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Figure 1. . The S4 sequences from α subunit of the rat skeletal muscle sodium channel were aligned with respect to their charged residues, shown in bold face. The residues (â², *, #) were mutated individually to cysteines and labeled with tetramethylrhodamine. The residues, shown in boxes, correspond to mutants whose fluorescence and electrophysiological properties were characterized in this study. Voltage-dependent fluorescence changes could be measured from additional sites (*) in each S4. The remaining mutants (#) did not show any voltage-dependent fluorescence signals.
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Figure 2. . Gating current characteristics of the wild type rat skeletal muscle sodium channel. (A) Gating currents of the wild type sodium channel were elicited by pulsing to â50, â30, â10, and 30 mV after 400 ms prepulse to â130 mV. The recorded traces were obtained by using a P/4 protocol at a subtracting holding potential of 50 mV. The arrowheads separate the fast and the slow component of the gating currents at potentials â50 and â30 mV. (B) Time constants of gating, activation, and inactivation. The data was obtained by fitting the decay phase of the gating current to the equation: A*exp(ât/Ï1) + B*exp(ât/Ï2). The slow gating component, Ïq(s), is shown with squares and the fast gating component, Ïq(f), with circles. The filled triangles correspond to inactivation time constants, Ïinact, obtained by fitting ionic currents. The activation time constants, (Ïact) filled circle, were obtained by fitting the ionic currents using a simplified version of the model proposed by Vandenberg and Bezanilla (1991).
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SCHEME I.
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SCHEME II.
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Figure 3. . Comparison of fluorescence, ionic currents, and the gating charge position for various cysteine mutants. Ionic currents (in blue), fluorescence (in red), and the gating charge position (time integral of the gating current) (in black) for TMRM labeled S216C (A), S660C (B), L1115C (C), and S1436C (D). Each panel is also marked with a domain name where the cysteine mutation is located. The ionic currents were elicited by pulsing to various test potentials following a 400 ms prepulse to â130 mV. The ionic traces were obtained by using P/â4 protocol with a subtracting holding potential of â130 mV. Fluorescence traces were obtained by making an average of 20 traces at each test potential with an interval of 2 s between pulses. The gating charge position was obtained by integrating the gating currents over the duration of the test pulse. The gating currents were obtained by subtracting at 50 mV using P/4 protocol. The data were scaled so that the peaks of ionic currents, fluorescence, and gating integrals have comparable amplitudes at 30 mV. Typical values of ÎF/F were between 1 and 2%. The time constant of membrane capacitance measured by small hyperpolarizing pulses from â130 mV to â140 mV was between 75 and 90 μs.
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Figure 4. . Steady-state fluorescence and gating characteristics of various mutants. (A) Normalized gating charge displacement as a function of pulse potential for S216C, S660C, L1115C, S1436C, and the wild-type Na channel. For each oocyte, the charge displaced was normalized to Qmax and the values were plotted as means ± SEM with n ⥠3 oocytes. The continuous lines represent best fits of the means to a single Boltzmann. (B) Normalized fluorescence change is plotted as a function pulse potential for S216C, S660C, L1115C, and S1436C. The steady-state fluorescence was determined by measuring the fluorescence change at the end of a 20-ms pulse. The fluorescence change recorded at various test potentials was normalized to the maximum fluorescence change recorded in the particular oocyte. The values were plotted as means ± SEM with n ⥠3 oocytes and the continuous lines are best fits to a Boltzmann distribution.
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Figure 5. . Kinetic characteristics of gating currents and fluorescence change for S216C, S660C, and L1115C. The gating currents and the fluorescence were fitted to a sum of two exponential functions: F(t) or q(t) = a + bexp(ât/Ï1) + cexp(ât/Ï2), where F(t) and q(t) are the fluorescence rise or the gating current decay as a function of time. The gating currents are shown as unfilled symbols and the fluorescence is shown as filled symbols. (A) Representative time constants of gating currents [slow component, Ïq(s) and the fast component, Ïq(f)] and fluorescence [slow component, ÏF(s) and the fast component, ÏF(f)] from a S216C-expressing oocyte. (B) Representative time constants of gating and fluorescence [slow component, ÏF(s) and the fast component, ÏF(f)] from a S660C-expressing oocyte. (C) Representative time constants of gating and fluorescence [slow component, ÏF(s) and the fast component, ÏF(f)] from a L1115C-expressing oocyte.
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Figure 6. . Kinetic characteristics of fluorescence change and gating currents for S1436C. (A) Gating currents traces (left) and the fluorescence traces (right) from a TMRM-labeled S1436C mutant. The superimposed fits (dashed black lines) were generated using a sum of two decaying exponentials. (B) Time constants of fluorescence (slow, ÏF(s) filled squares and fast, ÏF(f) circles), charge movement (slow, Ïq(s), unfilled squares and fast, Ïq(f), unfilled circles), inactivation (filled triangles), and activation (filled diamonds). The fluorescence and the gating currents were fitted to a sum of two exponentials. Inactivation time constants (ÏIN) were obtained by fitting the ionic current decay to single exponentials. The activation time constants (Ïact) were obtained by fitting the ionic current traces with the model shown in Scheme II. (C) Fluorescence decay traces (red) of labeled S1436C channels were compared with development of inactivation (black). The fluorescence was recorded for 20 ms at test potentials varying from â50 to 30 mV after 400 ms prepulse from â130 mV. The development of inactivation was followed by using a two-pulse protocol shown schematically on top of the panel. A conditioning pulse is given to potentials (â50 to 30 mV) where the development of inactivation is to be followed. Each data point (filled circle) represents the peak ionic current at a test potential (50 mV) elicited by varying the length of the conditioning pulse followed by a hyperpolarizing pulse of 1 ms to â130 mV. This 1 ms prepulse to â130 mV resets activation gates without significantly allowing channels to recover from inactivation. The length of the conditioning pulse was incremented by 0.1 ms for pulse lengths up to 1 ms, 0.5 ms for pulse lengths between 1 and 10 ms and 1 ms for pulse lengths between 10 and 20 ms. The fluorescence and the inactivation traces were scaled such that the peak amplitudes were comparable at 30 mV at the end of 20 ms.
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Figure 7. . Comparison of fluorescence traces obtained from S216C, S660C, L1115C, and S1436C. Fluorescence signals were recorded by pulsing to test potentials following a prepulse of 400 ms at â130 mV and are color coded in the following manner: S216C in blue, S660C in red, L1115C in green, and S1436C in black. For comparison, the fluorescence traces from various mutants were scaled to have identical steady-state amplitudes at 50 mV. For reference to the clamp speed, a representative capacity transient was obtained by pulsing to â140 mV from â130 mV (in magenta).
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Figure 8. . Limits on the rates to resolve sequential activation of S4 movement. (A) Sequential activation model for a hypothetical channel where the S4 in domain III activates later in the sequence. The state where all the S4s are deactivated is represented as C and the activation of any of the three S4s converts the channel into a state O. The number in the subscript represents the domain whose S4 is activated. α, β, and γ represent the rate constants of transition. (B) The fluorescence signals from S216C, S660C and L1115C were obtained by pulsing to â30 mV. The fluorescence signals of S4 domain I (smooth line) and S4 domain III (broken lines) representing transition of S4 segment from a resting to an activated state were simulated using a kinetic model described in Fig. 8 A. The fluorescence signals of S4 domain III were simulated for three different values of γ with respect to α.
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Figure 9. . Comparison of ionic currents and fluorescence signals from S1436C. (A) Ionic currents were obtained after subtracting the gating current component. Fluorescence and the ionic traces were scaled to have comparable peak amplitudes at every potential. The beginning of the pulse is marked with a dashed line. (B) Fluorescence traces recorded from three different sites (L1439C, S1436C, and R1441C) on S4 domain IV. The fluorescence data for S1436C and L1439C were recorded by pulsing to â30 mV and that for R1441C was recorded by pulsing to 70 mV.
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Figure 10. . Recordings of voltage-dependent fluorescence changes from additional sites. (Top) Fluorescence change recorded from labeled S216C and I215C by pulsing to 70 mV after a prepulse of 400 ms to â130 mV. (Middle) Fluorescence change recorded from labeled L1115C and S1113C using the same conditions as in A. The fluorescence trace for L1115C was inverted for comparison. (Bottom) Fluorescence change recorded from labeled L1439C and S1436C by pulsing to 70 mV (top) after a prepulse of 400 ms to â130 mV.
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Figure 11. . Proposed kinetic model of sodium channel activation. The S4 segments of each domain are shown as circles that are unfilled in closed state are filled in the activated state. Broadly, the channel exists in two sets of states; those without any inactivation (white bar) are shown in white and those with inactivation particle bound are shown in gray. The opening of the channel is indicated by open circle at the center. The inactivation particle can bind less tightly (slanted bar) or very tightly (horizontal bar). For details, see text.
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