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Figure 1. Fluorescence changes and gating currents for the M356C 5-aa linker mutant. (A) Fluorescence changes (upward deflections indicate a decrease in fluorescence) in response to 400-ms depolarizations from HP = â90 mV. (B) Integrated gating currents (smooth trace) and normalized fluorescence traces (jagged trace) during step hyperpolarizations to the membrane potentials indicated from HP = 0 mV. Hyperpolarizations occurred at the arrow. (C) Normalized deactivation charge displacement (Q) as a function of activation voltage for the M356C 5-aa linker mutation, normalized to the total charge measured from HP = 0 mV. The Q-Vs are marked with HP and duration of pulse. The gating currents were measured during the voltage steps for HP = 0 mV (on-gating charge), and after the return of the voltage to the resting potentials for HP = â90 mV (off-gating charge), so that in both cases deactivation gating charge is measured. (D) Time constants for the fluorescence changes in response to step depolarizations from HP = â90 mV (âª), and approximate time constants for activation gating charge movement obtained by fitting a single exponential to the fractional gating charge movements during voltage steps of variable durations (Fig. 1 C) as a function of voltage.
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Figure 2. Correlation of activation charge displacement and fluorescence in the M356C W434F 5-aa linker mutation (A) Gating currents (left) and fluorescence traces (right) during the test pulse to â180 mV as a function of prepulse duration at 0 mV. The start and end of the pulse to â180 mV are indicated by arrows. (B) Fractional gating charge displacement (Q) as a function of prepulse duration, and normalized fluorescence decrease (F) when stepping the voltage from â90 to 0 mV. The charge displacement was measured at â180 mV for variable durations at 0 mV (see A). The gating charge displacement was fitted with a double exponential decay function with the time constants indicated. The fluorescence signal was fitted with a single exponential and was normalized to the amplitude of the exponential fit.
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Figure 3. Slow down of the fluorescence signal during prolonged dwelling at â90 mV in the M356C W434F 5-aa linker mutation. From HP = 0 mV, the potential was prepulsed to â90 mV for variable durations (Ît), followed by a test pulse to 50 mV. Shown are the current traces (left) and fluorescence traces (right) during the test pulse. Start and end of the test pulse are indicated by arrows.
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Figure 4. Fluorescence changes for TMRM-stained A359C 5-aa linker mutation. Families of fluorescence traces are shown during voltage steps from the HPs to the test potentials indicated in the insets. Start and end of the voltage steps are shown by arrows (top).
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Figure 10. (A) Model used to account for biphasic fluorescence traces in the A359C 5-aa linker and the L361C wt linker channel. The rate constants α, β, γ, and δ were all assumed to be voltage dependent, in agreement with the demonstration that both interconversions carried gating charges. Thus α = α0 exp[zαeV/(kT)], β = β0 exp[âzβeV/(kT)], γ = γ0 exp[zγeV/(k T)], δ = δ0 exp[âzδeV/(kT)], where z is the fractional charge in elementary charge units, e is the elementary charge, k is Boltzmann's constant, V is the transmembrane voltage, and T is the absolute temperature. (B) Seven fluorescence traces (points) for the A359C 5-aa linker distributed between two holding potentials (â110 and 0 mV). The seven traces were fitted simultaneously with the three-state model in A and the fit displayed as lines. Fitted as free parameters were the eight rate constant parameters (α0, β0, γ0, δ0, zα, zβ, zγ, zδ), the three fluorescence intensities (negative values are higher fluorescence) associated with the three states (FS1, FS2, FS3), one parameter (Ws) for the interaction energy with the voltage sensor when the channel is C-type inactivated (Olcese et al. 1997), leading to a leftward displacement of the gating in the case of HP = 0 mV (Ws was set to zero for HP = â90 mV). Finally, one parameter (bleach) allowing the two holding potentials to have different absolute fluorescence intensities, due to the unavoidable bleaching of the fluorescence probe. The fitted values were: α0 = 59.7 sâ1, β0 = 0.216 sâ1, γ0 = 16.4 sâ1, δ0 = 5.00 sâ1, zα = 0.439, zβ = 1.32, zγ = 0.430, zδ = 0.279, FS1 = 0.892, FS2 = â12.2, FS3 = â4.80, WS = 0.155 (kT U), bleach = 1.23 (i.e., total fluorescence intensity was 23% higher at HP = â110 mV compared with 0 mV).
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Figure 5. Prepulse experiments for TMRM-stained A359C 5-aa linker mutation. The potential was prepulsed from HP = â90 to â40 mV for variable durations, followed by a test pulse to â180 mV. Given are the current traces (left) and the fluorescence traces (right) during the test pulse. Arrows indicate start and end of the voltage step to â180 mV.
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Figure 6. Biphasic fluorescence signals and gating charge movement. (A) Fluorescence signal for the TMRM-stained L361C W434F stepped from HP = 0 mV to membrane potentials indicated (bottom inset). Top inset shows the voltage step to â180 mV with biphasic signal indicated by the arrow. (B) Time constant for gating charge movement and time constants for decreasing (Ï1) and increasing (Ï2) part of the fluorescence trace for the L361C W434F, HP = 0 mV. (B) Fluorescence signal for the TMRM-stained A359C W434F 5-aa linker from HP = 0 mV to membrane potentials indicated (bottom inset). This panel is from the same oocyte as in Fig. 5, but with a fluorescence trace for every 10 mV, for comparison with L361C W434F. Top inset shows the voltage step to â180 mV with biphasic signal indicated by the arrow. (D) Time constants for fast (Ï1) and slow (Ï2) part of gating charge movement and time constants for decreasing (Ï1) and increasing (Ï2) part of the fluorescence trace for the A359C W434F 5-aa linker, HP = 0 mV.
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Figure 7. (A) Normalized gating charge movement (Q) and fluorescence (F) for the TMRM-stained L361C W434F from HP = 0 and â90 mV, respectively. Each Q-V was fitted with a sum of two Boltzmann distributions and each F-V with a single Boltzmann distribution. The broken lines show the Q-V for the wt W434F channel from HP = 0 mV (left) or â90 mV (right). (B) Prepulse experiment for the L361C W434F WT linker channel. The potential was prepulsed to â160 mV for variable durations from HP = â90 mV and gating charge measured during a subsequent test pulse to 0 mV (inset). Shown is the total gating charge (âª, left ordinate) measured at 0 mV as a function of prepulse duration and the scaled fluorescence trace taken during a pulse from HP = â90 to â160 mV (jagged trace, right ordinate). The smooth line is a single exponential decay function fitted to the gating charge with the time constant constrained to that obtained by fitting to the fluorescence trace (75.7 ms).
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Figure 8. pH-dependent modulation of TMRM fluorescence. Mean ± SEM for five oocytes of fluorescence intensity for M356C 5-aa linker taken at the end of 100-ms pulses from HP = â90 mV. The fluorescence intensities were normalized to the amplitude obtained at 160 mV for pH 7.4.
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Figure 9. Proposed model to explain the observed fluorescence changes for the M356C 5-aa linker, A359C 5-aa linker mutants, and the L361C wt linker. This is a visualization of the kinetic model in Fig. 10 with states A, B, and C, corresponding to the states S1, S2, and S3, respectively. It is proposed that the biphasic nature of the fluorescence signal for the A359C 5-aa linker and the L361C wt linker mutations is explained based on quenching by one group (QG1) at depolarized potentials (C) and another group (QG2) at hyperpolarized potentials. The middle state (B) has minimum quenching (maximum fluorescence) because the TMRM probes at A359C 5-aa linker and L361C wt linker reside in a relatively unquenched environment between QG1 and QG2. The M356C 5-aa linker probe does not interact noticeably with QG2, but interacts with QG1 at depolarized potentials The movement of the voltage sensor is displayed as a rotation, following the proposed model in Bezanilla 2000.
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