Peters CH et al. (2017), Depolarization of the conductance-voltage relat...

XB-ART-54024

PLoS One 2017 Sep 08;129:e0184605. doi: 10.1371/journal.pone.0184605.

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Depolarization of the conductance-voltage relationship in the NaV1.5 mutant, E1784K, is due to altered fast inactivation.

Peters CH, Yu A, Zhu W, Silva JR, Ruben PC.

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E1784K is the most common mixed long QT syndrome/Brugada syndrome mutant in the cardiac voltage-gated sodium channel NaV1.5. E1784K shifts the midpoint of the channel conductance-voltage relationship to more depolarized membrane potentials and accelerates the rate of channel fast inactivation. The depolarizing shift in the midpoint of the conductance curve in E1784K is exacerbated by low extracellular pH. We tested whether the E1784K mutant shifts the channel conductance curve to more depolarized membrane potentials by affecting the channel voltage-sensors. We measured ionic currents and gating currents at pH 7.4 and pH 6.0 in Xenopus laevis oocytes. Contrary to our expectation, the movement of gating charges is shifted to more hyperpolarized membrane potentials by E1784K. Voltage-clamp fluorimetry experiments show that this gating charge shift is due to the movement of the DIVS4 voltage-sensor being shifted to more hyperpolarized membrane potentials. Using a model and experiments on fast inactivation-deficient channels, we show that changes to the rate and voltage-dependence of fast inactivation are sufficient to shift the conductance curve in E1784K. Our results localize the effects of E1784K to DIVS4, and provide novel insight into the role of the DIV-VSD in regulating the voltage-dependencies of activation and fast inactivation.

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Species referenced: Xenopus laevis
Genes referenced: nav1

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	Fig 1. A Depolarizing shift in the C373F/E1784K conductance curve occurs in the presence of a hyperpolarized movement of the voltage-sensors.(A1-A4) Sample outward and inward gating currents recorded from C373F and C373F/E1784K NaV1.5 at pH 7.4 and pH 6.0. (B1-B4) Sample ionic currents recorded from C373F and C373F/E1784K NaV1.5 at pH 7.4 and pH 6.0. CF and CF/EK currents with relatively similar amplitudes were selected to highlight the effects of protons. (C) Average normalized peak ionic current plotted versus voltage for C373F and C373F/E1784K NaV1.5 at pH 7.4 and pH 6.0. Currents at pH 6.0 are normalized to the peak current recorded at pH 7.4 from the same cell. (D) Average peak conductance plotted versus pH in C373F and C373F/E1784K NaV1.5. Conductance at all pH values is normalized to the peak conductance recorded from the same cell at pH 8.0. The pH at which conductance is reduced by 50% is shifted to higher pH in CF/EK channels. (E) Conductance-voltage relationships from ionic currents recorded in C373F and C373F/E1784K NaV1.5 at pH 7.4 and pH 6.0. The E1784K mutant shifts the midpoint of the conductance-voltage relationship to more depolarized potentials. E1784K undergoes a larger depolarizing shift of the conductance curve when extracellular pH is lowered. (E inset) To measure current voltage relationships from which conductance was determined, cells were depolarized to membrane potentials between -100mV and +60mV (a) from a holding potential of -150mV. (F) Charge-voltage relationships for outward gating currents recorded from C373F and C373F/E1784K NaV1.5 at pH 7.4 and pH 6.0. The E1784K mutant shifts the midpoint of the charge-voltage relationship in the hyperpolarizing direction. (F inset) To measure gating current activation, cells were depolarized to membrane potentials between -150mV and +40mV (a) followed by a hyperpolarization to -150mV (b).
	Fig 2. E1784K shifts thevoltage-dependence of fast inactivation to more hyperpolarized potentials and accelerates the rate of onset and recovery.(A1-A4) Sample ionic currents elicited during steady-state fast inactivation recordings from C373F and C373F/E1784K NaV1.5 at pH 7.4 and pH 6.0. (B) Steady-state fast inactivation relationships for C373F and C373F/E1784K NaV1.5 at pH 7.4 and pH 6.0. The E1784K mutant shifts the midpoint of the fast inactivation voltage-dependence in the hyperpolarizing direction, whereas decreasing extracellular pH to pH 6.0 shifts the midpoint of the fast inactivation voltage-dependence to more depolarized membrane potentials. (B inset) To measure steady-state fast inactivation, cells were depolarized to -10mV (b) following a conditioning pulse to membrane potentials between -150mV and -10mV (a). (C) Recovery from fast inactivation time course at -90mV for C373F and C373F/E1784K NaV1.5 at pH 7.4 and pH 6.0. The E1784K mutant accelerates recovery from inactivation. (C inset) To measure recovery from inactivation, cells were depolarized to -10mV (c) following a conditioning pulse to 0mV (a) and a recovery pulse of variable duration to membrane potentials between -130mV and -70mV (b). (D) Time constants of fast inactivation recovery (-130mV to -70mV) and closed-state fast inactivation onset (-70mV and -50mV) plotted versus voltage for C373F and C373F/E1784K NaV1.5 at pH 7.4 and pH 6.0. The E1784K mutant accelerates fast inactivation recovery and closed-state fast inactivation onset at all potentials. (E) Time constants of open-state fast inactivation onset for C373F and C373F/E1784K NaV1.5 at pH 7.4 and pH 6.0. The E1784K mutant accelerates open-state inactivation at all membrane potentials excluding +20mV.
	Fig 3. Protons cause a greater increase in persistent sodium current in C373F/E1784K NaV1.5.(A) Sample persistent sodium current recordings at -20mV from C373F and C373F/E1784K NaV1.5 at pH 7.4 and pH 6.0. (A inset) Sample persistent sodium current recordings at -20mV normalized to peak current from C373F and C373F/E1784K NaV1.5 at pH 7.4 and pH 6.0. (B) Persistent current normalized to peak current for C373F and C373F/E1784K NaV1.5 at pH 7.4 and pH 6.0 at membrane potentials between -30mV and 0mV. The E1784K mutant increases the fraction of persistent current. When extracellular pH is lowered to pH 6.0 there is a larger increase in the fraction of persistent current in CF/EK compared with CF. (C) Sample gating currents recorded during a protocol to measure gating current deactivation voltage-dependence. (D) Sample gating currents recorded to measure the time course of voltage-sensor deactivation at -150mV. (E) Voltage-dependence of gating current deactivation for C373F and C373F/E1784K NaV1.5 at pH 7.4 and pH 6.0. The inverse of the amount of charge deactivating at a given voltage is plotted to facilitate comparisons with the outward charge-voltage relationship. The voltage-dependence of gating current deactivation is shifted in the depolarizing direction by the E1784K mutant. (E inset) To measure the voltage-dependence of gating current deactivation, cells were hyperpolarized to membrane potentials between -150mV and +20mV (b) following a depolarization pulse to +50mV (a). This was followed by depolarization to +50mV (c). (F) Time course of gating charge deactivation at -150mV for C373F and C373F/E1784K NaV1.5 at pH 7.4. Time courses at pH 6.0 overlap those recorded at pH 7.4 and are therefore not shown. The E1784K mutant increases the fraction of charge which recovers with the fast time constant of recovery and decreases the fraction of slow charge return. (F inset) To measure the rate of gating current deactivation, we depolarized cells to 0mV (c) following a conditioning pulse to 0mV (a) and a recovery pulse of variable length to -150mV (b).
	Fig 4. E1784K accelerates a component of slow inactivation with 1-10s time constants.(A1 and A2) Sample inward sodium currents recorded after 20ms to 60s repolarization pulses to -90mV following either a 500ms or 64s depolarization to -30mV. (B) Time course of slow inactivation recovery at -90mV in C373F NaV1.5 at pH 7.4 following depolarizations to -30mV for durations between 500ms (top curve) and 64s (bottom curve). (B inset) To measure onset and recovery time courses for slow inactivation, cells were depolarized to -10mV (c) following a depolarizing pulse of variable duration to membrane potentials between -30mV and +30mV (a) and a recovery pulse of variable duration to membrane potentials between -120mV and -80mV (b). (C) Time course of slow inactivation onset at -30mV in C373F NaV1.5 at pH 7.4 with recovery interpulses to -90mV for durations between 100ms (bottom curve) and 10s (top curve). (D) Time constants for the fast component of slow inactivation recovery (-120mV to -80mV) and slow inactivation onset (-30mV and 0mV) plotted versus voltage for C373F and C373F/E1784K NaV1.5 at pH 7.4 and pH 6.0. The E1784K mutant decelerates the fast component of inactivation recovery at -80mV and accelerates inactivation at 0mV. Decreasing extracellular pH slows the fast component of inactivation onset at 0mV. (E) Time constants for the slow component of slow inactivation recovery (-120mV to -80mV) and slow inactivation onset (-30mV to 30mV) plotted versus voltage for C373F and C373F/E1784K NaV1.5 at pH 7.4 and pH 6.0. The E1784K mutant accelerates the slow component of recovery at -120mV and -90mV and accelerates the slow component of onset at 0mV. Decreasing extracellular pH slows the slow component of inactivation onset at -30mV, 0mV, and +30mV.
	Fig 5. Kinetic and steady-state properties of fluorescence signals from the WT-DIII-VCF, E1784K-DIII-VCF, WT-DIV-VCF, and E1784K-DIV-VCF constructs.(A1-A4) Representative fluorescence signals for DIII-VCF, E1784K-DIII-VCF, DIV-VCF, and E1784K DIV-VCF constructs. Percentage of fluorescence change were calculated as ΔF/F0. (B) Voltage-dependence of steady state fluorescence signal change (FV) of WT-DIII-VCF and E1784K-DIII-VCF constructs. The E1784K mutant causes a shift in the DIII FV curve to more hyperpolarized membrane potentials. (B inset) To measure the voltage-dependence of voltage-sensor fluorescence, the membrane potential of cells was changed to between -180mV and +20mV (a) from a holding potential of -120mV. (C) Voltage-dependence of steady state fluorescence signal change (FV) of WT-DIV-VCF and E1784K-DIV-VCF constructs. The E1784K mutant shifts the DIV FV curve in the hyperpolarizing direction.
	Fig 6. Proposed model of NaV1.5 channels.Modelling scheme used to simulate C373F and C373F/E1784K channel ionic and gating currents.
	Fig 7. Modeled gating currents and ionic currents are similar to experimental recordings.(A1-4) Sample experimental and simulated gating currents and ionic currents for C373F NaV1.5 at pH 7.4. (B) Overlaps of outward gating current traces recorded from 6 different cells expressing C373F NaV1.5 at pH 7.4 with simulated outward gating currents at the same membrane potentials. Gating currents were normalized to the peak outward current at 40mV. (C) Overlaps of ionic current traces recorded from 6 different cells expressing C373F NaV1.5 at pH 7.4 with simulated ionic currents at the same membrane potentials. Ionic currents were normalized to the peak inward current at 20mV. Overall the model replicates the experimental ionic and gating currents at different membrane potentials.
	Fig 8. Modeled C373F and C373F/E1784K channels show similar gating parameters and persistent currents compared to experiments.(A1-A4) Simulated ionic currents from C373F and C373F/E1784K NaV1.5 models at pH 7.4 and pH 6.0. (B) Simulated outward charge-voltage relationships from C373F (solid circles) and C373F/E1784K (open circles) NaV1.5 models at pH 7.4 (black) and pH 6.0 (red) are overlapped with fits to data from C373F (solid lines) and C373F/E1784K (dashed lines) experiments at pH 7.4 (black) and pH 6.0 (red). In all cases, there is a small depolarizing shift in the simulated gating current voltage-dependence which is reviewed in the discussion section (C) Simulated fast inactivation steady-state and conductance-voltage relationships from C373F (solid circles) and C373F/E1784K (open circles) NaV1.5 models at pH 7.4 (black) and pH 6.0 (red) overlapped with fits to data from C373F (solid lines) and C373F/E1784K (dashed lines) experiments at pH 7.4 (black) and pH 6.0 (red). (D) Simulated time constants of fast inactivation recovery (-130mV to -70mV) and closed-state fast inactivation onset (-70mV and -50mV) from C373F (solid circles) and C373F/E1784K (open circles) NaV1.5 models at pH 7.4 (black) and pH 6.0 (red) overlapped with fits to data from C373F (solid lines) and C373F/E1784K (dashed lines) experiments at pH 7.4 (black) and pH 6.0 (red). (E) Simulated persistent currents at -20mV from C373F and C373F/E1784K NaV1.5 models at pH 7.4 and pH 6.0. (E inset) Sample experimental persistent current recordings at -20mV from C373F and C373F/E1784K NaV1.5 at pH 7.4 and pH 6.0. Overall the model accurately replicates the experimental conductance-voltage relationship, steady-state fast inactivation, time course of fast inactivation, and persistent current amplitudes.
	Fig 9. E1784K induced changes to channel fast inactivation are sufficient to depolarize the conductance-voltage relationship.(A1-A4) Sample ionic currents recorded from IFM/QQQ and IFM/QQQ-E1784K NaV1.5 at pH 7.4 and pH 6.0. (B) Simulated outward charge-voltage and conductance-voltage relationships from C373F and C373F/E1784K NaV1.5 models at pH 7.4. To replicate the hyperpolarizing shift in the midpoint of the charge-voltage relationship and the depolarizing shift in the midpoint of the conductance voltage-relationship in the E1784K model required only changes to DIVS4 movement to correspond to experimental data on fast inactivation (C) Conductance-voltage relationships of IFM/QQQ and IFM/QQQ-E1784K NaV1.5 at pH 7.4 and pH 6.0. In the absence of fast inactivation, the E1784K mutant does not significantly affect the conductance-voltage relationship.

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