Campos FV , Chanda B , Beirão PS , Bezanilla F .

GM30376 NIGMS NIH HHS , R01 GM030376 NIGMS NIH HHS , R37 GM030376 NIGMS NIH HHS

	Figure 1. Effect of Ts1 in the sodium currents activation. (A) Voltage protocol used to record the inward sodium currents. (B) Superimposed sodium currents obtained at −72 mV before (blue trace) and after (red trace) the treatment with 50 nM of Ts1. (C) Superimposed sodium currents obtained at −56 mV before (blue trace) and after (red trace) the treatment with Ts1 (50 nM). (D) Superimposed sodium currents obtained at 0 mV before (blue trace) and after (red trace) the treatment with Ts1 (50 nM). (E) Superimposed G-V curves obtained before (blue symbols) and after (red symbols) the treatment with Ts1 (25 nM).
	Figure 2. Effect of Ts1 in the Cole-Moore shift. (A) Voltage protocol used to measure the Cole-Moore shift. (B, top) Superimposed sodium currents obtained in the absence of Ts1, following a prepulse to −60 mV (lefthand trace) or −160 (righthand trace). (B, bottom) Superimposed sodium currents obtained in the presence of Ts1 (100 nM), elicited as described above for the control conditions. (C) Superimposed sodium currents (prepulses to −160 mV) recorded in control conditions (blue line) and in the presence of Ts1 (red line). Black lines are the fits to the function in Eq. 1 (see Materials and Methods). Arrows in A and B indicate the beginning of the pulse. (D) Time to the half-maximal sodium current for indicated potentials in control conditions (blue triangles) and in the presence of Ts1 (red circles). The inset shows the time shift necessary to superimpose the currents for the indicated potentials with respect to the currents at −160 mV (control, blue triangles; in presence of Ts1, red circles). Blue and red lines are the fits to the function shown in Eq. 2. The parameters obtained for the curve in the absence of Ts1 were z = 2.3; Vh = 0 mV; the parameters obtained in the presence of Ts1 were z = 4.4; Vh = −17.7 mV.
	Figure 3. Effect of Ts1 on gating currents. Superimposed gating current traces elicited by a test pulse to −150 mV (A), −100 mV (B), and 0 mV (C) in control conditions (blue line) and in the presence of 50 nM Ts1 (red line). (A, inset) Voltage protocol used to record the currents. (D) Charge–voltage (Q-V) relationships (normalized to the maximum charge in control conditions) in the presence of Ts1 (red circles) and in control conditions (blue triangles). Solid and dashed traces are the fits to the function in Eqs. 3 and 4, respectively (see Materials and Methods). See Table I for fitted parameters.
	Figure 4. Effect of Ts1 on S4-DII movement. Effect of Ts1 in R663C channels. Superimposed traces of the TMRM-labeled R663C channel recorded at −50 mV (first panel) and −40 mV (second panel) before (blue traces) and after the treatment with 1 μM of Ts1 (red traces).
	Figure 5. Effect of Ts1 on S4-DII movement: S660C channel. (A) Superimposed traces of the TMRM-labeled S660C channel in control conditions (blue traces) and in the presence of Ts1 (0.5 μM, red traces), recorded at −80 mV (first panel), −60 mV (second panel), −40 (third panel), and −20 mV (fourth panel). The currents were recorded by applying the voltage protocol described in Fig. 1 A. (B) G-V curves obtained before (blue triangles) and after (red circles) the treatment with Ts1 (0.5 μM).
	Figure 6. Effect of Ts1 on S4-DII movement. (A) Voltage protocol used to measure the fluorescence signals. Superimposed fluorescence traces recorded at +50 mV (C), 0 mV (D), −50 mV (E), and −100 mV (F) in control conditions (blue lines) and in the presence of Ts1 (0.5 μM, red lines). The fluorescence increases in the same direction of the arrow in the scale bar.
	Figure 7. Effect of Ts1 on S4-DII movement. (A) Fluorescence–voltage (F-V) relationships obtained before (blue triangles) and after (red circles) the treatment with Ts1 (mean ± SEM; n = 3). The protocol used to measure the fluorescence signals was described in Fig. 6 A. (B) Absolute fluorescence traces obtained at +60 mV before (blue lines) and after (red lines) the treatment with Ts1. When error bars are not visible they are smaller than the symbols.
	Figure 8. Effect of Ts1 on S4-DI, S4-DIII, and S4-DIV movements. (A) Voltage protocol used to measure the fluorescence signals. (B–D) F-V relationships for domains I (S216C) (B), III (L1115C) (C), and IV (S1436C) (D) in absence (blue triangles) and presence of Ts1 (50 nM, red circles). Solid lines are the fits to the function in Eq. 5 (see Materials and Methods). See Table II for fitted parameters.
	Figure 9. A general kinetic model for gating in a voltage-gated ion channel. C0 corresponds to the fully deactivated state, with all four S4 segments in the resting position. C1, C2, C3, and C4 correspond to the states where voltage sensors from domains I, II, III, and IV are activated. The numbers in the index refers to voltage sensors that are in activated position. C1234 corresponds to the fully activated state and the open channel form, with all the S4 segments in the activated position.
	Figure 10. Modulation of gating in independent and allosteric models. Simulations of the charge–voltage (A) and conductance–voltage curves (B) for the model with independent voltage sensors. Charge–voltage (C) and conductance–voltage (D) curves in the allosteric model, where all four voltage sensor movements are coupled. Toxin-modified data are shown as red lines, whereas the unmodified traces are shown as blue lines.

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