scholarly journals Interfacial gating triad is crucial for electromechanical transduction in voltage-activated potassium channels

2014 ◽  
Vol 144 (5) ◽  
pp. 457-467 ◽  
Author(s):  
Sandipan Chowdhury ◽  
Benjamin M. Haehnel ◽  
Baron Chanda
Keyword(s):  
Potassium Channels ◽  
Conformational Changes ◽  
Electromechanical Coupling ◽  
Structural Integrity ◽  
Voltage Sensor ◽  
Amino Acid Residues ◽  
Kv Channel ◽  
Electromechanical Transduction ◽  
Voltage Dependent ◽  
Graph Theoretical Approach

Voltage-dependent potassium channels play a crucial role in electrical excitability and cellular signaling by regulating potassium ion flux across membranes. Movement of charged residues in the voltage-sensing domain leads to a series of conformational changes that culminate in channel opening in response to changes in membrane potential. However, the molecular machinery that relays these conformational changes from voltage sensor to the pore is not well understood. Here we use generalized interaction-energy analysis (GIA) to estimate the strength of site-specific interactions between amino acid residues putatively involved in the electromechanical coupling of the voltage sensor and pore in the outwardly rectifying KV channel. We identified candidate interactors at the interface between the S4–S5 linker and the pore domain using a structure-guided graph theoretical approach that revealed clusters of conserved and closely packed residues. One such cluster, located at the intracellular intersubunit interface, comprises three residues (arginine 394, glutamate 395, and tyrosine 485) that interact with each other. The calculated interaction energies were 3–5 kcal, which is especially notable given that the net free-energy change during activation of the Shaker KV channel is ∼14 kcal. We find that this triad is delicately maintained by balance of interactions that are responsible for structural integrity of the intersubunit interface while maintaining sufficient flexibility at a critical gating hinge for optimal transmission of force to the pore gate.

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.

scholarly journals Interaction between tetraethylammonium and amino acid residues in the pore of cloned voltage-dependent potassium channels.

1991 ◽  
Vol 266 (12) ◽  
pp. 7583-7587
Author(s):  
M P Kavanaugh ◽  
M D Varnum ◽  
P B Osborne ◽  
M J Christie ◽  
A E Busch ◽  
...  
Keyword(s):  
Amino Acid ◽  
Potassium Channels ◽  
Amino Acid Residues ◽  
Voltage Dependent

scholarly journals Intermediate state trapping of a voltage sensor

2012 ◽  
Vol 140 (6) ◽  
pp. 635-652 ◽  
Author(s):  
Jérôme J. Lacroix ◽  
Stephan A. Pless ◽  
Luca Maragliano ◽  
Fabiana V. Campos ◽  
Jason D. Galpin ◽  
...  
Keyword(s):  
Ion Channels ◽  
Conformational Changes ◽  
Intermediate State ◽  
Molecular Mechanisms ◽  
Voltage Sensor ◽  
Functional Coupling ◽  
Kv Channel ◽  
New Information ◽  
Helical Turn ◽  
Voltage Gated Ion Channels

Voltage sensor domains (VSDs) regulate ion channels and enzymes by undergoing conformational changes depending on membrane electrical signals. The molecular mechanisms underlying the VSD transitions are not fully understood. Here, we show that some mutations of I241 in the S1 segment of the Shaker Kv channel positively shift the voltage dependence of the VSD movement and alter the functional coupling between VSD and pore domains. Among the I241 mutants, I241W immobilized the VSD movement during activation and deactivation, approximately halfway between the resting and active states, and drastically shifted the voltage activation of the ionic conductance. This phenotype, which is consistent with a stabilization of an intermediate VSD conformation by the I241W mutation, was diminished by the charge-conserving R2K mutation but not by the charge-neutralizing R2Q mutation. Interestingly, most of these effects were reproduced by the F244W mutation located one helical turn above I241. Electrophysiology recordings using nonnatural indole derivatives ruled out the involvement of cation-Π interactions for the effects of the Trp inserted at positions I241 and F244 on the channel’s conductance, but showed that the indole nitrogen was important for the I241W phenotype. Insight into the molecular mechanisms responsible for the stabilization of the intermediate state were investigated by creating in silico the mutations I241W, I241W/R2K, and F244W in intermediate conformations obtained from a computational VSD transition pathway determined using the string method. The experimental results and computational analysis suggest that the phenotype of I241W may originate in the formation of a hydrogen bond between the indole nitrogen atom and the backbone carbonyl of R2. This work provides new information on intermediate states in voltage-gated ion channels with an approach that produces minimum chemical perturbation.

scholarly journals A new mechanism of voltage-dependent gating exposed by KV10.1 channels interrupted between voltage sensor and pore

2017 ◽  
Vol 149 (5) ◽  
pp. 577-593 ◽  
Author(s):  
Adam P. Tomczak ◽  
Jorge Fernández-Trillo ◽  
Shashank Bharill ◽  
Ferenc Papp ◽  
Gyorgy Panyi ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Voltage Sensor ◽  
Ion Flow ◽  
Gating Model ◽  
Cryo Electron Microscopy ◽  
Channel Gate ◽  
Voltage Dependent ◽  
Voltage Gated Ion Channels ◽  
Voltage Sensing Domain ◽  
Pore Domain

Voltage-gated ion channels couple transmembrane potential changes to ion flow. Conformational changes in the voltage-sensing domain (VSD) of the channel are thought to be transmitted to the pore domain (PD) through an α-helical linker between them (S4–S5 linker). However, our recent work on channels disrupted in the S4–S5 linker has challenged this interpretation for the KCNH family. Furthermore, a recent single-particle cryo-electron microscopy structure of KV10.1 revealed that the S4–S5 linker is a short loop in this KCNH family member, confirming the need for an alternative gating model. Here we use “split” channels made by expression of VSD and PD as separate fragments to investigate the mechanism of gating in KV10.1. We find that disruption of the covalent connection within the S4 helix compromises the ability of channels to close at negative voltage, whereas disconnecting the S4–S5 linker from S5 slows down activation and deactivation kinetics. Surprisingly, voltage-clamp fluorometry and MTS accessibility assays show that the motion of the S4 voltage sensor is virtually unaffected when VSD and PD are not covalently bound. Finally, experiments using constitutively open PD mutants suggest that the presence of the VSD is structurally important for the conducting conformation of the pore. Collectively, our observations offer partial support to the gating model that assumes that an inward motion of the C-terminal S4 helix, rather than the S4–S5 linker, closes the channel gate, while also suggesting that control of the pore by the voltage sensor involves more than one mechanism.

scholarly journals The BK channel gating ring is strongly coupled to the voltage sensor

2018 ◽  
Author(s):  
Pablo Miranda ◽  
Miguel Holmgren ◽  
Teresa Giraldez
Keyword(s):  
Conformational Changes ◽  
Bk Channels ◽  
Bk Channel ◽  
Voltage Sensor ◽  
Voltage Sensitivity ◽  
Divalent Ions ◽  
Gating Currents ◽  
Open Probability ◽  
Voltage Dependent ◽  
Tightly Coupled

ABSTRACTThe open probability of large conductance voltage- and calcium-dependent potassium (BK) channels is regulated allosterically by changes in the transmembrane voltage and intracellular concentration of divalent ions (Ca2+ and Mg2+). The divalent cation sensors reside within the gating ring formed by eight Regulator of Conductance of Potassium (RCK) domains, two from each of the four channel subunits. Overall, the gating ring contains 12 sites that can bind Ca2+ with different affinities. Using patch-clamp fluorometry, we have shown robust changes in FRET signals within the gating ring in response to divalent ions and voltage, which do not directly track open probability. Only the conformational changes triggered through the RCK1 binding site are voltage-dependent in presence of Ca2+. Because the gating ring is outside the electric field, it must gain voltage sensitivity from coupling to the voltage-dependent channel opening, the voltage sensor or both. Here we demonstrate that alterations of voltage sensor dynamics known to shift gating currents produce a cognate shift in the gating ring voltage dependence, whereas changing BK channels’ relative probability of opening had little effect. These results strongly suggest that the conformational changes of the RCK1 domain of the gating ring are tightly coupled to the voltage sensor function, and this interaction is central to the allosteric modulation of BK channels.

scholarly journals Mg2+ Modulates Voltage-Dependent Activation in Ether-à-Go-Go Potassium Channels by Binding between Transmembrane Segments S2 and S3

2000 ◽  
Vol 116 (5) ◽  
pp. 663-678 ◽  
Author(s):  
William R. Silverman ◽  
Chih-Yung Tang ◽  
Allan F. Mock ◽  
Kyung-Bong Huh ◽  
Diane M. Papazian
Keyword(s):  
Potassium Channels ◽  
Binding Site ◽  
Voltage Sensor ◽  
Wild Type ◽  
Activation Kinetics ◽  
Fast Kinetics ◽  
K Channels ◽  
Transmembrane Segments ◽  
Acidic Residues ◽  
Voltage Dependent

Extracellular Mg2+ directly modulates voltage-dependent activation in ether-à-go-go (eag) potassium channels, slowing the kinetics of ionic and gating currents (Tang, C.-Y., F. Bezanilla, and D.M. Papazian. 2000. J. Gen. Physiol. 115:319-337). To exert its effect, Mg2+ presumably binds to a site in or near the eag voltage sensor. We have tested the hypothesis that acidic residues unique to eag family members, located in transmembrane segments S2 and S3, contribute to the Mg2+-binding site. Two eag-specific acidic residues and three acidic residues found in the S2 and S3 segments of all voltage-dependent K+ channels were individually mutated in Drosophila eag, mutant channels were expressed in Xenopus oocytes, and the effect of Mg2+ on ionic current kinetics was measured using a two electrode voltage clamp. Neutralization of eag-specific residues D278 in S2 and D327 in S3 eliminated Mg2+-sensitivity and mimicked the slowing of activation kinetics caused by Mg2+ binding to the wild-type channel. These results suggest that Mg2+ modulates activation kinetics in wild-type eag by screening the negatively charged side chains of D278 and D327. Therefore, these residues are likely to coordinate the bound ion. In contrast, neutralization of the widely conserved residues D284 in S2 and D319 in S3 preserved the fast kinetics seen in wild-type eag in the absence of Mg2+, indicating that D284 and D319 do not mediate the slowing of activation caused by Mg2+ binding. Mutations at D284 affected the eag gating pathway, shifting the voltage dependence of Mg2+-sensitive, rate limiting transitions in the hyperpolarized direction. Another widely conserved residue, D274 in S2, is not required for Mg2+ sensitivity but is in the vicinity of the binding site. We conclude that Mg2+ binds in a water-filled pocket between S2 and S3 and thereby modulates voltage-dependent gating. The identification of this site constrains the packing of transmembrane segments in the voltage sensor of K+ channels, and suggests a molecular mechanism by which extracellular cations modulate eag activation kinetics.

scholarly journals Voltage-Dependent Conformational Changes of the Voltage Sensor of KVAP Measured with LRET

2009 ◽  
Vol 96 (3) ◽  
pp. 484a
Author(s):  
Jerome J. Lacroix ◽  
Walter Sandtner ◽  
Clark Hyde ◽  
Francisco Bezanilla ◽  
Ana M. Correa
Keyword(s):  
Conformational Changes ◽  
Voltage Sensor ◽  
Voltage Dependent

scholarly journals Cadmium–cysteine coordination in the BK inner pore region and its structural and functional implications

2015 ◽  
Vol 112 (16) ◽  
pp. 5237-5242 ◽  
Author(s):  
Yu Zhou ◽  
Xiao-Ming Xia ◽  
Christopher J. Lingle
Keyword(s):  
Conformational Changes ◽  
State Dependence ◽  
Bk Channels ◽  
Bk Channel ◽  
Pore Region ◽  
Kv Channel ◽  
Kv Channels ◽  
Voltage Dependent ◽  
Channel Structures ◽  
Inner Pore

To probe structure and gating-associated conformational changes in BK-type potassium (BK) channels, we examined consequences of Cd2+ coordination with cysteines introduced at two positions in the BK inner pore. At V319C, the equivalent of valine in the conserved Kv proline-valine-proline (PVP) motif, Cd2+ forms intrasubunit coordination with a native glutamate E321, which would place the side chains of V319C and E321 much closer together than observed in voltage-dependent K+ (Kv) channel structures, requiring that the proline between V319C and E321 introduces a kink in the BK S6 inner helix sharper than that observed in Kv channel structures. At inner pore position A316C, Cd2+ binds with modest state dependence, suggesting the absence of an ion permeation gate at the cytosolic side of BK channel. These results highlight fundamental structural differences between BK and Kv channels in their inner pore region, which likely underlie differences in voltage-dependent gating between these channels.

scholarly journals Cryo-EM structure of the KvAP channel reveals a non-domain-swapped voltage sensor topology

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Xiao Tao ◽  
Roderick MacKinnon
Keyword(s):  
Ion Channels ◽  
Conformational Changes ◽  
Voltage Sensor ◽  
Structural Domains ◽  
Structural Class ◽  
Voltage Sensors ◽  
Voltage Gated ◽  
Voltage Dependent ◽  
Voltage Gated Ion Channels ◽  
Gated Ion Channels

Conductance in voltage-gated ion channels is regulated by membrane voltage through structural domains known as voltage sensors. A single structural class of voltage sensor domain exists, but two different modes of voltage sensor attachment to the pore occur in nature: domain-swapped and non-domain-swapped. Since the more thoroughly studied Kv1-7, Nav and Cav channels have domain-swapped voltage sensors, much less is known about non-domain-swapped voltage-gated ion channels. In this paper, using cryo-EM, we show that KvAP from Aeropyrum pernix has non-domain-swapped voltage sensors as well as other unusual features. The new structure, together with previous functional data, suggests that KvAP and the Shaker channel, to which KvAP is most often compared, probably undergo rather different voltage-dependent conformational changes when they open.

scholarly journals The Voltage Sensor in Voltage-Dependent Ion Channels

2000 ◽  
Vol 80 (2) ◽  
pp. 555-592 ◽  
Author(s):  
Francisco Bezanilla
Keyword(s):  
Membrane Potential ◽  
Conformational Changes ◽  
Structural Information ◽  
Noise Analysis ◽  
Voltage Sensor ◽  
Fluorescence Labeling ◽  
Gating Current ◽  
Gating Currents ◽  
Gating Charge ◽  
Voltage Dependent

In voltage-dependent Na, K, or Ca channels, the probability of opening is modified by the membrane potential. This is achieved through a voltage sensor that detects the voltage and transfers its energy to the pore to control its gate. We present here the theoretical basis of the energy coupling between the electric field and the voltage, which allows the interpretation of the gating charge that moves in one channel. Movement of the gating charge constitutes the gating current. The properties are described, along with macroscopic data and gating current noise analysis, in relation to the operation of the voltage sensor and the opening of the channel. Structural details of the voltage sensor operation were resolved initially by locating the residues that make up the voltage sensor using mutagenesis experiments and determining the number of charges per channel. The changes in conformation are then analyzed based on the differential exposure of cysteine or histidine-substituted residues. Site-directed fluorescence labeling is then analyzed as another powerful indicator of conformational changes that allows time and voltage correlation of local changes seen by the fluorophores with the global change seen by the electrophysiology of gating currents and ionic currents. Finally, we describe the novel results on lanthanide-based resonance energy transfer that show small distance changes between residues in the channel molecule. All of the electrophysiological and the structural information are finally summarized in a physical model of a voltage-dependent channel in which a change in membrane potential causes rotation of the S4 segment that changes the exposure of the basic residues from an internally connected aqueous crevice at hyperpolarized potentials to an externally connected aqueous crevice at depolarized potentials.

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