 mV. The voltage dependence of slow inactivation overlapped with that of activation: Eh = -25 +/- 4 mV and k = 3.7 +/- 0.7 (n = 14). The fully activated current-voltage relationship displayed outward rectification in 4 mM external K+ concentration, but was more linear at higher external K+ concentrations, changes that could be explained in part on the basis of constant field (Goldman-Hodgkin-Katz) rectification. Activation and inactivation kinetics displayed a marked temperature dependence, resulting in faster activation and enhanced inactivation at higher temperature. The current was sensitive to low concentrations of 4-aminopyridine, but relatively insensitive to external TEA and to high concentrations of dendrotoxin. The expressed current did not resemble either the rapid or the slow components of delayed rectification described in guinea pig myocytes. However, this channel has many similarities to the rapidly activating delayed rectifying currents described in adult rat atrial and neonatal canine epicardial myocytes.(ABSTRACT TRUNCATED AT 400 WORDS)

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