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Figure 1. Pore-lining residues in S6.Amino-acid sequence alignment of the S6 segments from Shaker, Kv1.2, four prokaryotic sodium channels, Nav1.4 and Cav2.1. Red highlighting indicates sites that display state-dependent accessibility to MTSET and those marked in blue denote positions that can be modified while channels are closed. Grey highlighting indicates positions that show no response to MTSET treatment. Residues predicted to form the hydrophobic gate are outlined in yellow and those that are underlined were mutated to cysteine and used for accessibility experiments. Asterisks indicate the location of residues that are part of the WSW triple mutant.
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Figure 2. Conductance–voltage relationships (G–Vs) for DI–DIV S6 cysteine mutants.Normalized G–Vs for (a) DI, (b) DII mutants, (c) DIII and (d) DIV mutants were generated by measuring the peak tail currents elicited from inside-out patches containing mutant channels over a range of potentials. All patches were held at −120 mV and subjected to varying ranges of depolarizations depending on the particular mutant. Conductance values were normalized by dividing the peak tail current value at a given voltage by the maximum tail current value obtained and fit to a Boltzmann function to determine V1/2 and Z values (Table 1).
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Figure 3. Reaction of intracellular MTSET with two cysteine residues within the DI S6.(a, top) Example MTSET perfusion and voltage protocol for a closed-state modification experiment. In this particular case, a 2-ms test pulse to 60 mV was used to evoke outward current; however, in some situations we used potentials to observe inward current. (a, bottom) Exemplar traces from a closed-state modification experiment using L437C channels. The concentration of MTSET was 1.4 mM. (b, top) Example MTSET perfusion and voltage protocol for an open-state modification experiment. Depending on the cysteine construct, different test pulse potentials were used to evoke either outward or inward current. (b, bottom) Exemplar traces from an open-state modification experiment using L437C channels. (c) Closed-state modification traces for V440C. (d) Open-state modification traces for V440C. The concentration of MTSET was 180 μM and the test pulse was −40 mV for 10 s.
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Figure 4. Accessibility experiments for two cysteine residues within the DII S6.(a) Closed-state modification experiment for L792C using 5-ms test pulses to 60 mV. (b) Open-state modification experiment for L792C. The concentration of MTSET was 744 μM (c) Closed-state modification traces for L796C using 20-ms test pulses to −30 mV. The concentration of MTSET was 1.3 mM. (d) Open-state modification traces for L796C using 12-ms test pulses to −30 mV. The concentration of MTSET was 1.8 mM.
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Figure 5. Modification time course of two cysteine residues within the DIII S6.(a) Exemplar traces from a closed-state modification experiment using I1284C channels. (b) Exemplar traces from an open-state modification experiment using I1284C channels. One-millisecond test pulses to 60 mV were used. (c) Time course of the reaction of MTSET with I1287C while channels are held closed. The concentration of MTSET was 19.6 mM and test pulses were at −30 mV for 12 ms. (d) Open-state modification traces for I1287C. The concentration of MTSET was 100 μM and test pulses were at −30 mV for 20 ms.
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Figure 6. State-dependent accessibility of lower DI–DIV S6 cysteine mutants.Cysteine modification rates in the closed (filled black circles) and open (filled red circles) states are plotted on a logarithmic scale for 5–6 positions within each domain. Each point is the mean of at least three experiments; standard errors are smaller than symbols. The length of the bar represents the fold change in rate between the closed and open states. Sites above V440C, L795C, I1287C and I1590C have closed-state reactivity values of <1 M−1 s−1. L794C and V1286C displayed no change in peak current after MTSET exposure (red cross filled circles). Depending on the rates of modification, the concentration of MTSET used to test each site ranged from 100 μM to 20 mM.
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Figure 7. Accessibility data mapped onto structural data.(a) Closed-state homology model showing only DII–DIV (left) and only DI, DII and DIV (right). Hydrophobic gating residues (orange) are found at the transition from sites that can only react with MTSET when the channel is open (red) to sites that can be modified even when the channel is closed (blue). (b) The same data represented on an open-state homology model showing only DII–DIV (left) and only DI, DII and DIV (right) highlights how movement of the hydrophobic gating residues on channel activation allows for ionic conduction.
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Figure 8. Zinc accessibility experiments with V440C–I1287C channels.(a) Inward sodium current from V440C–I1287C channels in response to 40-ms test pulses to −20 mV was recorded before and after multiple treatments of 10 μM zinc for 2 s each while the channels were held closed. Ten seconds elapsed between each test pulse. The red trace indicates the final test pulse after the cumulative exposure time. The decrease in current is comparable to that seen for WSW in both closed- and open-state experiments. (b) The effect of zinc application onto open channels was assessed by giving a test pulse to −20 mV for 20 ms before zinc treatment (black trace), a 5-s recovery at −120 mV, a 100-ms exposure to 10 μM zinc while the channels were held open at −20 mV (orange line), a 40-s washout and recovery period at −120 mV, followed by another test pulse to −20 mV for 20 ms (red trace). No further decrease in current is observed after a second round of zinc treatment. Sodium current recorded during the first exposure to zinc shows no sign of inhibition, suggesting that zinc does not form a bridge with V440C and I1287C while channels are open, but does so after entering the pore and channel closure. The data are representative of three separate experiments.
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Figure 9. Change in current amplitude after MTSET treatment.By plotting the current amplitude after complete MTSET modification by the initial amplitude, the extent of current inhibition induced by MTSET can be visualized for DI–DIV cysteine mutants and compared with the WSW background. Except for V440C, L794C and V1286C, all mutants display a reduction in current after MTSET application. The current amplitude increases for V440C, while L794C and V1286C (red crosses) are either inaccessible to MTSET or their reaction with MTSET produces no functional change in current properties. Error bars represent s.e.m. of at least three independent experiments.
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