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 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 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 Heme Regulates Allosteric Activation of the Slo1 BK Channel

2005 ◽  
Vol 126 (1) ◽  
pp. 7-21 ◽  
Author(s):  
Frank T. Horrigan ◽  
Stefan H. Heinemann ◽  
Toshinori Hoshi
Keyword(s):  
Divalent Cations ◽  
Ionic Currents ◽  
Bk Channels ◽  
Bk Channel ◽  
Voltage Sensor ◽  
Gating Currents ◽  
Calcium Dependent ◽  
Channel Opening ◽  
Activation Gate ◽  
Voltage Dependent

Large conductance calcium-dependent (Slo1 BK) channels are allosterically activated by membrane depolarization and divalent cations, and possess a rich modulatory repertoire. Recently, intracellular heme has been identified as a potent regulator of Slo1 BK channels (Tang, X.D., R. Xu, M.F. Reynolds, M.L. Garcia, S.H. Heinemann, and T. Hoshi. 2003. Nature. 425:531–535). Here we investigated the mechanism of the regulatory action of heme on heterologously expressed Slo1 BK channels by separating the influences of voltage and divalent cations. In the absence of divalent cations, heme generally decreased ionic currents by shifting the channel's G–V curve toward more depolarized voltages and by rendering the curve less steep. In contrast, gating currents remained largely unaffected by heme. Simulations suggest that a decrease in the strength of allosteric coupling between the voltage sensor and the activation gate and a concomitant stabilization of the open state account for the essential features of the heme action in the absence of divalent ions. At saturating levels of divalent cations, heme remained similarly effective with its influence on the G–V simulated by weakening the coupling of both Ca2+ binding and voltage sensor activation to channel opening. The results thus show that heme dampens the influence of allosteric activators on the activation gate of the Slo1 BK channel. To account for these effects, we consider the possibility that heme binding alters the structure of the RCK gating ring and thereby disrupts both Ca2+- and voltage-dependent gating as well as intrinsic stability of the open state.

The 1997 Stevenson Award Lecture. Cardiac K+channel gating: cloned delayed rectifier mechanisms and drug modulation

1998 ◽  
Vol 76 (2) ◽  
pp. 77-89 ◽  
Author(s):  
David Fedida ◽  
Fred SP Chen ◽  
Xue Zhang
Keyword(s):  
Channel Gating ◽  
State Dependence ◽  
Structural Features ◽  
K Channel ◽  
Delayed Rectifier ◽  
Specific Reference ◽  
Channel Blockers ◽  
Antiarrhythmic Agents ◽  
Gating Currents ◽  
Voltage Gated

K+ channels are ubiquitous membrane proteins, which have a central role in the control of cell excitability. In the heart, voltage-gated delayed rectifier K+ channels, like Kv1.5, determine repolarization and the cardiac action potential plateau duration. Here we review the broader properties of cloned voltage-gated K+ channels with specific reference to the hKv1.5 channel in heart. We discuss the basic structural components of K+ channels such as the pore, voltage sensor, and fast inactivation, all of which have been extensively studied. Slow, or C-type, inactivation and the structural features that control pore opening are less well understood, although recent studies have given new insight into these problems. Information about channel transitions that occur prior to opening is provided by gating currents, which reflect charge-carrying transitions between kinetic closed states. By studying modulation of the gating properties of K+ channels by cations and with drugs, we can make a more complete interpretation of the state dependence of drug and ion interactions with the channel. In this way we can uncover the detailed mechanisms of action of K+ channel blockers such as tetraethylammonium ions and 4-aminopyridine, and antiarrhythmic agents such as nifedipine and quinidine.Key words: potassium channel, Kv1.5, channel gating, inactivation, pore region, gating currents.

scholarly journals Molecular Template for a Voltage Sensor in a Novel K+ Channel. I. Identification and Functional Characterization of KvLm, a Voltage-gated K+ Channel from Listeria monocytogenes

2006 ◽  
Vol 128 (3) ◽  
pp. 283-292 ◽  
Author(s):  
Jose S. Santos ◽  
Alicia Lundby ◽  
Cecilia Zazueta ◽  
Mauricio Montal
Keyword(s):  
Listeria Monocytogenes ◽  
Voltage Sensor ◽  
K Channel ◽  
Open Probability ◽  
Voltage Gated ◽  
Kv Channel ◽  
Kv Channels ◽  
Sensor Module ◽  
Single Channel Currents ◽  
Hallmark Feature

The fundamental principles underlying voltage sensing, a hallmark feature of electrically excitable cells, are still enigmatic and the subject of intense scrutiny and controversy. Here we show that a novel prokaryotic voltage-gated K+ (Kv) channel from Listeria monocytogenes (KvLm) embodies a rudimentary, yet robust, sensor sufficient to endow it with voltage-dependent features comparable to those of eukaryotic Kv channels. The most conspicuous feature of the KvLm sequence is the nature of the sensor components: the motif is recognizable; it appears, however, to contain only three out of eight charged residues known to be conserved in eukaryotic Kv channels and accepted to be deterministic for folding and sensing. Despite the atypical sensor sequence, flux assays of KvLm reconstituted in liposomes disclosed a channel pore that is highly selective for K+ and is blocked by conventional Kv channel blockers. Single-channel currents recorded in symmetric K+ solutions from patches of enlarged Escherichia coli (spheroplasts) expressing KvLm showed that channel open probability sharply increases with depolarization, a hallmark feature of Kv channels. The identification of a voltage sensor module in KvLm with a voltage dependence comparable to that of other eukaryotic Kv channels yet encoded by a sequence that departs significantly from the consensus sequence of a eukaryotic voltage sensor establishes a molecular blueprint of a minimal sequence for a voltage sensor.

scholarly journals Can Shaker Potassium Channels be Locked in the Deactivated State?

2004 ◽  
Vol 124 (2) ◽  
pp. 163-171 ◽  
Author(s):  
Youshan Yang ◽  
Yangyang Yan ◽  
Fred J. Sigworth
Keyword(s):  
Xenopus Oocytes ◽  
Voltage Dependence ◽  
Voltage Sensor ◽  
Structural Studies ◽  
Gating Currents ◽  
Shaker Potassium Channel ◽  
Voltage Sensors ◽  
Voltage Gated ◽  
Gated Channel ◽  
Shaker Potassium Channels

For structural studies it would be useful to constrain the voltage sensor of a voltage-gated channel in its deactivated state. Here we consider one Shaker potassium channel mutant and speculate about others that might allow the channel to remain deactivated at zero membrane potential. Ionic and gating currents of F370C Shaker, expressed in Xenopus oocytes, were recorded in patches with internal application of the methanethiosulfonate reagent MTSET. It appears that the voltage dependence of voltage sensor movement is strongly shifted by reaction with internal MTSET, such that the voltage sensors appear to remain deactivated even at positive potentials. A disadvantage of this construct is that the rate of modification of voltage sensors by MTSET is quite low, ∼0.17 mM−1·s−1 at −80 mV, and is expected to be much lower at depolarized potentials.

scholarly journals Specificity of Charge-carrying Residues in the Voltage Sensor of Potassium Channels

2004 ◽  
Vol 123 (3) ◽  
pp. 205-216 ◽  
Author(s):  
Christopher A. Ahern ◽  
Richard Horn
Keyword(s):  
Potassium Channels ◽  
Electric Field ◽  
Protein A ◽  
Membrane Lipid ◽  
Voltage Sensor ◽  
Charge Movement ◽  
Gating Currents ◽  
Gating Charge ◽  
Channel Opening ◽  
Positively Charged

Positively charged voltage sensors of sodium and potassium channels are driven outward through the membrane's electric field upon depolarization. This movement is coupled to channel opening. A recent model based on studies of the KvAP channel proposes that the positively charged voltage sensor, christened the “voltage-sensor paddle”, is a peripheral domain that shuttles its charged cargo through membrane lipid like a hydrophobic cation. We tested this idea by attaching charged adducts to cysteines introduced into the putative voltage-sensor paddle of Shaker potassium channels and measuring fractional changes in the total gating charge from gating currents. The only residues capable of translocating attached charges through the membrane-electric field are those that serve this function in the native channel. This remarkable specificity indicates that charge movement involves highly specialized interactions between the voltage sensor and other regions of the protein, a mechanism inconsistent with the paddle model.

scholarly journals Interpreting the functional role of a novel interaction motif in prokaryotic sodium channels

2017 ◽  
Vol 149 (6) ◽  
pp. 613-622 ◽  
Author(s):  
Altin Sula ◽  
B.A. Wallace
Keyword(s):  
Sodium Channels ◽  
Functional Role ◽  
Structural Features ◽  
Channel Activation ◽  
Voltage Gated ◽  
Voltage Gated Sodium Channels ◽  
Channel Opening ◽  
Activated State ◽  
Electrical Signaling

Voltage-gated sodium channels enable the translocation of sodium ions across cell membranes and play crucial roles in electrical signaling by initiating the action potential. In humans, mutations in sodium channels give rise to several neurological and cardiovascular diseases, and hence they are targets for pharmaceutical drug developments. Prokaryotic sodium channel crystal structures have provided detailed views of sodium channels, which by homology have suggested potentially important functionally related structural features in human sodium channels. A new crystal structure of a full-length prokaryotic channel, NavMs, in a conformation we proposed to represent the open, activated state, has revealed a novel interaction motif associated with channel opening. This motif is associated with disease when mutated in human sodium channels and plays an important and dynamic role in our new model for channel activation.

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 Fast and slow voltage sensor rearrangements during activation gating in Kv1.2 channels detected using tetramethylrhodamine fluorescence

2010 ◽  
Vol 136 (1) ◽  
pp. 83-99 ◽  
Author(s):  
Andrew James Horne ◽  
Christian Joseph Peters ◽  
Thomas William Claydon ◽  
David Fedida
Keyword(s):  
Conformational Changes ◽  
Time Course ◽  
Slow Component ◽  
Ionic Current ◽  
Voltage Sensor ◽  
Activation Process ◽  
Dependent Manner ◽  
Gating Currents ◽  
Kv Channel ◽  
Voltage Dependent

The Kv1.2 channel, with its high resolution crystal structure, provides an ideal model for investigating conformational changes associated with channel gating, and fluorescent probes attached at the extracellular end of S4 are a powerful way to gain a more complete understanding of the voltage-dependent activity of these dynamic proteins. Tetramethylrhodamine-5-maleimide (TMRM) attached at A291C reports two distinct rearrangements of the voltage sensor domains, and a comparative fluorescence scan of the S4 and S3–S4 linker residues in Shaker and Kv1.2 shows important differences in their emission at other homologous residues. Kv1.2 shows a rapid decrease in A291C emission with a time constant of 1.5 ± 0.1 ms at 60 mV (n = 11) that correlates with gating currents and reports on translocation of the S4 and S3–S4 linker. However, unlike any Kv channel studied to date, this fast component is dwarfed by a larger, slower quenching of TMRM emission during depolarizations between −120 and −50 mV (τ = 21.4 ± 2.1 ms at 60 mV, V1/2 of −73.9 ± 1.4 mV) that is not seen in either Shaker or Kv1.5 and that comprises >60% of the total signal at all activating potentials. The slow fluorescence relaxes after repolarization in a voltage-dependent manner that matches the time course of Kv1.2 ionic current deactivation. Fluorophores placed directly in S1 and S2 at I187 and T219 recapitulate the time course and voltage dependence of slow quenching. The slow component is lost when the extracellular S1–S2 linker of Kv1.2 is replaced with that of Kv1.5 or Shaker, suggesting that it arises from a continuous internal rearrangement within the voltage sensor, initiated at negative potentials but prevalent throughout the activation process, and which must be reversed for the channel to close.

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