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Figure 1. Effect of Ts3 on sodium currents from Xenopus oocytes expressing Nav1.4. (A) I-V curves obtained before (white circles) and after (black circles) the treatment with 200 nM of Ts3. Both curves were normalized by the maximal current obtained in the presence of toxin. Data shown as mean ± SEM (n = 4). Sodium currents were recorded with 20-ms pulses varying from â100 to +40 mV, which were followed and preceded by a 100-ms pulse to â100 mV. The holding potential was â90 mV. (B) Representative traces obtained in control conditions. (C) Representative traces obtained after the treatment with 200 nM of Ts3. (D) Superimposed recordings obtained at â20 mV before and after the treatment with Ts3. The traces were normalized by the peak value. Experiments were performed at 14°C.
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Figure 2. Ts3 voltage-dependent displacement. (A) Pulse protocol used to remove the bound Ts3. A strong depolarizing pulse, varying from +60 to +180 mV during 20 ms, was applied just after a â20 mV test pulse. Between pulses the oocytes were held at â90 mV, and the protocol was repeated at least 15 times. (B) Traces (gray lines) obtained in control conditions, in the presence of Ts3 (Pulse #0) and after 14 successive depolarization to +120 mV, by applying the protocol described above (Pulse #14) in the absence of Ts3. Black lines shows the curves obtained by fitting the data with function 1 (see Materials and methods). (C) Voltage dependence of toxin removal, obtained by applying the pulse protocol described in A. The graph shows the data obtained from a representative experiment. The number of pulses needed to an e-fold displacement was calculated by fitting the slow component contribution decay with a single exponential function.
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Figure 3. Effects of Ts3 on Nav 1.4 sodium channel gating currents. (A) Superimposed representative gating currents obtained in control conditions. Holding potential was â80 mV. ON gating currents were recorded with 40-ms pulses varying from â150 to +40 mV following a 100-ms prepulse to â120 mV. OFF gating currents were recorded with 40-ms pulses to â120 mV, preceded by pulses varying from â150 to +40 mV. (B) Superimposed representative gating currents obtained after the treatment with 200 nM of Ts3, by applying the protocol described in A. Experiments were performed at 14°C. (C) Comparison of traces of the ON gating currents obtained at â40 mV (gray lines) before and after the treatment with Ts3. Solid lines show the best fits obtained with function 1. (D) Comparison of traces of the OFF gating currents obtained at â120 mV after a pulse to â40 mV (gray lines) before and after the treatment with Ts3. Solid lines show the best fits obtained with function 1.
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Figure 4. Effects of Ts3 on the kinetic parameters of the ON gating currents. The parameters were obtained by fitting the ON gating current decay with function 1. Gating currents were recorded as described in Fig. 3 A. (A) Fast and slow time constants of the gating current decay. (B) Contribution of the slow component to the gating current decay. White circles show the slow component in control conditions and the black circles show the slow component in the presence of Ts3. Data shown as mean ± SEM, n = 4. *, existence of statistical difference (P < 0.05) between compared groups.
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Figure 5. Effects of Ts3 on the kinetic parameters of the OFF gating currents. The parameters were obtained by fitting the OFF gating current decay with function 1. Gating currents were recorded as described in Fig. 3 A. (A) Fast and slow time constants of the gating current decay. (B) Fraction of charge carried by the fast component of the OFF gating current decay. (C) Fraction of charge carried by the slow component of the decay. White triangles show the fast component in control conditions, black triangles show the fast component in the presence of Ts3, white circles show the slow component in control conditions, and the black circles show the slow component in the presence of Ts3. Note that for some of the potentials, the error bars are smaller than the symbols. Data shown as mean ± SEM, n = 4. *, existence of statistical difference (P < 0.05) between compared groups.
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Figure 6. Effects of Ts3 in the chargeâvoltage relationship. White symbols represent the Q-V curve obtained in control conditions (mean ± SEM, n = 3), and black symbols the curve obtained after the treatment with 200 nM of Ts3 (mean ± SEM, n = 3). The amount of charge recorded in each potential was normalized to the maximum charge recorded before the treatment with Ts3. Solid black lines are the curves obtained by fitting the data with a double Boltzmann function (function 2). Dotted gray lines are the curves obtained by fitting the data with a single Boltzmann function. Experiments were performed at 14°C.
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Figure 7. Effects of Ts3 in the fluorescence changes that track the movement of the S4 segment of domain IV of mutant channels S1436C stained with TMRM. Holding potential was â80 mV and the test pulse was preceded by a 200-ms pulse to â90 mV. (A) Representative sodium currents recorded at â20 mV before and after the treatment with 2 μM Ts3. (B) Representative fluorescence signals obtained at â20 mV before and after the treatment with 2 μM of Ts3. The signals are shown as ÎF/F (%), where F is the fluorescence background. The arrow indicates the direction in which fluorescence increases. These experiments were performed at room temperature.
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Figure 8. Effects of Ts3 on the fluorescence changes that track the movement of the S4 segment of domain IV of mutant channels L1439C stained with TMRM. Traces were recorded as described in Fig. 6. (A) Sodium currents recorded at â20 mV before and after the treatment with 200 nM Ts3. (B) Fluorescence signals obtained at â20 mV before (black trace) and after (gray trace) the treatment with 200 nM of Ts3. The signals are shown as ÎF/F (%), where F is the fluorescence background. The arrow indicates the direction in which fluorescence increases. These experiments were performed at room temperature. (C) F-V curves obtained before (white symbols) and after (black symbols) the treatment with 200 nM of Ts3 (mean ± SEM, n = 3; paired experiments). Solid lines are the curves obtained by fitting the data with the function 3.
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Figure 9. Effects of Ts3 in the fluorescence changes that track the movement of the S4 segment of domain I of mutant channel S216C stained with TMRM. Traces were recorded as described in Fig. 7. (A) F-V curves obtained before (white symbols) and after (black symbols) the treatment with 200 nM Ts3 (mean ± SEM, n = 3). The fluorescence recorded at each potential was normalized to the maximal fluorescence. Solid lines are the curves obtained by fitting the data with function 3. (BâD) Superimposed representative traces obtained at +60 (B), â60 (C), and â120 (D) before (gray traces) and after (black traces) the treatment with 200 nM of Ts3. The signals are shown as ÎF/F (%), where F is the fluorescence background. The arrow indicates the direction in which fluorescence increases. These experiments were performed at room temperature.
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Figure 10. Effects of Ts3 in the fluorescence changes that track the movement of the S4 segment of domains II and III, using the mutant channel S660C and L1115C stained with TMRM. Traces were obtained as described in Fig. 7. (A) F-V curves obtained from S660C channels before (white symbols) and after (black symbols) the treatment with 200 nM Ts3 (mean ± SEM, n = 3). The fluorescence recorded at each potential was normalized to the maximal fluorescence. Solid lines are the curves obtained by fitting the data with the function 3. (B) F-V curves obtained from L1115C channels before (white symbols) and after (black symbols) the treatment with 200 nM Ts3 (mean ± SEM, n = 3). The fluorescence recorded at each potential was normalized to the maximal fluorescence. Solid lines are the curves obtained by fitting the data with function 3.These experiments were performed at room temperature.
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Figure 11. Kinetic model of inactivation. (A) Kinetic diagram showing the open (O) and inactivated (I) states of the channel. Closed states are not shown and are assumed not to participate on the decay phase of the current at 0 mV (see Discussion). O1 is invariably the first activated state. The numbers indicate the rate constants (in msâ1) obtained by fitting the decay of the sodium current in the presence and absence of Ts3. In the presence of Ts3 the transitions between O1 and O2, and between I1 and I2, are blocked. (B) Experimental records in the presence and absence of Ts3 are superimposed with the theoretical curve calculated using the model and its rate constants.
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