Biophysical analysis of an HCN1 epilepsy variant suggests a critical role for S5 helix Met-305 in voltage sensor to pore domain coupling

Slow deactivation of Kv11.1 channels is critical for its function in the heart. The S4-S5 linker, which joins the voltage sensor and pore domains, plays a critical role in this slow deactivation gating. Here, we use NMR spectroscopy to identify the membrane-bound surface of the S4S5 linker, and we show that two highly conserved tyrosine residues within the KCNH subfamily of channels are membrane-associated. Site-directed mutagenesis and electrophysiological analysis indicates that Tyr-542 interacts with both the pore domain and voltage sensor residues to stabilize activated conformations of the channel, whereas Tyr-545 contributes to the slow kinetics of deactivation by primarily stabilizing the transition state between the activated and closed states. Thus, the two tyrosine residues in the Kv11.1 S4S5 linker play critical but distinct roles in the slow deactivation phenotype, which is a hallmark of Kv11.1 channels.

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Key Residues in the Interface between Voltage Sensor and Pore Domain in Shaker Potassium Channels

Biophysical Journal ◽

10.1016/j.bpj.2017.11.2619 ◽

2018 ◽

Vol 114 (3) ◽

pp. 476a

Author(s):

João L. Carvalho-de-Souza ◽

Francisco Bezanilla

Keyword(s):

Potassium Channels ◽

Voltage Sensor ◽

Key Residues ◽

Pore Domain ◽

Shaker Potassium Channels

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Upgraded molecular models of the human KCNQ1 potassium channel

10.1101/648634 ◽

2019 ◽

Author(s):

Georg Kuenze ◽

Amanda M. Duran ◽

Hope Woods ◽

Kathryn R. Brewer ◽

Eli Fritz McDonald ◽

...

Keyword(s):

Voltage Sensor ◽

Cardiac Action Potential ◽

Molecular Models ◽

Loss Of Function ◽

Voltage Sensing ◽

Common Cause ◽

Cardiac Action ◽

Electrical Impulses ◽

Voltage Sensing Domain ◽

Pore Domain

AbstractThe voltage-gated potassium channel KCNQ1 (KV7.1) assembles with the KCNE1 accessory protein to generate the slow delayed rectifier current, IKS, which is critical for membrane repolarization as part of the cardiac action potential. Loss-of-function (LOF) mutations in KCNQ1 are the most common cause of congenital long QT syndrome (LQTS), type 1 LQTS, an inherited genetic predisposition to cardiac arrhythmia and sudden cardiac death. A detailed structural understanding of KCNQ1 is needed to elucidate the molecular basis for KCNQ1 LOF in disease and to enable structure-guided design of new anti-arrhythmic drugs. In this work, advanced structural models of human KCNQ1 in the resting/closed and activated/open states were developed by Rosetta homology modeling guided by newly available experimentally-based templates: X. leavis KCNQ1 and resting voltage sensor structures. Using molecular dynamics (MD) simulations, the models’ capability to describe experimentally established channel properties including state-dependent voltage sensor gating charge interactions and pore conformations, PIP2 binding sites, and voltage sensor – pore domain interactions were validated. Rosetta energy calculations were applied to assess the models’ utility in interpreting mutation-evoked KCNQ1 dysfunction by predicting the change in protein thermodynamic stability for 50 characterized KCNQ1 variants with mutations located in the voltage-sensing domain. Energetic destabilization was successfully predicted for folding-defective KCNQ1 LOF mutants whereas wild type-like mutants had no significant energetic frustrations, which supports growing evidence that mutation-induced protein destabilization is an especially common cause of KCNQ1 dysfunction. The new KCNQ1 Rosetta models provide helpful tools in the study of the structural mechanisms of KCNQ1 function and can be used to generate structure-based hypotheses to explain KCNQ1 dysfunction.Author SummaryCardiac rhythm is maintained by synchronized electrical impulses conducted throughout the heart. The potassium ion channel KCNQ1 is important for the repolarization phase of the cardiac action potential that underlies these electrical impulses. Heritable mutations in KCNQ1 can lead to channel loss-of-function (LOF) and predisposition to a life-threatening cardiac arrhythmia. Knowledge of the three-dimensional structure of KCNQ1 is important to understand how mutations lead to LOF and to support structurally-guided design of new anti-arrhythmic drugs. In this work, we present the development and validation of molecular models of human KCNQ1 inferred by homology from the structure of frog KCNQ1. Models were developed for the open channel state in which potassium ions can pass through the channel and the closed state in which the channel is not conductive. Using molecular dynamics simulations, interactions in the voltage-sensing and pore domain of KCNQ1 and with the membrane lipid PIP2 were analyzed. Energy calculations for KCNQ1 mutations in the voltage-sensing domain reveled that most of the mutations that lead to LOF cause energetic destabilization of the KCNQ1 protein. The results support both the utility of the new models and growing evidence that mutation-induced protein destabilization is a common cause of KCNQ1 dysfunction.

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Residues Connecting Voltage Sensor Domain to Pore Domain in Shaker K+ Channel by Noncanonical Coupling Mechanism

Biophysical Journal ◽

10.1016/j.bpj.2019.11.1860 ◽

2020 ◽

Vol 118 (3) ◽

pp. 333a

Author(s):

Carlos Alberto Z. Bassetto Jr ◽

Joao L. Carvalho-de-Souza ◽

Francisco Bezanilla

Keyword(s):

Voltage Sensor ◽

K Channel ◽

Coupling Mechanism ◽

Pore Domain ◽

Voltage Sensor Domain

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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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Voltage-insensitive Gating after Charge-neutralizing Mutations in the S4 Segment of Shaker Channels

The Journal of General Physiology ◽

10.1085/jgp.113.1.139 ◽

1999 ◽

Vol 113 (1) ◽

pp. 139-151 ◽

Cited By ~ 25

Author(s):

Hongxia Bao ◽

Atiya Hakeem ◽

Mark Henteleff ◽

John G. Starkus ◽

Martin D. Rayner

Keyword(s):

Single Channel ◽

Voltage Sensor ◽

Control Voltage ◽

Shaker Channels ◽

Open Time ◽

Channel Opening ◽

Electrostatic Coupling ◽

And Control ◽

Pore Domain ◽

S4 Segment

Shaker channel mutants, in which the first (R362), second (R365), and fourth (R371) basic residues in the S4 segment have been neutralized, are found to pass potassium currents with voltage-insensitive kinetics when expressed in Xenopus oocytes. Single channel recordings clarify that these channels continue to open and close from −160 to +80 mV with a constant opening probability (Po). Although Po is low (∼0.15) in these mutants, mean open time is voltage independent and similar to that of control Shaker channels. Additionally, these mutant channels retain characteristic Shaker channel selectivity, sensitivity to block by 4-aminopyridine, and are partially blocked by external Ca2+ ions at very negative potentials. Furthermore, mean open time is approximately doubled, in both mutant channels and control Shaker channels, when Rb+ is substituted for K+ as the permeant ion species. Such strong similarities between mutant channels and control Shaker channels suggests that the pore region has not been substantially altered by the S4 charge neutralizations. We conclude that single channel kinetics in these mutants may indicate how Shaker channels would behave in the absence of voltage sensor input. Thus, mean open times appear primarily determined by voltage-insensitive transitions close to the open state rather than by voltage sensor movement, even in control, voltage-sensitive Shaker channels. By contrast, the low and voltage-insensitive Po seen in these mutant channels suggests that important determinants of normal channel opening derive from electrostatic coupling between S4 charges and the pore domain.

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Mode shift of the voltage sensors in Shaker K+ channels is caused by energetic coupling to the pore domain

The Journal of General Physiology ◽

10.1085/jgp.201010573 ◽

2011 ◽

Vol 137 (5) ◽

pp. 455-472 ◽

Cited By ~ 48

Author(s):

Georges A. Haddad ◽

Rikard Blunck

Keyword(s):

Conformational Change ◽

Mechanical Load ◽

Voltage Sensor ◽

Charge Movement ◽

Gating Currents ◽

Mode Shift ◽

Voltage Sensors ◽

Pore Opening ◽

Voltage Gated Ion Channels ◽

Pore Domain

The voltage sensors of voltage-gated ion channels undergo a conformational change upon depolarization of the membrane that leads to pore opening. This conformational change can be measured as gating currents and is thought to be transferred to the pore domain via an annealing of the covalent link between voltage sensor and pore (S4-S5 linker) and the C terminus of the pore domain (S6). Upon prolonged depolarizations, the voltage dependence of the charge movement shifts to more hyperpolarized potentials. This mode shift had been linked to C-type inactivation but has recently been suggested to be caused by a relaxation of the voltage sensor itself. In this study, we identified two ShakerIR mutations in the S4-S5 linker (I384N) and S6 (F484G) that, when mutated, completely uncouple voltage sensor movement from pore opening. Using these mutants, we show that the pore transfers energy onto the voltage sensor and that uncoupling the pore from the voltage sensor leads the voltage sensors to be activated at more negative potentials. This uncoupling also eliminates the mode shift occurring during prolonged depolarizations, indicating that the pore influences entry into the mode shift. Using voltage-clamp fluorometry, we identified that the slow conformational change of the S4 previously correlated with the mode shift disappears when uncoupling the pore. The effects can be explained by a mechanical load that is imposed upon the voltage sensors by the pore domain and allosterically modulates its conformation. Mode shift is caused by the stabilization of the open state but leads to a conformational change in the voltage sensor.

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S1–S3 counter charges in the voltage sensor module of a mammalian sodium channel regulate fast inactivation

The Journal of General Physiology ◽

10.1085/jgp.201210935 ◽

2013 ◽

Vol 141 (5) ◽

pp. 601-618 ◽

Cited By ~ 14

Author(s):

James R. Groome ◽

Vern Winston

Keyword(s):

Sodium Channel ◽

Sodium Channels ◽

Negative Charge ◽

Critical Role ◽

Voltage Sensor ◽

Mammalian Skeletal Muscle ◽

Fast Inactivation ◽

Domain Specific ◽

Sensor Module ◽

Charge Cluster

The movement of positively charged S4 segments through the electric field drives the voltage-dependent gating of ion channels. Studies of prokaryotic sodium channels provide a mechanistic view of activation facilitated by electrostatic interactions of negatively charged residues in S1 and S2 segments, with positive counterparts in the S4 segment. In mammalian sodium channels, S4 segments promote domain-specific functions that include activation and several forms of inactivation. We tested the idea that S1–S3 countercharges regulate eukaryotic sodium channel functions, including fast inactivation. Using structural data provided by bacterial channels, we constructed homology models of the S1–S4 voltage sensor module (VSM) for each domain of the mammalian skeletal muscle sodium channel hNaV1.4. These show that side chains of putative countercharges in hNaV1.4 are oriented toward the positive charge complement of S4. We used mutagenesis to define the roles of conserved residues in the extracellular negative charge cluster (ENC), hydrophobic charge region (HCR), and intracellular negative charge cluster (INC). Activation was inhibited with charge-reversing VSM mutations in domains I–III. Charge reversal of ENC residues in domains III (E1051R, D1069K) and IV (E1373K, N1389K) destabilized fast inactivation by decreasing its probability, slowing entry, and accelerating recovery. Several INC mutations increased inactivation from closed states and slowed recovery. Our results extend the functional characterization of VSM countercharges to fast inactivation, and support the premise that these residues play a critical role in domain-specific gating transitions for a mammalian sodium channel.

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