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

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.

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

The Journal of General Physiology ◽

10.1085/jgp.201611742 ◽

2017 ◽

Vol 149 (5) ◽

pp. 577-593 ◽

Cited By ~ 20

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.

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

10.1101/381319 ◽

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.

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

eLife ◽

10.7554/elife.52164 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 5

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.

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The Voltage Sensor in Voltage-Dependent Ion Channels

Physiological Reviews ◽

10.1152/physrev.2000.80.2.555 ◽

2000 ◽

Vol 80 (2) ◽

pp. 555-592 ◽

Cited By ~ 580

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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Voltage-dependent motion of the catalytic region of voltage-sensing phosphatase monitored by a fluorescent amino acid

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1604218113 ◽

2016 ◽

Vol 113 (27) ◽

pp. 7521-7526 ◽

Cited By ~ 22

Author(s):

Souhei Sakata ◽

Yuka Jinno ◽

Akira Kawanabe ◽

Yasushi Okamura

Keyword(s):

Plasma Membrane ◽

Catalytic Activity ◽

Amino Acid ◽

Conformational Changes ◽

Unnatural Amino Acid ◽

Voltage Sensor ◽

Voltage Sensing ◽

Cytoplasmic Region ◽

Voltage Dependent ◽

Voltage Gated Ion Channels

The cytoplasmic region of voltage-sensing phosphatase (VSP) derives the voltage dependence of its catalytic activity from coupling to a voltage sensor homologous to that of voltage-gated ion channels. To assess the conformational changes in the cytoplasmic region upon activation of the voltage sensor, we genetically incorporated a fluorescent unnatural amino acid, 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (Anap), into the catalytic region of Ciona intestinalis VSP (Ci-VSP). Measurements of Anap fluorescence under voltage clamp in Xenopus oocytes revealed that the catalytic region assumes distinct conformations dependent on the degree of voltage-sensor activation. FRET analysis showed that the catalytic region remains situated beneath the plasma membrane, irrespective of the voltage level. Moreover, Anap fluorescence from a membrane-facing loop in the C2 domain showed a pattern reflecting substrate turnover. These results indicate that the voltage sensor regulates Ci-VSP catalytic activity by causing conformational changes in the entire catalytic region, without changing their distance from the plasma membrane.

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

The Journal of General Physiology ◽

10.1085/jgp.201010413 ◽

2010 ◽

Vol 136 (1) ◽

pp. 83-99 ◽

Cited By ~ 9

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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Fast and Slow Voltage Sensor Movements in HERG Potassium Channels

The Journal of General Physiology ◽

10.1085/jgp.20028534 ◽

2002 ◽

Vol 119 (3) ◽

pp. 275-293 ◽

Cited By ~ 76

Author(s):

Paula L. Smith ◽

Gary Yellen

Keyword(s):

Conformational Changes ◽

Voltage Dependence ◽

Voltage Sensor ◽

Inward Rectification ◽

Dependent Inactivation ◽

Activation Gating ◽

Voltage Dependent ◽

Probe Voltage ◽

Inactivation Gating ◽

Rapid Kinetics

HERG encodes an inwardly-rectifying potassium channel that plays an important role in repolarization of the cardiac action potential. Inward rectification of HERG channels results from rapid and voltage-dependent inactivation gating, combined with very slow activation gating. We asked whether the voltage sensor is implicated in the unusual properties of HERG gating: does the voltage sensor move slowly to account for slow activation and deactivation, or could the voltage sensor move rapidly to account for the rapid kinetics and intrinsic voltage dependence of inactivation? To probe voltage sensor movement, we used a fluorescence technique to examine conformational changes near the positively charged S4 region. Fluorescent probes attached to three different residues on the NH2-terminal end of the S4 region (E518C, E519C, and L520C) reported both fast and slow voltage-dependent changes in fluorescence. The slow changes in fluorescence correlated strongly with activation gating, suggesting that the slow activation gating of HERG results from slow voltage sensor movement. The fast changes in fluorescence showed voltage dependence and kinetics similar to inactivation gating, though these fluorescence signals were not affected by external tetraethylammonium blockade or mutations that alter inactivation. A working model with two types of voltage sensor movement is proposed as a framework for understanding HERG channel gating and the fluorescence signals.

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Voltage-dependent dynamics of the BK channel cytosolic gating ring are coupled to the membrane-embedded voltage sensor

eLife ◽

10.7554/elife.40664 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 8

Author(s):

Pablo Miranda ◽

Miguel Holmgren ◽

Teresa Giraldez

Keyword(s):

Conformational Changes ◽

Bk Channels ◽

Bk Channel ◽

Voltage Sensor ◽

Cation Binding ◽

Voltage Sensitivity ◽

Open Probability ◽

Calcium Dependent ◽

Transmembrane Voltage ◽

Voltage Dependent

In humans, large conductance voltage- and calcium-dependent potassium (BK) channels are regulated allosterically by transmembrane voltage and intracellular Ca2+. Divalent cation binding sites reside within the gating ring formed by two Regulator of Conductance of Potassium (RCK) domains per subunit. Using patch-clamp fluorometry, we show that Ca2+ binding to the RCK1 domain triggers gating ring rearrangements that depend on transmembrane voltage. Because the gating ring is outside the electric field, this voltage sensitivity must originate from coupling to the voltage-dependent channel opening, the voltage sensor or both. Here we demonstrate that alterations of the voltage sensor, either by mutagenesis or regulation by auxiliary subunits, are paralleled by changes in the voltage dependence of the gating ring movements, whereas modifications of the relative open probability are not. These results strongly suggest that conformational changes of RCK1 domains are specifically coupled to the voltage sensor function during allosteric modulation of BK channels.

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Voltage-Dependent Structural Interactions in the Shaker K+ Channel

The Journal of General Physiology ◽

10.1085/jgp.115.2.123 ◽

2000 ◽

Vol 115 (2) ◽

pp. 123-138 ◽

Cited By ~ 112

Author(s):

Seema K. Tiwari-Woodruff ◽

Meng-chin A. Lin ◽

Christine T. Schulteis ◽

Diane M. Papazian

Keyword(s):

Conformational Changes ◽

Voltage Sensor ◽

K Channel ◽

Charge Reversal ◽

K Channels ◽

Closed Conformation ◽

Preliminary Model ◽

Structural Interactions ◽

Voltage Dependent ◽

Double Charge

Using a strategy related to intragenic suppression, we previously obtained evidence for structural interactions in the voltage sensor of Shaker K+ channels between residues E283 in S2 and R368 and R371 in S4 (Tiwari-Woodruff, S.K., C.T. Schulteis, A.F. Mock, and D.M. Papazian. 1997. Biophys. J. 72:1489–1500). Because R368 and R371 are involved in the conformational changes that accompany voltage-dependent activation, we tested the hypothesis that these S4 residues interact with E283 in S2 in a subset of the conformational states that make up the activation pathway in Shaker channels. First, the location of residue 283 at hyperpolarized and depolarized potentials was inferred by substituting a cysteine at that position and determining its reactivity with hydrophilic, sulfhydryl-specific probes. The results indicate that position 283 reacts with extracellularly applied sulfhydryl reagents with similar rates at both hyperpolarized and depolarized potentials. We conclude that E283 is located near the extracellular surface of the protein in both resting and activated conformations. Second, we studied the functional phenotypes of double charge reversal mutations between positions 283 and 368 and between 283 and 371 to gain insight into the conformations in which these positions approach each other most closely. We found that combining charge reversal mutations at positions 283 and 371 stabilized an activated conformation of the channel, and dramatically slowed transitions into and out of this state. In contrast, charge reversal mutations at positions 283 and 368 stabilized a closed conformation, which by virtue of the inferred position of 368 corresponds to a partially activated (intermediate) closed conformation. From these results, we propose a preliminary model for the rearrangement of structural interactions of the voltage sensor during activation of Shaker K+ channels.

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