Ion-pair interactions between voltage-sensing domain IV and pore domain I regulate CaV1.1 channel gating

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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Molecular Coupling of S4 to a K+ Channel's Slow Inactivation Gate

The Journal of General Physiology ◽

10.1085/jgp.116.5.623 ◽

2000 ◽

Vol 116 (5) ◽

pp. 623-636 ◽

Cited By ~ 84

Author(s):

Eli Loots ◽

Ehud Y. Isacoff

Keyword(s):

Transmembrane Domain ◽

Direct Interaction ◽

K Channel ◽

Slow Inactivation ◽

Domain Interaction ◽

Voltage Sensing ◽

Molecular Gates ◽

Voltage Sensing Domain ◽

Pore Domain ◽

Bond Disruption

The mechanism by which physiological signals regulate the conformation of molecular gates that open and close ion channels is poorly understood. Voltage clamp fluorometry was used to ask how the voltage-sensing S4 transmembrane domain is coupled to the slow inactivation gate in the pore domain of the Shaker K+ channel. Fluorophores attached at several sites in S4 indicate that the voltage-sensing rearrangements are followed by an additional inactivation motion. Fluorophores attached at the perimeter of the pore domain indicate that the inactivation rearrangement projects from the selectivity filter out to the interface with the voltage-sensing domain. Some of the pore domain sites also sense activation, and this appears to be due to a direct interaction with S4 based on the finding that S4 comes into close enough proximity to the pore domain for a pore mutation to alter the nanoenvironment of an S4-attached fluorophore. We propose that activation produces an S4–pore domain interaction that disrupts a bond between the S4 contact site on the pore domain and the outer end of S6. Our results indicate that this bond holds the slow inactivation gate open and, therefore, we propose that this S4-induced bond disruption triggers inactivation.

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The isolated voltage sensing domain of the Shaker potassium channel forms a voltage-gated cation channel

eLife ◽

10.7554/elife.18130 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 24

Author(s):

Juan Zhao ◽

Rikard Blunck

Keyword(s):

Transmembrane Helix ◽

Voltage Sensing ◽

Shaker Potassium Channel ◽

Voltage Gated ◽

Kv Channel ◽

C Terminus ◽

Ion Conducting ◽

Voltage Gated Ion Channels ◽

Voltage Sensing Domain ◽

Pore Domain

Domains in macromolecular complexes are often considered structurally and functionally conserved while energetically coupled to each other. In the modular voltage-gated ion channels the central ion-conducting pore is surrounded by four voltage sensing domains (VSDs). Here, the energetic coupling is mediated by interactions between the S4-S5 linker, covalently linking the domains, and the proximal C-terminus. In order to characterize the intrinsic gating of the voltage sensing domain in the absence of the pore domain, the Shaker Kv channel was truncated after the fourth transmembrane helix S4 (Shaker-iVSD). Shaker-iVSD showed significantly altered gating kinetics and formed a cation-selective ion channel with a strong preference for protons. Ion conduction in Shaker-iVSD developed despite identical primary sequence, indicating an allosteric influence of the pore domain. Shaker-iVSD also displays pronounced 'relaxation'. Closing of the pore correlates with entry into relaxation suggesting that the two processes are energetically related.

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Dual role of Ca(V)1.1 voltage-sensing domain I in determining kinetics and voltage dependence of calcium channel activation

Intrinsic Activity ◽

10.25006/ia.7.s1-a3.11 ◽

2019 ◽

Vol 7 (Suppl. 1) ◽

pp. A3.11

Author(s):

Yousra El Ghaleb

Keyword(s):

Calcium Channel ◽

Voltage Dependence ◽

Dual Role ◽

Voltage Sensing ◽

Channel Activation ◽

Domain I ◽

Voltage Sensing Domain

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Abstract 17379: Atrial Fibrillation Mutations in β3 Subunits Enhance DIII Voltage Sensing Domain Coupling to Channel Activation Which Inhibits Lidocaine Blockade

Circulation ◽

10.1161/circ.132.suppl_3.17379 ◽

2015 ◽

Vol 132 (suppl_3) ◽

Author(s):

Wandi Zhu ◽

Eric J Hsu ◽

Bicong Li ◽

Angela R Schubert ◽

Zoltan Varga ◽

...

Keyword(s):

Atrial Fibrillation ◽

Channel Gating ◽

Ionic Current ◽

Fluorescence Emission ◽

Post Translational Modification ◽

Voltage Sensing ◽

Channel Activation ◽

Α Subunit ◽

Heart Chamber ◽

Voltage Sensing Domain

Background: Co-assembly of cardiac Na + channels (Na v 1.5) with β subunits modifies channel gating, expression, and post-translational modification. β subunit mutations have been linked to the Brugada and Long QT Syndromes, and atrial fibrillation (AF). Hypothesis: We tested whether β 3 subunits regulate Nav.1.5 ionic current and drug response by modulating the voltage sensing domains (VSDs). Methods: The Na V 1.5 α subunit contains four domains (DI-DIV), each with its own voltage sensing domain (VSD). We previously created four DNA constructs that carried a cysteine within a single VSD. Channels expressed in Xenopus oocytes and these cysteines were labeled with TAMRA-MTS fluorophores. Ionic current and fluorescence emission that tracked VSD conformation were simultaneously recorded using the cut-open configuration with and without β 3 . Results: Steady state inactivation is significantly right shifted by β 3 (V 1/2 = -88.9 ± 1.1 SEM (with, +β 3 ) and -97.8 ± 1.5 (without, -β 3 ), p=0.002, n=4). β 3 also right shifts DIII-VSD activation (V 1/2 = -93.0±2.3 +β 3 , V 1/2 = --114.8±0.8 -β 3 , p=0.001, n=4), while modestly left-shifting channel activation, suggesting enhanced DIII-VSD to pore coupling (V 1/2GV -V 1/2FV =55.0 ± 4.0 +β 3 , V 1/2GV -V 1/2FV =73.6 ± 2.4 -β 3 , n=4). DI and DII were not affected, while DIV was modestly shifted, consistent with DIII/DIV cooperativity. Extracellular domain AF-linked β 3 mutations, R6K and L10P, further enhance DIII-VSD to pore coupling (V 1/2GV -V 1/2FV =34.1 ± 5.8, (R6K), V 1/2GV -V 1/2FV =41.5 ± 4.7 (L10P), n=4). β 3 nearly abolishes stabilization of the DIII-VSD by lidocaine (DIII FV shift by lidocaine: ΔV 1/2 lido=-27.71 ± 12.23 (+β 3 ), ΔV 1/2 lido=-65.44 ± 3.83 (-β 3 ), n=3). The conservative R6K mutation exacerbates this effect, suggesting a cation-pi interaction with Na V 1.5. W1684 is co-localized with the DIII-VSD, and W1684A disrupted β 3 modification of channel gating and the DIII lidocaine interaction. Conclusions: β 3 modifies Nav1.5 gating by increasing DIII-VSD coupling to the pore via interaction with W1684. AF β 3 mutants further enhance DIII-VSD to pore coupling. The differential lidocaine response caused by WT and AF β 3 mutants suggests a molecular mechanism whereby the lidocaine response is patient and heart-chamber specific.

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New Molecular Scaffolds for Fluorescent Voltage Indicators

10.26434/chemrxiv.7338683.v1 ◽

2018 ◽

Author(s):

Steven Boggess ◽

Shivaani Gandhi ◽

Brian Siemons ◽

Nathaniel Huebsch ◽

Kevin Healy ◽

...

Keyword(s):

Membrane Potential ◽

Action Potentials ◽

Induced Pluripotent Stem Cell ◽

Molecular Wire ◽

Voltage Sensing ◽

Design Synthesis ◽

Cardiac Action ◽

Action Potential Waveform ◽

Induced Pluripotent ◽

Voltage Sensing Domain

<div> <p>The ability to non-invasively monitor membrane potential dynamics in excitable cells like neurons and cardiomyocytes promises to revolutionize our understanding of the physiology and pathology of the brain and heart. Here, we report the design, synthesis, and application of a new class of fluorescent voltage indicator that makes use of a fluorene-based molecular wire as a voltage sensing domain to provide fast and sensitive measurements of membrane potential in both mammalian neurons and human-derived cardiomyocytes. We show that the best of the new probes, fluorene VoltageFluor 2 (fVF 2) readily reports on action potentials in mammalian neurons, detects perturbations to cardiac action potential waveform in human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes, shows a substantial decrease in phototoxicity compared to existing molecular wire-based indicators, and can monitor cardiac action potentials for extended periods of time. Together, our results demonstrate the generalizability of a molecular wire approach to voltage sensing and highlights the utility of fVF 2 for interrogating membrane potential dynamics.</p> </div>

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Quantum Calculation of Proton and Other Charge Transfer Steps in Voltage Sensing in the Kv1.2 Channel

10.26434/chemrxiv.8251073 ◽

2019 ◽

Author(s):

Alisher M Kariev ◽

Michael Green

Keyword(s):

Charge Transfer ◽

Energy Charge ◽

Quantum Calculation ◽

Quantum Calculations ◽

Gating Current ◽

Transmembrane Segment ◽

Voltage Sensing ◽

Standard Models ◽

Charge Displacement ◽

Voltage Sensing Domain

Quantum calculations on 976 atoms of the voltage sensing domain of the K<sub>v</sub>1.2 channel, with protons in several positions, give energy, charge transfer, and other properties. Motion of the S4 transmembrane segment that accounts for gating current in standard models is shown not to occur; there is H<sup>+ </sup>transfer instead. The potential at which two proton positions cross in energy approximately corresponds to the gating potential for the channel. The charge displacement seems approximately correct for the gating current. Two mutations are accounted for (Y266F, R300cit, cit =citrulline). The primary conclusion is that voltage sensing depends on H<sup>+</sup> transfer, not motion of arginine charges.

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