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.

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 Mutations in the S4 Region Isolate the Final Voltage-dependent Cooperative Step in Potassium Channel Activation

1999 ◽  
Vol 113 (3) ◽  
pp. 389-414 ◽  
Author(s):  
Jennifer L. Ledwell ◽  
Richard W. Aldrich
Keyword(s):  
Voltage Dependence ◽  
Ionic Current ◽  
Amino Acid Sequences ◽  
Total Charge ◽  
Charge Movement ◽  
Activation Pathway ◽  
Channel Opening ◽  
Voltage Dependent ◽  
Rate Limiting ◽  
Cooperative Transition

Charged residues in the S4 transmembrane segment play a key role in determining the sensitivity of voltage-gated ion channels to changes in voltage across the cell membrane. However, cooperative interactions between subunits also affect the voltage dependence of channel opening, and these interactions can be altered by making substitutions at uncharged residues in the S4 region. We have studied the activation of two mutant Shaker channels that have different S4 amino acid sequences, ILT (V369I, I372L, and S376T) and Shaw S4 (the S4 of Drosophila Shaw substituted into Shaker), and yet have very similar ionic current properties. Both mutations affect cooperativity, making a cooperative transition in the activation pathway rate limiting and shifting it to very positive voltages, but analysis of gating and ionic current recordings reveals that the ILT and Shaw S4 mutant channels have different activation pathways. Analysis of gating currents suggests that the dominant effect of the ILT mutation is to make the final cooperative transition to the open state of the channel rate limiting in an activation pathway that otherwise resembles that of Shaker. The charge movement associated with the final gating transition in ILT activation can be measured as an isolated component of charge movement in the voltage range of channel opening and accounts for 13% (∼1.8 e0) of the total charge moved in the ILT activation pathway. The remainder of the ILT gating charge (87%) moves at negative voltages, where channels do not open, and confirms the presence of Shaker-like conformational changes between closed states in the activation pathway. In contrast to ILT, the activation pathway of Shaw S4 seems to involve a single cooperative charge-moving step between a closed and an open state. We cannot detect any voltage-dependent transitions between closed states for Shaw S4. Restoring basic residues that are missing in Shaw S4 (R1, R2, and K7) rescues charge movement between closed states in the activation pathway, but does not alter the voltage dependence of the rate-limiting transition in activation.

scholarly journals Uncharged S4 Residues and Cooperativity in Voltage-dependent Potassium Channel Activation

1998 ◽  
Vol 111 (3) ◽  
pp. 421-439 ◽  
Author(s):  
Catherine J. Smith-Maxwell ◽  
Jennifer L. Ledwell ◽  
Richard W. Aldrich
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Potassium Channel ◽  
Voltage Dependence ◽  
Channel Gating ◽  
Ionic Currents ◽  
Channel Activation ◽  
Functional Changes ◽  
Activation Pathway ◽  
Voltage Dependent

Substitution of the S4 of Shaw into Shaker alters cooperativity in channel activation by slowing a cooperative transition late in the activation pathway. To determine the amino acids responsible for the functional changes in Shaw S4, we created several mutants by substituting amino acids from Shaw S4 into Shaker. The S4 amino acid sequences of Shaker and Shaw S4 differ at 11 positions. Simultaneous substitution of just three noncharged residues from Shaw S4 into Shaker (V369I, I372L, S376T; ILT) reproduces the kinetic and voltage-dependent properties of Shaw S4 channel activation. These substitutions cause very small changes in the structural and chemical properties of the amino acid side chains. In contrast, substituting the positively charged basic residues in the S4 of Shaker with neutral or negative residues from the S4 of Shaw S4 does not reproduce the shallow voltage dependence or other properties of Shaw S4 opening. Macroscopic ionic currents for ILT could be fit by modifying a single set of transitions in a model for Shaker channel gating (Zagotta, W.N., T. Hoshi, and R.W. Aldrich. 1994. J. Gen. Physiol. 103:321–362). Changing the rate and voltage dependence of a final cooperative step in activation successfully reproduces the kinetic, steady state, and voltage-dependent properties of ILT ionic currents. Consistent with the model, ILT gating currents activate at negative voltages where the channel does not open and, at more positive voltages, they precede the ionic currents, confirming the existence of voltage-dependent transitions between closed states in the activation pathway. Of the three substitutions in ILT, the I372L substitution is primarily responsible for the changes in cooperativity and voltage dependence. These results suggest that noncharged residues in the S4 play a crucial role in Shaker potassium channel gating and that small steric changes in these residues can lead to large changes in cooperativity within the channel protein.

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 The Role of Potassium Channel Activation in Celecoxib-Induced Analgesic Action

PLoS ONE ◽  
2013 ◽  
Vol 8 (1) ◽  
pp. e54797 ◽  
Author(s):  
Yao Mi ◽  
Xuan Zhang ◽  
Fan Zhang ◽  
Jinlong Qi ◽  
Haixia Gao ◽  
...  
Keyword(s):  
Potassium Channel ◽  
Analgesic Action ◽  
Channel Activation

scholarly journals Role of the S3-S4 Linker in Shaker Potassium Channel Activation

1997 ◽  
Vol 109 (2) ◽  
pp. 191-199 ◽  
Author(s):  
Rajesh Mathur ◽  
Jie Zheng ◽  
Yangyang Yan ◽  
Fred J. Sigworth
Keyword(s):  
Conformational Changes ◽  
Xenopus Oocytes ◽  
Channel Activation ◽  
Activation Kinetics ◽  
Shaker Potassium Channel ◽  
Large Conformational Change ◽  
Voltage Gated Channels ◽  
Mutant Construct

Structural models of voltage-gated channels suggest that flexibility of the S3-S4 linker region may be important in allowing the S4 region to undergo large conformational changes in its putative voltage-sensing function. We report here the initial characterization of 18 mutations in the S3-S4 linker of the Shaker channel, including deletions, insertions, charge changes, substitution of prolines, and chimeras replacing the 25-residue Shaker linker with 7- or 9-residue sequences from Shab, Shaw, or Shal. As measured in Xenopus oocytes with a two-microelectrode voltage clamp, each mutant construct yielded robust currents. Changes in the voltage dependence of activation were small, with activation voltage shifts of 13 mV or less. Substitution of linkers from the slowly activating Shab and Shaw channels resulted in a three- to fourfold slowing of activation and deactivation. It is concluded that the S3-S4 linker is unlikely to participate in a large conformational change during channel activation. The linker, which in some channel subfamilies has highly conserved sequences, may however be a determinant of activation kinetics in potassium channels, as previously has been suggested in the case of calcium channels.

scholarly journals Role of Tyrosine Phosphorylation in Potassium Channel Activation

1995 ◽  
Vol 270 (41) ◽  
pp. 24292-24299 ◽  
Author(s):  
Natalia B. Prevarskaya ◽  
Roman N. Skryma ◽  
Pierre Vacher ◽  
Nathalie Daniel ◽  
Jean Djiane ◽  
...  
Keyword(s):  
Potassium Channel ◽  
Tyrosine Phosphorylation ◽  
Channel Activation

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.

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