Calmodulin acts as a state-dependent switch to control a cardiac potassium channel opening

Calmodulin (CaM) and phosphatidylinositol 4,5-bisphosphate (PIP2) are potent regulators of the voltage-gated potassium channel KCNQ1 (KV7.1), which conducts the cardiac IKs current. Although cryo–electron microscopy structures revealed intricate interactions between the KCNQ1 voltage-sensing domain (VSD), CaM, and PIP2, the functional consequences of these interactions remain unknown. Here, we show that CaM-VSD interactions act as a state-dependent switch to control KCNQ1 pore opening. Combined electrophysiology and molecular dynamics network analysis suggest that VSD transition into the fully activated state allows PIP2 to compete with CaM for binding to VSD. This leads to conformational changes that alter VSD-pore coupling to stabilize open states. We identify a motif in the KCNQ1 cytosolic domain, which works downstream of CaM-VSD interactions to facilitate the conformational change. Our findings suggest a gating mechanism that integrates PIP2 and CaM in KCNQ1 voltage-dependent activation, yielding insights into how KCNQ1 gains the phenotypes critical for its physiological function.

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Calmodulin acts as a state-dependent switch to control a cardiac potassium channel opening

10.1101/2020.07.04.187161 ◽

2020 ◽

Author(s):

Po Wei Kang ◽

Annie M. Westerlund ◽

Jingyi Shi ◽

Kelli McFarland White ◽

Alex K. Dou ◽

...

Keyword(s):

Potassium Channel ◽

Conformational Change ◽

State Dependent ◽

Gating Mechanism ◽

Channel Opening ◽

Activated State ◽

Voltage Dependent ◽

Functional Consequences ◽

Pore Opening ◽

Voltage Sensing Domain

AbstractCalmodulin (CaM) and PIP2 are potent regulators of the voltage-gated potassium channel KCNQ1 (KV7.1), which conducts the IKs current important for repolarization of cardiac action potentials. Although cryo-EM structures revealed intricate interactions between the KCNQ1 voltage-sensing domain (VSD), CaM, and PIP2, the functional consequences of these interactions remain unknown. Here, we show that CaM-VSD interactions act as a state-dependent switch to control KCNQ1 pore opening. Combined electrophysiology and molecular dynamics network analysis suggest that VSD transition into the fully-activated state allows PIP2 to compete with CaM for binding to VSD, leading to the conformational change that alters the VSD-pore coupling. We identify a motif in the KCNQ1 cytosolic domain which works downstream of CaM-VSD interactions to facilitate the conformational change. Our findings suggest a gating mechanism that integrates PIP2 and CaM in KCNQ1 voltage-dependent activation, yielding insights into how KCNQ1 gains the phenotypes critical for its function in the heart.

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Two-stage electro-mechanical coupling of a KV channel in voltage-dependent activation

10.1101/798207 ◽

2019 ◽

Author(s):

Panpan Hou ◽

Po Wei Kang ◽

Audrey Deyawe Kongmeneck ◽

Nien-Du Yang ◽

Yongfeng Liu ◽

...

Keyword(s):

Coupling Mechanism ◽

Two Stage ◽

Mechanical Coupling ◽

Kv Channel ◽

Gating Mechanism ◽

C Terminus ◽

Activated State ◽

Voltage Dependent ◽

Open States ◽

Voltage Sensing Domain

AbstractIn voltage-gated potassium (KV) channels, the voltage-sensing domain (VSD) undergoes sequential activation from the resting state to the intermediate state and activated state to trigger pore opening via electro-mechanical (E-M) coupling. However, the spatial and temporal details underlying E-M coupling remain elusive. Here, we leverage KV7.1’s unique two open states associated with the VSD adopting the intermediate and activated conformations to report a two-stage E-M coupling mechanism in voltage-dependent gating of KV7.1 as triggered by VSD activations to the intermediate and then activated state. When the S4 segment transitions to the intermediate state, the hand-like C-terminus of the VSD-pore linker (S4-S5L) interacts with the pore in the same subunit. When S4 then proceeds on to the fully-activated state, the elbow-like hinge between S4 and S4-S5L engages with the pore to activate conductance. This two-stage “hand-and-elbow” gating mechanism elucidates distinct tissue-specific modulations, pharmacology, and disease pathogenesis of KV7.1, and likely applies to numerous KV channels.

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Status of the Intracellular Gate in the Activated-not-open State of Shaker K+ Channels

The Journal of General Physiology ◽

10.1085/jgp.200509385 ◽

2005 ◽

Vol 126 (5) ◽

pp. 419-428 ◽

Cited By ~ 47

Author(s):

Donato del Camino ◽

Max Kanevsky ◽

Gary Yellen

Keyword(s):

Conformational Changes ◽

Charge Movement ◽

Ion Flow ◽

K Channels ◽

Closed State ◽

Channel Opening ◽

Activated State ◽

Form Part ◽

Voltage Dependent ◽

Flow Through

Voltage-dependent K+ channels like Shaker use an intracellular gate to control ion flow through the pore. When the membrane voltage becomes more positive, these channels traverse a series of closed conformations before the final opening transition. Does the intracellular gate undergo conformational changes before channel opening? To answer this question we introduced cysteines into the intracellular end of the pore and studied their chemical modification in conditions favoring each of three distinct states, the open state, the resting closed state, and the activated-not-open state (the closed state adjacent to the open state). We used two independent ways to isolate the channels in the activated-not-open state. First, we used mutations in S4 (ILT; Smith-Maxwell, C.J., J.L. Ledwell, and R.W. Aldrich. 1998. J. Gen. Physiol. 111:421–439; Ledwell, J.L., and R.W. Aldrich. 1999. J. Gen. Physiol. 113:389–414) that separate the final opening step from earlier charge-movement steps. Second, we used the open channel blocker 4-aminopyridine (4-AP), which has been proposed to promote closure of the intracellular gate and thus specifically to stabilize the activated-not-open state of the channels. Supporting this proposed mechanism, we found that 4-AP enters channels only after opening, remaining trapped in closed channels, and that in the open state it competes with tetraethylammonium for binding. Using these tools, we found that in the activated-not-open state, a cysteine located at a position considered to form part of the gate (Shaker 478) showed higher reactivity than in either the open or the resting closed states. Additionally, we have found that in this activated state the intracellular gate continued to prevent access to the pore by molecules as small as Cd2+ ions. Our results suggest that the intracellular opening to the pore undergoes some rearrangements in the transition from the resting closed state to the activated-not-open state, but throughout this process the intracellular gate remains an effective barrier to the movement of potassium ions through the pore.

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Hydrophobic gasket mutation produces gating pore currents in closed human voltage-gated proton channels

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1905462116 ◽

2019 ◽

Vol 116 (38) ◽

pp. 18951-18961 ◽

Cited By ~ 6

Author(s):

Richard Banh ◽

Vladimir V. Cherny ◽

Deri Morgan ◽

Boris Musset ◽

Sarah Thomas ◽

...

Keyword(s):

Proton Channel ◽

Voltage Gated ◽

Closed State ◽

Channel Opening ◽

Proton Current ◽

Voltage Dependent ◽

Current Flows ◽

Voltage Gated Ion Channels ◽

Dynamics Simulations ◽

Voltage Sensing Domain

The hydrophobic gasket (HG), a ring of hydrophobic amino acids in the voltage-sensing domain of most voltage-gated ion channels, forms a constriction between internal and external aqueous vestibules. Cationic Arg or Lys side chains lining the S4 helix move through this “gating pore” when the channel opens. S4 movement may occur during gating of the human voltage-gated proton channel, hHV1, but proton current flows through the same pore in open channels. Here, we replaced putative HG residues with less hydrophobic residues or acidic Asp. Substitution of individuals, pairs, or all 3 HG positions did not impair proton selectivity. Evidently, the HG does not act as a secondary selectivity filter. However, 2 unexpected functions of the HG in HV1 were discovered. Mutating HG residues independently accelerated channel opening and compromised the closed state. Mutants exhibited open–closed gating, but strikingly, at negative voltages where “normal” gating produces a nonconducting closed state, the channel leaked protons. Closed-channel proton current was smaller than open-channel current and was inhibited by 10 μM Zn2+. Extreme hyperpolarization produced a deeper closed state through a weakly voltage-dependent transition. We functionally identify the HG as Val109, Phe150, Val177, and Val178, which play a critical and exclusive role in preventing H+ influx through closed channels. Molecular dynamics simulations revealed enhanced mobility of Arg208 in mutants exhibiting H+ leak. Mutation of HG residues produces gating pore currents reminiscent of several channelopathies.

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Conformational Changes in the Pore of CLC-0

The Journal of General Physiology ◽

10.1085/jgp.200308834 ◽

2003 ◽

Vol 122 (3) ◽

pp. 277-294 ◽

Cited By ~ 68

Author(s):

Alessio Accardi ◽

Michael Pusch

Keyword(s):

Conformational Changes ◽

Single Channel ◽

General Structure ◽

Point Mutations ◽

Clofibric Acid ◽

Selectivity Filter ◽

Side Chain ◽

Closed State ◽

State Dependent ◽

Channel Opening

The Torpedo Cl− channel, CLC-0, is inhibited by clofibric acid derivatives from the intracellular side. We used the slow gate-deficient mutant CLC-0C212S to investigate the mechanism of block by the clofibric acid–derivative p-chlorophenoxy-acetic acid (CPA). CPA blocks open channels with low affinity (KDO= 45 mM at 0 mV) and shows fast dissociation (koff = 490 s−1 at −140 mV). In contrast, the blocker binds to closed channels with higher affinity and with much slower kinetics. This state-dependent block coupled with the voltage dependence of the gating transitions results in a highly voltage-dependent inhibition of macroscopic currents (KD ∼1 mM at −140 mV; KD ∼65 mM at 60 mV). The large difference in CPA affinity of the open and closed state suggests that channel opening involves more than just a local conformational rearrangement. On the other hand, in a recent work (Dutzler, R., E.B. Campbell, and R. MacKinnon. 2003. Science. 300:108–112) it was proposed that the conformational change underlying channel opening is limited to a movement of a single side chain. A prediction of this latter model is that mutations that influence CPA binding to the channel should affect the affinities for an open and closed channel in a similar manner since the general structure of the pore remains largely unchanged. To test this hypothesis we introduced point mutations in four residues (S123, T471, Y512, and K519) that lie close to the intracellular pore mouth or to the putative selectivity filter. Mutation T471S alters CPA binding exclusively to closed channels. Pronounced effects on the open channel block are observed in three other mutants, S123T, Y512A, and K519Q. Together, these results collectively suggest that the structure of the CPA binding site is different in the open and closed state. Finally, replacement of Tyr 512, a residue directly coordinating the central Cl− ion in the crystal structure, with Phe or Ala has very little effect on single channel conductance and selectivity. These observations suggest that channel opening in CLC-0 consists in more than a movement of a side chain and that other parts of the channel and of the selectivity filter are probably involved.

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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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Conformational changes in a pore-forming region underlie voltage-dependent “loop gating” of an unapposed connexin hemichannel

The Journal of General Physiology ◽

10.1085/jgp.200910207 ◽

2009 ◽

Vol 133 (6) ◽

pp. 555-570 ◽

Cited By ~ 52

Author(s):

Qingxiu Tang ◽

Terry L. Dowd ◽

Vytas K. Verselis ◽

Thaddeus A. Bargiello

Keyword(s):

Conformational Changes ◽

Disulfide Bonds ◽

Molecular Mechanisms ◽

Extracellular Loop ◽

Connexin Gene ◽

Gating Mechanism ◽

Biochemical Assays ◽

Voltage Dependent ◽

Tm Domain ◽

Connexin Hemichannel

The structure of the pore is critical to understanding the molecular mechanisms underlying selective permeation and voltage-dependent gating of channels formed by the connexin gene family. Here, we describe a portion of the pore structure of unapposed hemichannels formed by a Cx32 chimera, Cx32*Cx43E1, in which the first extracellular loop (E1) of Cx32 is replaced with the E1 of Cx43. Cysteine substitutions of two residues, V38 and G45, located in the vicinity of the border of the first transmembrane (TM) domain (TM1) and E1 are shown to react with the thiol modification reagent, MTSEA–biotin-X, when the channel resides in the open state. Cysteine substitutions of flanking residues A40 and A43 do not react with MTSEA–biotin-X when the channel resides in the open state, but they react with dibromobimane when the unapposed hemichannels are closed by the voltage-dependent “loop-gating” mechanism. Cysteine substitutions of residues V37 and A39 do not appear to be modified in either state. Furthermore, we demonstrate that A43C channels form a high affinity Cd2+ site that locks the channel in the loop-gated closed state. Biochemical assays demonstrate that A43C can also form disulfide bonds when oocytes are cultured under conditions that favor channel closure. A40C channels are also sensitive to micromolar Cd2+ concentrations when closed by loop gating, but with substantially lower affinity than A43C. We propose that the voltage-dependent loop-gating mechanism for Cx32*Cx43E1 unapposed hemichannels involves a conformational change in the TM1/E1 region that involves a rotation of TM1 and an inward tilt of either each of the six connexin subunits or TM1 domains.

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Conformational heterogeneity in closed and open states of the KcsA potassium channel in lipid bicelles

The Journal of General Physiology ◽

10.1085/jgp.201611602 ◽

2016 ◽

Vol 148 (2) ◽

pp. 119-132 ◽

Cited By ~ 15

Author(s):

Dorothy M. Kim ◽

Igor Dikiy ◽

Vikrant Upadhyay ◽

David J. Posson ◽

David Eliezer ◽

...

Keyword(s):

Ion Channel ◽

Potassium Channel ◽

Conformational Changes ◽

Ph Dependence ◽

Channel Gating ◽

Conformational Heterogeneity ◽

Ion Channel Gating ◽

Potassium Channel Kcsa ◽

Functional States ◽

Open States

The process of ion channel gating—opening and closing—involves local and global structural changes in the channel in response to external stimuli. Conformational changes depend on the energetic landscape that underlies the transition between closed and open states, which plays a key role in ion channel gating. For the prokaryotic, pH-gated potassium channel KcsA, closed and open states have been extensively studied using structural and functional methods, but the dynamics within each of these functional states as well as the transition between them is not as well understood. In this study, we used solution nuclear magnetic resonance (NMR) spectroscopy to investigate the conformational transitions within specific functional states of KcsA. We incorporated KcsA channels into lipid bicelles and stabilized them into a closed state by using either phosphatidylcholine lipids, known to favor the closed channel, or mutations designed to trap the channel shut by disulfide cross-linking. A distinct state, consistent with an open channel, was uncovered by the addition of cardiolipin lipids. Using selective amino acid labeling at locations within the channel that are known to move during gating, we observed at least two different slowly interconverting conformational states for both closed and open channels. The pH dependence of these conformations and the predictable disruptions to this dependence observed in mutant channels with altered pH sensing highlight the importance of conformational heterogeneity for KcsA gating.

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Structural basis for activation of voltage sensor domains in an ion channel TPC1

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1805651115 ◽

2018 ◽

Vol 115 (39) ◽

pp. E9095-E9104 ◽

Cited By ~ 14

Author(s):

Alexander F. Kintzer ◽

Evan M. Green ◽

Pawel K. Dominik ◽

Michael Bridges ◽

Jean-Paul Armache ◽

...

Keyword(s):

Ion Channel ◽

Conformational Changes ◽

Electrical Potential ◽

Structural Basis ◽

Ion Conductance ◽

Activated State ◽

Voltage Dependent ◽

Internal Side ◽

Transmembrane Electrical Potential ◽

Gating Charges

Voltage-sensing domains (VSDs) couple changes in transmembrane electrical potential to conformational changes that regulate ion conductance through a central channel. Positively charged amino acids inside each sensor cooperatively respond to changes in voltage. Our previous structure of a TPC1 channel captured an example of a resting-state VSD in an intact ion channel. To generate an activated-state VSD in the same channel we removed the luminal inhibitory Ca2+-binding site (Cai2+), which shifts voltage-dependent opening to more negative voltage and activation at 0 mV. Cryo-EM reveals two coexisting structures of the VSD, an intermediate state 1 that partially closes access to the cytoplasmic side but remains occluded on the luminal side and an intermediate activated state 2 in which the cytoplasmic solvent access to the gating charges closes, while luminal access partially opens. Activation can be thought of as moving a hydrophobic insulating region of the VSD from the external side to an alternate grouping on the internal side. This effectively moves the gating charges from the inside potential to that of the outside. Activation also requires binding of Ca2+ to a cytoplasmic site (Caa2+). An X-ray structure with Caa2+ removed and a near-atomic resolution cryo-EM structure with Cai2+ removed define how dramatic conformational changes in the cytoplasmic domains may communicate with the VSD during activation. Together four structures provide a basis for understanding the voltage-dependent transition from resting to activated state, the tuning of VSD by thermodynamic stability, and this channel’s requirement of cytoplasmic Ca2+ ions for activation.

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