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

Figures

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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…

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  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…

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  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…

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  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…

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  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.

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  Fig 6. Proposed model of NaV1.5 channels. Modelling scheme used to simulate C373F and C373F/E1784K channel ionic and gating currents.

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  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.

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  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…

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  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.