Trapping Charge Mechanism in Hv1 Channels (CiHv1)

Figures

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  Figure 1. N264R promotes charge trapping. Representative gating currents for (A) ΔNΔC N264R, (B) ΔNΔC D160N and (C) ΔNΔC D160N N264R. Colored rectangles represent modifications in OFF-gating currents produced by the arginine in the 264 position. (D) Q(V) relation was fitted using two-state Boltzmann equation. The values obtained were V0.5 = 48.6 ± 1.2 mV and zδ =1.04 ± 0.10 for ΔNΔC N264R (N = 4), V0.5 = 124.1 ± 6.2 mV and zδ =1.14 ± 0.03 for ΔNΔC D160N (N = 8) and V0.5 = 117.0 ± 2.2 mV and zδ = 1.18 ± 0.02 for ΔNΔC D160N N264R (N = 7). (E) Mean ON-gating current decay time constants of ΔNΔC D160N and ΔNΔC D160N N264R mutants as a function of voltage, fitted to a single exponential function Ig(t) = I0 exp(−t/τ). Data are shown as MEAN ± SEM.

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  Figure 2. Time course of voltage sensor gating charge trapping due to N264R mutation. (A–C) represents traces of currents produced by time-varying depolarizations for ΔNΔC N264R, ΔNΔC D160N and ΔNΔC D160N N264R, respectively, with the length of the pulse is represented by darker colors. (D) Maximum peaks of fast transient OFF-gating currents in function of time depolarizations for ΔNΔC D160N and ΔNΔC D160N N264R. A double-exponential function was fitted, as follows: Ig = A (1 − exp(−t/τ1)) + B (1 − exp(−t/τ2)). The time constants calculated were τ1 = 0.69 ± 0.09 ms and τ2 = 15.66 ± 0.74 for ΔNΔC D160N (N = 4) and τ1 = 0.70 ± 0.06 ms and τ2 = 1175.13 ± 247.19 for ΔNΔC D160N N264R (N = 5). (E) The QOFF/QON as a function of time for ΔNΔC D160N and ΔNΔC D160N N264R mutants. A straight line near QOFF/QON = 1.05 ± 0.00 was fitted for ΔNΔC D160N and ΔNΔC D160N N264R was fitted to an exponential function (A exp((−t/τ1)) with parameters τ1 = 121.77 ± 10.32 ms. Time course of charge trapping shown as ratio of Qoff/Qon as a function of time. The ON-gating charge was calculated by integrating each depolarizing pulse of the ON-gating current at successive times of 2, 7, 12, 17, 22, 27, 32, 37, 42, 47…

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  Figure 3. 2GBI mimics ΔNΔC N264R OFF-gating current. (A,B) Representative gating currents of ΔNΔC D160N before (A) and after (B) perfusion of 1.2 mM 2GBI. (C) Q(V) relation was fitted using two-state Boltzmann equation in ΔNΔC D160N (purple) and ΔNΔC D160N + 2GBI (pink). The parameters obtained were V0.5 = 127.3 ± 4.8 mV and zδ =1.18 ± 0.08 (N = 9). (D) ON-gating current time decay of ΔNΔC D160N mutants + 2GBI as function of voltage, fitted as a single-exponential function Ig(t) = I0 exp(−t/τ). Data shown as MEAN ± SEM.

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  Figure 4. 2GBI modifies properties of voltage sensor kinetics. (A,B) Representative traces of currents produced by time-varying depolarizations for ΔNΔC D160N before (A) and after (B) perfusion of 1.2 mM of 2GBI. (C) Maximum peaks of fast transient OFF-gating currents in function of time depolarizations for ΔNΔC D160N in presence of 2GBI perfusion (darker color-schemes represents longer depolarization intervals). A double-exponential function was fitted as follows: Ig = A (1 − exp(−t/τ1)) + B (1 − exp(−t/τ2)). The time constants calculated were τ1 = 1.51 ± 0.08 ms and τ2 = 333.12 ± 29.22 ms for ΔNΔC D160N (N = 5). (D) The QOFF/QON as a function of time for ΔNΔC D160N + 1.2 mM 2GBI was fitted to A exp(−t/τ1) + B exp(−t/τ2), and the parameters found were τ1 = 185.25 ± 18.13 ms and τ2 = 5.00 ± 0.78 ms. The depolarization time of the ON-gating current at successive times of 2, 7, 12, 17, 22, 27, 32, 37, 42, 47 and 52 ms while the OFF-gating charge was calculated from the OFF-gating current during the first 20 ms. Data shown as MEAN ± SEM.

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  Figure 5. Changes in the hydrophobicity of the selectivity filter result in changes in the voltage sensor displacement. (A) Gating currents produced in patches expressing different mutants in D160 (selectivity filter) position. From top to bottom, the hydrophobicity index has changed; the more negative the energy value, the more hydrophobic the amino acid. (B) Normalized Q(V) curves for different mutations in D160, a two-state Boltzmann distribution was adjusted for each one (see fit values in Table S1) at same symmetrical pH 7/7 (ΔpH = 0). (C) Linear correlation between the energy to move from an active to an inactive state of the voltage sensor and the hydrophobicity index for each amino acid. Data shown as MEAN ± S.D.

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  Figure 6. Acidity modifies OFF-gating currents. (A,B) Superimposed traces of gating currents of (A) ΔNΔC D160N and (B) ΔNΔC D160N N264R to ΔpH = 2 (pHo = 7 and pHi = 5). The upper inset of (A) shows how the ΔNΔC D160N mutant carries a small amount of macroscopic current (green) whereas, in the ΔNΔC D160N N264R mutant, this current disappears (blue). In both mutants, it can be seen that the OFF-gating current becomes smaller compared to the ON-gating current (lower insets). (C) Q(V) curves of ΔNΔC D160N (green) and ΔNΔC D160N N264R (blue) to pH 7/5. Curves were fitted using a two-state Boltzmann distribution. Data shown as MEAN ± S.E.M.

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  Figure 7. Acidity also promotes charge trapping. (A) Representative traces of currents (in colors) produced by time-varying depolarizations for ΔNΔC D160N N264R. The depolarization time of the ON-gating current at successive times of 2, 7, 12, 17, 22, 27, 32, 37, 42, 47 and 52 ms. (B) Maximum peaks of fast transient OFF-gating currents in function of time depolarizations using ΔpH = 2 (pHo = 7 and pHi = 5) for ΔNΔC D160N N264R. A double-exponential function was fitted to obtain the time constants, Ig = A (1 − exp(−t/τ1)) + B (1 − exp(−t/τ2)); the values found were τ1 = 0.0632 ± 0.00 ms and τ2 = 8.40 ± 0.73 ms (N = 4). (C) QOFF/QON time depolarization course of ΔNΔC D160N N264R ΔpH = 2, charge ratio was fitted as follow: A exp(−t/t1) + B exp(−t/t2), the parameters found were τ1 = 6.39 ± 1.59 ms and τ2 = 87.57 ± 32.90 ms. Data shown as MEAN ± SEM.

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  Figure 8. Molecular mechanism of charge trapping. (A) Distance probability distributions of AI model of ΔNΔC CiHv1. The right panel shows a representative time-series interaction of position D160 (blue) with arginines 258 and 261 (red). (B) When the N264R mutation is placed, the distance probability distributions in the AI model collapse at values corresponding approximately to salt bridge interactions. The right panel shows how the time series remains, on average, much longer at values close to 4 Å. (C) Representative structure of a cluster of structures showing that the selectivity filter D160 and R258 and R261 are located at a position near 4 Å when the N264R mutation is placed.

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  Figure 9. 2GBI-ΔNΔC D160N interaction recovers the gating charge via a voltage pulse protocol. (A) ON-gating current recovery protocol. The protocol consists in two depolarizing pulses from a holding potential of −90 mV to 200 mV which were separated by an interval of time between the depolarizing pulses that increased 5 ms between traces (shown in different colors for differentiation). (B) The charge displaced during the second depolarization as a function of the time interval between the contiguous depolarizations. The data were fitted using a single increasing exponential function Q [exp(−t/τ)] where Q is the maximum value of amplitude 3872.52 ± 37.42 fC and τ =35.63 ± 1.1 ms is the time constant.