scholarly journals How Does the W434F Mutation Block Current in Shaker Potassium Channels?

1997 ◽  
Vol 109 (6) ◽  
pp. 779-789 ◽  
Author(s):  
Youshan Yang ◽  
Yangyang Yan ◽  
Fred J. Sigworth
Keyword(s):  
Potassium Channels ◽  
Potassium Current ◽  
Xenopus Oocytes ◽  
Single Channel ◽  
Ion Selectivity ◽  
Independent Component ◽  
Single Channel Recordings ◽  
Shaker Channels ◽  
Rapid Inactivation ◽  
Shaker Potassium Channels

The mutation W434F produces an apparently complete block of potassium current in Shaker channels expressed in Xenopus oocytes. Tandem tetrameric constructs containing one or two subunits with this mutation showed rapid inactivation, although the NH2-terminal inactivation domain was absent from these constructs. The inactivation showed a selective dependence on external cations and was slowed by external TEA; these properties are characteristic of C-type inactivation. Inactivation was, however, incompletely relieved by hyperpolarization, suggesting the presence of a voltage-independent component. The hybrid channels had near-normal conductance and ion selectivity. Single-channel recordings from patches containing many W434F channels showed occasional channel openings, consistent with open probabilities of 10−5 or less. We conclude that the W434F mutation produces a channel that is predominantly found in an inactivated state.

scholarly journals Ion Conduction through C-Type Inactivated Shaker Channels

1997 ◽  
Vol 110 (5) ◽  
pp. 539-550 ◽  
Author(s):  
John G. Starkus ◽  
Lioba Kuschel ◽  
Martin D. Rayner ◽  
Stefan H. Heinemann
Keyword(s):  
Potassium Channels ◽  
Xenopus Oocytes ◽  
Ion Selectivity ◽  
Complete Removal ◽  
Ion Conduction ◽  
Internal Solution ◽  
Shaker Channels ◽  
Na Ions ◽  
Shaker Potassium Channels ◽  
Inside Out

C-type inactivation of Shaker potassium channels involves entry into a state (or states) in which the inactivated channels appear nonconducting in physiological solutions. However, when Shaker channels, from which fast N-type inactivation has been removed by NH2-terminal deletions, are expressed in Xenopus oocytes and evaluated in inside-out patches, complete removal of K+ ions from the internal solution exposes conduction of Na+ and Li+ in C-type inactivated conformational states. The present paper uses this observation to investigate the properties of ion conduction through C-type inactivated channel states, and demonstrates that both activation and deactivation can occur in C-type states, although with slower than normal kinetics. Channels in the C-type states appear “inactivated” (i.e., nonconducting) in physiological solutions due to the summation of two separate effects: first, internal K+ ions prevent Na+ ions from permeating through the channel; second, C-type inactivation greatly reduces the permeability of K+ relative to the permeability of Na+, thus altering the ion selectivity of the channel.

scholarly journals Hidden Markov Model Analysis of Intermediate Gating Steps Associated with the Pore Gate of Shaker Potassium Channels

2001 ◽  
Vol 118 (5) ◽  
pp. 547-564 ◽  
Author(s):  
Jie Zheng ◽  
Lalitha Vankataramanan ◽  
Fred J. Sigworth
Keyword(s):  
Potassium Channels ◽  
Conformational Changes ◽  
Time Course ◽  
Single Channel ◽  
Hidden Markov ◽  
Voltage Sensitivity ◽  
Open Probability ◽  
Shaker Channels ◽  
Voltage Dependent ◽  
Shaker Potassium Channels

Cooperativity among the four subunits helps give rise to the remarkable voltage sensitivity of Shaker potassium channels, whose open probability changes tenfold for a 5-mV change in membrane potential. The cooperativity in these channels is thought to arise from a concerted structural transition as the final step in opening the channel. Recordings of single-channel ionic currents from certain other channel types, as well as our previous recordings from T442S mutant Shaker channels, however, display intermediate conductance levels in addition to the fully open and closed states. These sublevels might represent stepwise, rather than concerted, transitions in the final steps of channel activation. Here, we report a similar fine structure in the closing transitions of Shaker channels lacking the mutation. Describing the deactivation time course with hidden Markov models, we find that two subconductance levels are rapidly traversed during most closing transitions of chimeric, high conductance Shaker channels. The lifetimes of these levels are voltage-dependent, with maximal values of 52 and 22 μs at −100 mV, and the voltage dependences of transitions among these states suggest that they arise from equivalent conformational changes occurring in individual subunits. At least one subconductance level is found to be traversed in normal conductance Shaker channels. We speculate that voltage-dependent conformational changes in the subunits give rise to changes in a “pore gate” associated with the selectivity filter region of the channel, producing the subconductance states. As a control for the hidden Markov analysis, we applied the same procedures to recordings of the recovery from N-type inactivation in Shaker channels. These transitions are found to be instantaneous in comparison.

scholarly journals Function of Shaker Potassium Channels Produced by Cell-Free Translation Upon Injection into Xenopus Oocytes

2013 ◽  
Vol 104 (2) ◽  
pp. 197a
Author(s):  
Brian W. Jarecki ◽  
Shin-ichi Makino ◽  
Emily T. Beebe ◽  
Brian G. Fox ◽  
Baron Chanda
Keyword(s):  
Potassium Channels ◽  
Xenopus Oocytes ◽  
Free Translation ◽  
Shaker Potassium Channels

scholarly journals Function of Shaker potassium channels produced by cell-free translation upon injection into Xenopus oocytes

2013 ◽  
Vol 3 (1) ◽  
Author(s):  
Brian W. Jarecki ◽  
Shin-ichi Makino ◽  
Emily T. Beebe ◽  
Brian G. Fox ◽  
Baron Chanda
Keyword(s):  
Potassium Channels ◽  
Xenopus Oocytes ◽  
Free Translation ◽  
Shaker Potassium Channels

scholarly journals Intermediate Conductances during Deactivation of Heteromultimeric Shaker Potassium Channels

1998 ◽  
Vol 112 (4) ◽  
pp. 457-474 ◽  
Author(s):  
Jie Zheng ◽  
Fred J. Sigworth
Keyword(s):  
Voltage Dependence ◽  
Ion Selectivity ◽  
Mutant Channel ◽  
Shaker Channels ◽  
Mean Lifetime ◽  
The Mean ◽  
Time Courses ◽  
Shaker Potassium Channels ◽  
Ion Selectivities ◽  
Linear Voltage

A previous study of the T442S mutant Shaker channel revealed activation-coupled subconductance levels that apparently represent kinetic intermediates in channel activation (Zheng, J., and F.J. Sigworth. 1997. J. Gen. Physiol. 110:101–117). We have now extended the study to heteromultimeric channels consisting of various numbers of mutant subunits as well as channels without mutant subunits, all in the background of a chimeric Shaker channel having increased conductance. It has been found that activation-coupled sublevels exist in all these channel types, and are traversed in at least 80% of all deactivation time courses. In symmetric K+ solutions, the currents in the two sublevels have a linear voltage dependence, being 23–44% and 54–70% of the fully open conductance. Sublevels in different channel types share similar voltage dependence of the mean lifetime and similar ion selectivity properties. However, the mean lifetime of each current level depends approximately geometrically on the number of mutant subunits in the channel, becoming shorter in channels having fewer mutant subunits. Each mutant subunit appears to stabilize all of the conducting states by ∼0.5 kcal/mol. Consistent with previous results in the mutant channel, sublevels in channels with two or no mutant subunits also showed ion selectivities that differ from that of the fully open level, having relatively higher K+ than Rb+ conductances. A model is presented in which Shaker channels have two coupled activation gates, one associated with the selectivity filter and a second associated with the S6 helix bundle.

scholarly journals Potassium current activated by intracellular sodium in quail trigeminal ganglion neurons.

1990 ◽  
Vol 95 (5) ◽  
pp. 961-979 ◽  
Author(s):  
C Haimann ◽  
L Bernheim ◽  
D Bertrand ◽  
C R Bader
Keyword(s):  
Voltage Clamp ◽  
Trigeminal Ganglion ◽  
Potassium Current ◽  
Single Channel ◽  
Sodium Concentration ◽  
Intracellular Sodium ◽  
Single Channel Recordings ◽  
Trigeminal Ganglion Neurons ◽  
Ganglion Neurons ◽  
The Relationship

Whole-cell voltage clamp and single-channel recordings were performed on cultured trigeminal ganglion neurons from quail embryos in order to study a sodium-activated potassium current (KNa). When KNa was activated by a step depolarization in voltage clamp, there was a proportionality between KNa and INa at all voltages between the threshold of INa and ENa. Single-channel recordings indicated that KNa could be activated already by 12 mM intracellular sodium and was almost fully activated at 50 mM sodium. 100 mM lithium, 100 mM choline, or 5 microM calcium did not activate KNa. The relationship between the probability for the channel to be open (Po) vs. the sodium concentration and the relationship of KNa open time-distributions vs. the sodium concentration suggest that two to three sodium ions bind cooperatively before KNa channels open. KNa channels were sensitive to depolarization; at 12 mM sodium, a 42-mV depolarization caused an e-fold increase in Po. Under physiological conditions, the conductance of the KNa channel was 50 pS. This conductance increased to 174 pS when the intra- and extracellular potassium concentrations were 75 and 150 mM, respectively.

scholarly journals Activation of Shaker Potassium Channels

1998 ◽  
Vol 111 (2) ◽  
pp. 295-311 ◽  
Author(s):  
N.E. Schoppa ◽  
F.J. Sigworth
Keyword(s):  
Potassium Channels ◽  
Single Channel ◽  
Total Charge ◽  
Gating Currents ◽  
Mutant Channel ◽  
General Properties ◽  
Gating Model ◽  
Activation Gating ◽  
Shaker Potassium Channels ◽  
Gating Process

This second of three papers, in which we functionally characterize activation gating in Shaker potassium channels, focuses on the properties of a mutant channel (called V2), in which the leucine at position 382 (in the Shaker B sequence) is mutated to valine. The general properties of V2's ionic and gating currents are consistent with changes in late gating transitions, in particular, with V2 disrupting the positively cooperative gating process of the normally activating wild type (WT) channel. An analysis of forward and backward rate constants, analogous to that used for WT in the previous paper, indicates that V2 causes little change in the rates for most of the transitions in the activation path, but causes large changes in the backward rates of the final two transitions. Single channel data indicate that the V2 mutation causes moderate changes in the rates of transitions to states that are not in the activation path, but little change in the rates from these states. V2's data also yield insights into the general properties of the activation gating process that could not be readily obtained from the WT channel, including evidence that intermediate transitions have rapid backward rates, and an estimate of a total charge 2 e0 for the final two transitions. Taken together, these data will help constrain an activation gating model in the third paper of this series, while also providing an explanation for V2's effects.

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