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Figure 1. The L689I mutation does not significantly affect fast gating transitions. Ionic and gating currents of NaV1.4 channels bearing the L689I mutation are studied as described in our companion paper (Silva and Goldstein, 2013). Changes in the fluorescence magnitude (ÎF/F) of tetramethylrhodamine maleimide conjugated to each domain (via DI-S216C, DII-S660C, DIII-L115C, or DIV-S1436C) are recorded during fast voltage-dependent gating transitions to follow induction of SI and recovery. The mean ± SEM for groups of three to six cells is reported. (A) Drawing of the NaV1.4 channel subunit indicating the location of the L689I change on the DII S4âS5 linker. (B) Sodium currents. Activation and fast inactivation of ionic current are apparent with steps from a holding voltage of â100 mV to test potentials of â90 to +60 mV for 60 ms (10 ms shown) in 10-mV steps with a 10-s interpulse interval. For all recordings of ionic currents, gating currents were subtracted. (C) Gating current recorded as in A, with 2 µM TTX to block ionic current. (D) Currentâvoltage (circles) and chargeâvoltage (squares) relationships. Dotted-dashed lines correspond to previously reported data with WT (Silva and Goldstein, 2013). Protocol as in B and C. Conductance was calculated by normalizing the current to the driving force (Erev = 7.9 mV) and was fit with a Boltzmann of the form 1/(1+ezFRT(VâVmid)) (Vmid = â19.1 mV and z = 2.4). For the chargeâvoltage relationship, the fit gave Vmid = â23.3 mV and z = 1.0. (E) Fluorescence. VCF assessment of ÎF/F for DI-S216C L689I channels using steps from a holding potential of â120 to test potentials of â140 to 40 mV in 10-mV steps. Shown are traces from â140 to 20 mV in 40-mV steps. (F) L689I channel F-V relationship for each domain by protocol and data analysis as in D. The fits gave the following: DI: Vmid = â65.1 mV and z = 0.95; DII: Vmid = â70.2 mV and z = 1.1; DIII: Vmid = â83.1 mV and z = 2.3; DIV: Vmid = â79.1 mV and z = 1.5. Dotted-dashed lines correspond to data reported previously with WT (Silva and Goldstein, 2013).
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Figure 2. SI onset and recovery for L689I channels: Ionic and gating currents. SI induction and recovery of ionic and gating currents measured as described in our companion paper (Silva and Goldstein, 2013). Groups of four to seven cells reported as mean ± SEM. (A) SI is probed by 320 repetitions of a triple-pulse protocol from a holding potential of â100 mV: a 5-ms test pulse to +45 mV was used to measure peak current (phase a); SI was induced by a 500-ms pulse at â100, â75, â45, â15, 15, or 45 mV (phase b); and a 30-ms pulse at â100 mV used (phase c) to allow for recovery from fast inactivation before the next test pulse. Above the protocol, sample traces are shown for cycles 1, 2, 80, and 320 and demonstrate progressive decrease in peak current with SI at +45 mV. Dotted-dashed lines correspond to data reported previously with WT (Silva and Goldstein, 2013). Parameters for L689I SI onset are reported in Table 1. Insets compare L689I ionic current (circles) at 160 s to WT (triangles). (B) Loss of gating current with SI induced as in A. (Top) Sample traces from cycles 1, 2, 80, and 320 show progressive reduction in peak gating current with SI at +45 mV. (Bottom) Plot of the gating charge (Q, the integral of the gating current) with time at different potentials. Insets compare L689I gating current at 160 s to WT. (C) Sodium current. (Top) Recovery from SI is probed after induction pulses to +45 mV for 5, 40, or 160 s (phase a) during 5,000 cycles of 20 ms at â100 mV (phase b), followed by 4 ms at +45 mV when peak current is recorded (phase c). Sample traces from cycles 1, 2, 500, and 5,000 show the recovery of peak current. (Bottom) Voltage and cycle dependence of recovery from SI on logâlog plots of peak current. Parameters for recovery from SI are shown in Table 2. Dotted-dashed lines correspond to data reported previously with WT (Silva and Goldstein, 2013). (D) Gating current. Recovery from SI is probed as in A, with 2 µM TTX.
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Figure 3. L689I alters sensor mobility. Slow voltage-dependent changes in L689I channel fluorescence magnitude (ÎF/F0) during SI induction and recovery studied with tetramethylrhodamine maleimide linked via DI-S216C, DII-S660C, DIII-L115C, or DIV-S1436C. Groups of three to eight cells are reported as mean ± SEM. (A) Fluorescence. (Top) Application of the triple-pulse protocol reveals reduction in the ÎF/F0 with increasing cycle number; 4 ms before and 4 ms after the test step to +45 mV (phase a) is shown. (B) Time-dependent change in the magnitude of ÎF/F0 for the four channels using the triple-pulse protocol shown in Fig. 2 A. Parameters are shown in Table 1. Voltage and cycle dependence of recovery from SI of ÎF/F0 for the four channels are presented on logâlog plots. Dotted-dashed lines correspond to data reported previously with WT (Silva and Goldstein, 2013). Insets compare L689I (triangle) to WT (circle). For DI, the mutant reaches 0.55 ± 0.03 immobile versus 0.70 ± 0.04 for WT; for DII mutant, the mutant is 0.39 ± 0.05 immobile versus 0.77 ± 0.06 for WT channels. (C) Changes in ÎF/F0 with DI-S216C by the protocol shown in Fig. 2 C with a duration of 40 ms in phase b. (D) Voltage and cycle dependence of recovery from SI of ÎF/F0 for the four channels on logâlog plots. Parameters are shown in Table 2.
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Figure 4. Models of L689I mutant voltage-sensor slow immobilization. Experimental data from this paper are shown with symbols, and solid lines represent models. The effect of the protocol was removed for model fitting by subtracting the change with pulses to â100 mV, which accounts for any effects on the measurement caused by the short pulses to +45 mV. For model fitting, experimentally determined recovery is scaled so that it begins where onset terminated. Ionic current recovery is simulated using the same parameters and models used to simulate ionic onset. (A) Model of WT channel voltage-sensor slow immobilization for all four domains. (B) Recovery of WT channels.
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Figure 5. Reconstruction of SI based on voltage-sensor models. Models of the voltage sensors from our companion paper (Fig. 8 in Silva and Goldstein, 2013) and Fig. 4 are linked in various ways in an effort to recapitulate SI kinetics. (A) WT ionic SI from our companion paper (Fig. 2 in Silva and Goldstein, 2013). Axes adjusted for comparison to other panels. (B) Simulated ionic SI requiring all four sensors makes a slow transition. (C) Simulated ionic SI requiring any one voltage sensor makes a slow transition. (D) Simulated ionic SI requiring DI, DII, or DIII, but not DIV. (E) Simulated ionic SI requiring the L689I/TTX-sensitive component of DI and DII coupled with the sensor transition of DIII. (F) Simulated ionic SI requiring the L689I/TTX-sensitive component of DI and DII with the slow I1âI2 and I2âI3 movements of DIII. (G) Experiment from our companion paper (Fig. 6 in Silva and Goldstein, 2013) showing ionic current recovery from SI for comparison to the model. (H) Simulated recovery from SI using the same mathematical model applied to simulate SI onset in F.
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Figure 6. Proposed participation of DI/DII and DIII components in SI. (A) Simulation of L689I/TTX-sensitive component of DI/DII and the slow components of DIII during SI onset at +45 mV (arrows). Dark trace (Product) is the combination of both components at +45 (also plotted in Fig. 5 F). (B) Simulation of L689I/TTX-sensitive component of DI/DII and the slow components of DIII during SI recovery after a 160-s pulse to +45 mV. Dark trace (Product) is the combination of both components after a 160-s pulse (also plotted in Fig. 5 H).
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