scholarly journals Investigating the Putative Glycine Hinge in Shaker Potassium Channel

2005 ◽  
Vol 126 (3) ◽  
pp. 213-226 ◽  
Author(s):  
Shinghua Ding ◽  
Lindsey Ingleby ◽  
Christopher A. Ahern ◽  
Richard Horn
Keyword(s):  
Potassium Channel ◽  
Functional Expression ◽  
Shaker Potassium Channel ◽  
Glycine Residue ◽  
Shaker Channels ◽  
Channel Opening ◽  
Activation Gating ◽  
Voltage Dependent ◽  
Shaker Potassium Channels

The crystal structure of an open potassium channel reveals a kink in the inner helix that lines the pore (Jiang, Y.X., A. Lee, J.Y. Chen, M. Cadene, B.T. Chait, and R. MacKinnon. 2002. Nature 417:523–526). The putative hinge point is a highly conserved glycine residue. We examined the role of the homologous residue (Gly466) in the S6 transmembrane segment of Shaker potassium channels. The nonfunctional alanine mutant G466A will assemble, albeit poorly, with wild-type (WT) subunits, suppressing functional expression. To test if this glycine residue is critical for activation gating, we did a glycine scan along the S6 segment in the background of G466A. Although all of these double mutants lack the higher-level glycosylation that is characteristic of mature Shaker channels, one (G466A/V467G) is able to generate voltage-dependent potassium current. Surface biotinylation shows that functional and nonfunctional constructs containing G466A express at comparable levels in the plasma membrane. Compared with WT channels, the shifted-glycine mutant has impairments in voltage-dependent channel opening, including a right-shifted activation curve and a decreased rate of activation. The double mutant has relatively normal open-channel properties, except for a decreased affinity for intracellular blockers, a consequence of the loss of the side chain of Val467. Control experiments with the double mutants M440A/G466A and G466A/V467A suggest that the flexibility provided by Gly466 is more important for channel function than its small size. Our results support roles for Gly466 both in biogenesis of the channel and as a hinge in activation gating.

scholarly journals Role of the S4 in Cooperativity of Voltage-dependent Potassium Channel Activation

1998 ◽  
Vol 111 (3) ◽  
pp. 399-420 ◽  
Author(s):  
Catherine J. Smith-Maxwell ◽  
Jennifer L. Ledwell ◽  
Richard W. Aldrich
Keyword(s):  
Potassium Channel ◽  
Amino Acid Sequences ◽  
Channel Activation ◽  
Shaker Potassium Channel ◽  
Activation Pathway ◽  
Activation Gating ◽  
Voltage Dependent ◽  
Rate Limiting ◽  
Cooperative Transition

Charged residues in the S4 transmembrane segment of voltage-gated cation channels play a key role in opening channels in response to changes in voltage across the cell membrane. However, the molecular mechanism of channel activation is not well understood. To learn more about the role of the S4 in channel gating, we constructed chimeras in which S4 segments from several divergent potassium channels, Shab, Shal, Shaw, and Kv3.2, were inserted into a Shaker potassium channel background. These S4 donor channels have distinctly different voltage-dependent gating properties and S4 amino acid sequences. None of the S4 chimeras have the gating behavior of their respective S4 donor channels. The conductance–voltage relations of all S4 chimeras are shifted to more positive voltages and the slopes are decreased. There is no consistent correlation between the nominal charge content of the S4 and the slope of the conductance–voltage relation, suggesting that the mutations introduced by the S4 chimeras may alter cooperative interactions in the gating process. We compared the gating behavior of the Shaw S4 chimera with its parent channels, Shaker and Shaw, in detail. The Shaw S4 substitution alters activation gating profoundly without introducing obvious changes in other channel functions. Analysis of the voltage-dependent gating kinetics suggests that the dominant effect of the Shaw S4 substitution is to alter a single cooperative transition late in the activation pathway, making it rate limiting. This interpretation is supported further by studies of channels assembled from tandem heterodimer constructs with both Shaker and Shaw S4 subunits. Activation gating in the heterodimer channels can be predicted from the properties of the homotetrameric channels only if it is assumed that the mutations alter a cooperative transition in the activation pathway rather than independent transitions.

Use of voltage clamp fluorimetry in understanding potassium channel gating: a review of Shaker fluorescence data

2009 ◽  
Vol 87 (6) ◽  
pp. 411-418 ◽  
Author(s):  
A.J. Horne ◽  
D. Fedida
Keyword(s):  
Potassium Channels ◽  
Potassium Channel ◽  
Voltage Clamp ◽  
Channel Gating ◽  
Specific Protein ◽  
Channel Activation ◽  
Shaker Potassium Channel ◽  
Inactivation Gating ◽  
Nanosecond Time Resolution ◽  
Shaker Potassium Channels

Voltage clamp fluorimetry (VCF) utilizes fluorescent probes that covalently bind to cysteine residues introduced into proteins and emit light as a function of their environment. Measurement of this emitted light during membrane depolarization reveals changes in the emission level as the environment of the labelled residue changes. This allows for the correlation of channel gating events with movement of specific protein moieties, at nanosecond time resolution. Since the pioneering use of this technique to investigate Shaker potassium channel activation movements, VCF has become an invaluable technique used to understand ion channel gating. This review summarizes the theory and some of the data on the application of the VCF technique. Although its usage has expanded beyond voltage-gated potassium channels and VCF is now used in a number of other voltage- and ligand-gated channels, we will focus on studies conducted in Shaker potassium channels, and what they have told us about channel activation and inactivation gating.

scholarly journals Shaker potassium channel gating. I: Transitions near the open state.

1994 ◽  
Vol 103 (2) ◽  
pp. 249-278 ◽  
Author(s):  
T Hoshi ◽  
W N Zagotta ◽  
R W Aldrich
Keyword(s):  
Time Course ◽  
Single Channel ◽  
Activation Process ◽  
Time Data ◽  
Shaker Potassium Channel ◽  
Voltage Dependent ◽  
Average Current ◽  
Shaker Potassium Channels ◽  
Kinetics Of ◽  
Deactivation Process

Kinetics of single voltage-dependent Shaker potassium channels expressed in Xenopus oocytes were studied in the absence of fast N-type inactivation. Comparison of the single-channel first latency distribution and the time course of the ensemble average current showed that the activation time course and its voltage dependence are largely determined by the transitions before first opening. The open dwell time data are consistent with a single kinetically distinguishable open state. Once the channel opens, it can enter at least two closed states which are not traversed frequently during the activation process. The rate constants for the transitions among these closed states and the open state are nearly voltage-independent at depolarized voltages (> -30 mV). During the deactivation process at more negative voltages, the channel can close directly to a closed state in the activation pathway in a voltage-dependent fashion.

scholarly journals Desensitization to ANG II in guinea pig ileum depends on membrane repolarization: role of maxi-K+ channel

1999 ◽  
Vol 277 (4) ◽  
pp. C739-C745 ◽  
Author(s):  
Bagnólia A. Silva ◽  
Viviane L. A. Nouailhetas ◽  
Jeannine Aboulafia
Keyword(s):  
Guinea Pig ◽  
Time Course ◽  
K Channel ◽  
Ang Ii ◽  
Guinea Pig Ileum ◽  
Tonic Component ◽  
Channel Opening ◽  
Voltage Dependent ◽  
Sustained Activation

Desensitization of ANG II tonic contractile response of the guinea pig ileum is related to membrane repolarization determined by Ca2+-activated K+(maxi-K+) channel opening. ANG II-stimulated depolarized myocytes presented sustained activation of maxi-K+ channels, characterized by reduction from 415 to 12 ms of the closed time constant. ANG II desensitization was prevented by 100 nM iberiotoxin, being reversible within 30 min. Depolarization by KCl, higher than 4 mM, impaired desensitization, suggesting that the membrane potential must attain a threshold to counteract the repolarization induced by maxi-K+ channel opening. Once this value is attained, there is no time dependency because the desensitization process was shut off by addition of KCl along the time course of the tonic response. In contrast, the sustained ACh tonic component was not altered by these maneuvers. We conclude that desensitization of the ANG II tonic component is foremost due to the opening of maxi-K+ channels, leading to membrane repolarization, thus closing the voltage-dependent Ca2+ channels responsible for the Ca2+ influx that sustains the tonic component in this muscle.

scholarly journals Calmodulin acts as a state-dependent switch to control a cardiac potassium channel opening

2020 ◽  
Author(s):  
Po Wei Kang ◽  
Annie M. Westerlund ◽  
Jingyi Shi ◽  
Kelli McFarland White ◽  
Alex K. Dou ◽  
...  
Keyword(s):  
Potassium Channel ◽  
Conformational Change ◽  
State Dependent ◽  
Gating Mechanism ◽  
Channel Opening ◽  
Activated State ◽  
Voltage Dependent ◽  
Functional Consequences ◽  
Pore Opening ◽  
Voltage Sensing Domain

AbstractCalmodulin (CaM) and PIP2 are potent regulators of the voltage-gated potassium channel KCNQ1 (KV7.1), which conducts the IKs current important for repolarization of cardiac action potentials. Although cryo-EM structures revealed intricate interactions between the KCNQ1 voltage-sensing domain (VSD), CaM, and PIP2, the functional consequences of these interactions remain unknown. Here, we show that CaM-VSD interactions act as a state-dependent switch to control KCNQ1 pore opening. Combined electrophysiology and molecular dynamics network analysis suggest that VSD transition into the fully-activated state allows PIP2 to compete with CaM for binding to VSD, leading to the conformational change that alters the VSD-pore coupling. We identify a motif in the KCNQ1 cytosolic domain which works downstream of CaM-VSD interactions to facilitate the conformational change. Our findings suggest a gating mechanism that integrates PIP2 and CaM in KCNQ1 voltage-dependent activation, yielding insights into how KCNQ1 gains the phenotypes critical for its function in the heart.

scholarly journals Independence and Cooperativity in Rearrangements of a Potassium Channel Voltage Sensor Revealed by Single Subunit Fluorescence

2000 ◽  
Vol 115 (3) ◽  
pp. 257-268 ◽  
Author(s):  
Lidia M. Mannuzzu ◽  
Ehud Y. Isacoff
Keyword(s):  
Action Potential ◽  
Potassium Channel ◽  
Voltage Gated ◽  
Channel Opening ◽  
Physiological Relevance ◽  
Voltage Dependent ◽  
Action Potential Threshold ◽  
Single Subunit ◽  
S4 Segment ◽  
Potential Threshold

Voltage-gated potassium channels are composed of four subunits. Voltage-dependent activation of these channels consists of a depolarization-triggered series of charge-carrying steps that occur in each subunit. These major charge-carrying steps are followed by cooperative step(s) that lead to channel opening. Unlike the late cooperative steps, the major charge-carrying steps have been proposed to occur independently in each of the channel subunits. In this paper, we examine this further. We showed earlier that the two major charge-carrying steps are associated with two sequential outward transmembrane movements of the charged S4 segment. We now use voltage clamp fluorometry to monitor these S4 movements in individual subunits of heterotetrameric channels. In this way, we estimate the influence of one subunit's S4 movement on another's when the energetics of their transmembrane movements differ. Our results show that the first S4 movement occurs independently in each subunit, while the second occurs cooperatively. At least part of the cooperativity appears to be intrinsic to the second S4 charge-carrying rearrangement. Such cooperativity in gating of voltage-dependent channels has great physiological relevance since it can affect both action potential threshold and rate of propagation.

scholarly journals Macroscopic Na+ Currents in the “Nonconducting” Shaker Potassium Channel Mutant W434F

1998 ◽  
Vol 112 (1) ◽  
pp. 85-93 ◽  
Author(s):  
John G. Starkus ◽  
Lioba Kuschel ◽  
Martin D. Rayner ◽  
Stefan H. Heinemann
Keyword(s):  
Structural Changes ◽  
Ionic Current ◽  
Outer Region ◽  
Ionic Currents ◽  
Shaker Potassium Channel ◽  
Shaker Channels ◽  
Gating Charge ◽  
Na Currents ◽  
Shaker Potassium Channels ◽  
Kinetics Of

C-type inactivation in Shaker potassium channels inhibits K+ permeation. The associated structural changes appear to involve the outer region of the pore. Recently, we have shown that C-type inactivation involves a change in the selectivity of the Shaker channel, such that C-type inactivated channels show maintained voltage-sensitive activation and deactivation of Na+ and Li+ currents in K+-free solutions, although they show no measurable ionic currents in physiological solutions. In addition, it appears that the effective block of ion conduction produced by the mutation W434F in the pore region may be associated with permanent C-type inactivation of W434F channels. These conclusions predict that permanently C-type inactivated W434F channels would also show Na+ and Li+ currents (in K+-free solutions) with kinetics similar to those seen in C-type-inactivated Shaker channels. This paper confirms that prediction and demonstrates that activation and deactivation parameters for this mutant can be obtained from macroscopic ionic current measurements. We also show that the prolonged Na+ tail currents typical of C-type inactivated channels involve an equivalent prolongation of the return of gating charge, thus demonstrating that the kinetics of gating charge return in W434F channels can be markedly altered by changes in ionic conditions.

scholarly journals On the role of ball and chain interactions in recovery from the inactivation of the shaker potassium channel

2008 ◽  
Vol 13 (4) ◽  
Author(s):  
Przemysław Borys ◽  
Zbigniew Grzywna
Keyword(s):  
Potassium Channel ◽  
Binding Site ◽  
Chain Model ◽  
Shaker Potassium Channel ◽  
Recovery From Inactivation

AbstractWe describe a new factor in the recovery from inactivation in the ball and chain model. We propose a model in which the tension from the chain may help pull the ball away from its binding site, reducing the duration of the inactivation period. A corresponding model was built and analysed.

scholarly journals Calmodulin acts as a state-dependent switch to control a cardiac potassium channel opening

2020 ◽  
Vol 6 (50) ◽  
pp. eabd6798
Author(s):  
Po Wei Kang ◽  
Annie M. Westerlund ◽  
Jingyi Shi ◽  
Kelli McFarland White ◽  
Alex K. Dou ◽  
...  
Keyword(s):  
Potassium Channel ◽  
Conformational Changes ◽  
State Dependent ◽  
Gating Mechanism ◽  
Channel Opening ◽  
Activated State ◽  
Voltage Dependent ◽  
Functional Consequences ◽  
Open States ◽  
Voltage Sensing Domain

Calmodulin (CaM) and phosphatidylinositol 4,5-bisphosphate (PIP2) are potent regulators of the voltage-gated potassium channel KCNQ1 (KV7.1), which conducts the cardiac IKs current. Although cryo–electron microscopy structures revealed intricate interactions between the KCNQ1 voltage-sensing domain (VSD), CaM, and PIP2, the functional consequences of these interactions remain unknown. Here, we show that CaM-VSD interactions act as a state-dependent switch to control KCNQ1 pore opening. Combined electrophysiology and molecular dynamics network analysis suggest that VSD transition into the fully activated state allows PIP2 to compete with CaM for binding to VSD. This leads to conformational changes that alter VSD-pore coupling to stabilize open states. We identify a motif in the KCNQ1 cytosolic domain, which works downstream of CaM-VSD interactions to facilitate the conformational change. Our findings suggest a gating mechanism that integrates PIP2 and CaM in KCNQ1 voltage-dependent activation, yielding insights into how KCNQ1 gains the phenotypes critical for its physiological function.

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