scholarly journals The ladder-shaped polyether toxin gambierol anchors the gating machinery of Kv3.1 channels in the resting state

2013 ◽  
Vol 141 (3) ◽  
pp. 359-369 ◽  
Author(s):  
Ivan Kopljar ◽  
Alain J. Labro ◽  
Tessa de Block ◽  
Jon D. Rainier ◽  
Jan Tytgat ◽  
...  
Keyword(s):  
Binding Site ◽  
Resting State ◽  
Ionic Current ◽  
Dissociation Kinetics ◽  
Gating Currents ◽  
Voltage Range ◽  
High Affinity ◽  
Kv Channels ◽  
Channel Opening ◽  
Nav Channels

Voltage-gated potassium (Kv) and sodium (Nav) channels are key determinants of cellular excitability and serve as targets of neurotoxins. Most marine ciguatoxins potentiate Nav channels and cause ciguatera seafood poisoning. Several ciguatoxins have also been shown to affect Kv channels, and we showed previously that the ladder-shaped polyether toxin gambierol is a potent Kv channel inhibitor. Most likely, gambierol acts via a lipid-exposed binding site, located outside the K+ permeation pathway. However, the mechanism by which gambierol inhibits Kv channels remained unknown. Using gating and ionic current analysis to investigate how gambierol affected S6 gate opening and voltage-sensing domain (VSD) movements, we show that the resting (closed) channel conformation forms the high-affinity state for gambierol. The voltage dependence of activation was shifted by >120 mV in the depolarizing direction, precluding channel opening in the physiological voltage range. The (early) transitions between the resting and the open state were monitored with gating currents, and provided evidence that strong depolarizations allowed VSD movement up to the activated-not-open state. However, for transition to the fully open (ion-conducting) state, the toxin first needed to dissociate. These dissociation kinetics were markedly accelerated in the activated-not-open state, presumably because this state displayed a much lower affinity for gambierol. A tetrameric concatemer with only one high-affinity binding site still displayed high toxin sensitivity, suggesting that interaction with a single binding site prevented the concerted step required for channel opening. We propose a mechanism whereby gambierol anchors the channel’s gating machinery in the resting state, requiring more work from the VSD to open the channel. This mechanism is quite different from the action of classical gating modifier peptides (e.g., hanatoxin). Therefore, polyether toxins open new opportunities in structure–function relationship studies in Kv channels and in drug design to modulate channel function.

scholarly journals Basis for allosteric open-state stabilization of voltage-gated potassium channels by intracellular cations

2012 ◽  
Vol 140 (5) ◽  
pp. 495-511 ◽  
Author(s):  
Samuel J. Goodchild ◽  
Hongjian Xu ◽  
Zeineb Es-Salah-Lamoureux ◽  
Christopher A. Ahern ◽  
David Fedida
Keyword(s):  
Ionic Current ◽  
Structural Features ◽  
Voltage Sensor ◽  
Gating Currents ◽  
Voltage Gated ◽  
Kv Channels ◽  
Deactivation Kinetics ◽  
Channel Opening ◽  
In The Wild ◽  
Pore Closure

The open state of voltage-gated potassium (Kv) channels is associated with an increased stability relative to the pre-open closed states and is reflected by a slowing of OFF gating currents after channel opening. The basis for this stabilization is usually assigned to intrinsic structural features of the open pore. We have studied the gating currents of Kv1.2 channels and found that the stabilization of the open state is instead conferred largely by the presence of cations occupying the inner cavity of the channel. Large impermeant intracellular cations such as N-methyl-d-glucamine (NMG+) and tetraethylammonium cause severe slowing of channel closure and gating currents, whereas the smaller cation, Cs+, displays a more moderate effect on voltage sensor return. A nonconducting mutant also displays significant open state stabilization in the presence of intracellular K+, suggesting that K+ ions in the intracellular cavity also slow pore closure. A mutation in the S6 segment used previously to enlarge the inner cavity (Kv1.2-I402C) relieves the slowing of OFF gating currents in the presence of the large NMG+ ion, suggesting that the interaction site for stabilizing ions resides within the inner cavity and creates an energetic barrier to pore closure. The physiological significance of ionic occupation of the inner cavity is underscored by the threefold slowing of ionic current deactivation in the wild-type channel compared with Kv1.2-I402C. The data suggest that internal ions, including physiological concentrations of K+, allosterically regulate the deactivation kinetics of the Kv1.2 channel by impairing pore closure and limiting the return of voltage sensors. This may represent a primary mechanism by which Kv channel deactivation kinetics is linked to ion permeation and reveals a novel role for channel inner cavity residues to indirectly regulate voltage sensor dynamics.

scholarly journals Transfer of ion binding site from ether-à-go-go to Shaker: Mg2+ binds to resting state to modulate channel opening

2010 ◽  
Vol 135 (5) ◽  
pp. 415-431 ◽  
Author(s):  
Meng-chin A. Lin ◽  
Jeff Abramson ◽  
Diane M. Papazian
Keyword(s):  
Binding Site ◽  
Resting State ◽  
Ionic Current ◽  
Voltage Sensor ◽  
Ion Binding ◽  
Charge Movement ◽  
X Ray ◽  
Cation Binding Site ◽  
Channel Opening ◽  
Voltage Dependent

In ether-à-go-go (eag) K+ channels, extracellular divalent cations bind to the resting voltage sensor and thereby slow activation. Two eag-specific acidic residues in S2 and S3b coordinate the bound ion. Residues located at analogous positions are ∼4 Å apart in the x-ray structure of a Kv1.2/Kv2.1 chimera crystallized in the absence of a membrane potential. It is unknown whether these residues remain in proximity in Kv1 channels at negative voltages when the voltage sensor domain is in its resting conformation. To address this issue, we mutated Shaker residues I287 and F324, which correspond to the binding site residues in eag, to aspartate and recorded ionic and gating currents in the presence and absence of extracellular Mg2+. In I287D+F324D, Mg2+ significantly increased the delay before ionic current activation and slowed channel opening with no readily detectable effect on closing. Because the delay before Shaker opening reflects the initial phase of voltage-dependent activation, the results indicate that Mg2+ binds to the voltage sensor in the resting conformation. Supporting this conclusion, Mg2+ shifted the voltage dependence and slowed the kinetics of gating charge movement. Both the I287D and F324D mutations were required to modulate channel function. In contrast, E283, a highly conserved residue in S2, was not required for Mg2+ binding. Ion binding affected activation by shielding the negatively charged side chains of I287D and F324D. These results show that the engineered divalent cation binding site in Shaker strongly resembles the naturally occurring site in eag. Our data provide a novel, short-range structural constraint for the resting conformation of the Shaker voltage sensor and are valuable for evaluating existing models for the resting state and voltage-dependent conformational changes that occur during activation. Comparing our data to the chimera x-ray structure, we conclude that residues in S2 and S3b remain in proximity throughout voltage-dependent activation.

scholarly journals Kinetics of the multitasking high-affinity Win binding site of WDR5 in restricted and unrestricted conditions

2021 ◽  
Author(s):  
Ali Imran ◽  
Brandon S. Moyer ◽  
Ashley J. Canning ◽  
Dan Kalina ◽  
Thomas M Duncan ◽  
...  
Keyword(s):  
Binding Site ◽  
Primary Role ◽  
Dissociation Kinetics ◽  
High Affinity ◽  
Histone 3 ◽  
Quantitative Understanding ◽  
Slow Dissociation ◽  
Kinetics Of ◽  
Association Kinetics

Recent advances in quantitative proteomics show that WD40 proteins play a pivotal role in numerous cellular networks. Yet, they have been fairly unexplored and their physical associations with other proteins are ambiguous. A quantitative understanding of these interactions has wide-ranging significance. WD40 repeat protein 5 (WDR5) interacts with all members of human SET1/MLL methyltransferases, which regulate methylation of the histone 3 lysine 4 (H3K4). Here, using real-time binding measurements in a high-throughput setting, we identified the kinetic fingerprint of  transient associations between WDR5 and 14-residue WDR5 interaction (Win) motif peptides of each SET1 protein (SET1Win). Our results reveal that the high-affinity WDR5-SET1Win interactions feature slow association kinetics. This finding is likely due to the requirement of SET1Win to insert into the narrow WDR5 cavity, also named the Win binding site. Furthermore, our explorations indicate fairly slow dissociation kinetics. This conclusion is in accordance with the primary role of WDR5 in maintaining the functional integrity of a large multisubunit complex, which regulates the histone methylation. Because the Win binding site is considered a key therapeutic target, the immediate outcomes of this study could form the basis for accelerated developments in medical biotechnology.

scholarly journals Voltage Clamp Fluorimetry Reveals a Novel Outer Pore Instability in a Mammalian Voltage-gated Potassium Channel

2008 ◽  
Vol 132 (2) ◽  
pp. 209-222 ◽  
Author(s):  
Moninder Vaid ◽  
Thomas W. Claydon ◽  
Saman Rezazadeh ◽  
David Fedida
Keyword(s):  
Voltage Clamp ◽  
Time Course ◽  
Ionic Current ◽  
Voltage Sensor ◽  
Selectivity Filter ◽  
Similar Time ◽  
Voltage Gated ◽  
Kv Channels ◽  
Channel Opening ◽  
Outer Pore

Voltage-gated potassium (Kv) channel gating involves complex structural rearrangements that regulate the ability of channels to conduct K+ ions. Fluorescence-based approaches provide a powerful technique to directly report structural dynamics underlying these gating processes in Shaker Kv channels. Here, we apply voltage clamp fluorimetry, for the first time, to study voltage sensor motions in mammalian Kv1.5 channels. Despite the homology between Kv1.5 and the Shaker channel, attaching TMRM or PyMPO fluorescent probes to substituted cysteine residues in the S3–S4 linker of Kv1.5 (M394C-V401C) revealed unique and unusual fluorescence signals. Whereas the fluorescence during voltage sensor movement in Shaker channels was monoexponential and occurred with a similar time course to ionic current activation, the fluorescence report of Kv1.5 voltage sensor motions was transient with a prominent rapidly dequenching component that, with TMRM at A397C (equivalent to Shaker A359C), represented 36 ± 3% of the total signal and occurred with a τ of 3.4 ± 0.6 ms at +60 mV (n = 4). Using a number of approaches, including 4-AP drug block and the ILT triple mutation, which dissociate channel opening from voltage sensor movement, we demonstrate that the unique dequenching component of fluorescence is associated with channel opening. By regulating the outer pore structure using raised (99 mM) external K+ to stabilize the conducting configuration of the selectivity filter, or the mutations W472F (equivalent to Shaker W434F) and H463G to stabilize the nonconducting (P-type inactivated) configuration of the selectivity filter, we show that the dequenching of fluorescence reflects rapid structural events at the selectivity filter gate rather than the intracellular pore 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 Isolation of myocardial L-type calcium channel gating currents with the spider toxin omega-Aga-IIIA.

1994 ◽  
Vol 103 (5) ◽  
pp. 731-753 ◽  
Author(s):  
E A Ertel ◽  
M M Smith ◽  
M D Leibowitz ◽  
C J Cohen
Keyword(s):  
Guinea Pig ◽  
Time Course ◽  
Slow Component ◽  
Ionic Current ◽  
Reversal Potential ◽  
Ca Channel ◽  
Ca Channels ◽  
Gating Currents ◽  
Voltage Range ◽  
Charge Movements

The peptide omega-agatoxin-IIIA (omega-Aga-IIIA) blocks ionic current through L-type Ca channels in guinea pig atrial cells without affecting the associated gating currents. omega-Aga-IIIA permits the study of L-type Ca channel ionic and gating currents under nearly identical ionic conditions. Under conditions that isolate L-type Ca channel currents, omega-Aga-IIIA blocks all ionic current during a test pulse and after repolarization. This block reveals intramembrane charge movements of equal magnitude and opposite sign at the beginning of the pulse (Q(on)) and after repolarization (Q(off)). Q(on) and Q(off) are suppressed by 1 microM felodipine, saturate with increasing test potential, and are insensitive to Cd. The decay of the transient current associated with Q(on) is composed of fast and slow exponential components. The slow component has a time constant similar to that for activation of L-type Ca channel ionic current, over a broad voltage range. The current associated with Q(off) decays monoexponentially and more slowly than ionic current. Similar charge movements are found in guinea pig tracheal myocytes, which lack Na channels and T-type Ca channels. The kinetic and pharmacological properties of Q(on) and Q(off) indicate that they reflect gating currents associated with L-type Ca channels. omega-Aga-IIIA has no effect on gating currents when ionic current is eliminated by stepping to the reversal potential for Ca or by Cd block. Gating currents constitute a significant component of total current when physiological concentrations of Ca are present and they obscure the activation and deactivation of L-type Ca channels. By using omega-Aga-IIIA, we resolve the entire time course of L-type Ca channel ionic and gating currents. We also show that L- and T-type Ca channel ionic currents can be accurately quantified by tail current analysis once gating currents are taken into account.

Influence of L-type Ca channel alpha 2/delta-subunit on ionic and gating current in transiently transfected HEK 293 cells

1996 ◽  
Vol 270 (5) ◽  
pp. H1521-H1528 ◽  
Author(s):  
R. Bangalore ◽  
G. Mehrke ◽  
K. Gingrich ◽  
F. Hofmann ◽  
R. S. Kass
Keyword(s):  
Ionic Current ◽  
Charge Movement ◽  
Ca Channel ◽  
Gating Currents ◽  
Maximal Conductance ◽  
Hek 293 ◽  
Voltage Range ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Delta Subunit

We have measured ionic and gating currents in human embryonic kidney (HEK 293) cells transiently transfected with cDNAs encoding subunits of the cardiac voltage-gated L-type Ca2+ channel. Robust recombinant ionic current and associated nonlinear charge movement could be measured over a broad voltage range without contamination by endogenous channel activity. Coexpression of the alpha 2/delta-subunit along with alpha 1- and beta 2-subunits speeded activation and deactivation kinetics and significantly increased the maximal conductance of ionic current. Charge movement was measured at voltages negative to the threshold for activation of ionic current, and gating charge could be immobilized at positive holding potentials that did not inactivate ionic current. The ratio of maximal ionic conductance to maximal charge moved remained the same in the absence or presence of the alpha 2/delta-subunit. However, the maximal amount of charge moved was increased about twofold in the presence of the alpha 2/delta-subunit. These results suggest that coexpression of the alpha 2/delta-subunit enhances the expression of functional L-type channels and, in addition, provide evidence that most of the L-type channel-associated nonlinear charge movement is caused by transitions between nonconducting states of the channel protein that precede the open and inactivated states.

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