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Figure 1. Schematic cartoon showing the S3–S4 linker and the S4 N-terminal region of the Kv7.1 channel where the cysteine scan was performed.In red labels are shown the three endogenous cysteines accessible from the external solution, which could potentially form metal or disulfide bridges (C136, C214 and C331, located in the S1, S3 and S6 transmembrane segments, respectively).
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Figure 2. Effects of external Cd2+ or Cu-Phen on WT Kv7.1 (A and B), non-injected oocytes (C) and mutants F222C (D) and A226C (E).Oocytes were bathed in ND96 in the absence and presence of either 100 µM CdCl2 or 100 µM Cu-Phen. Currents were evoked by depolarizing steps from −140 mV to +40 mV in 15 mV increments (holding potential, −80 mV; tail potentials −60 mV), as shown in the scheme protocol between panels A and B. Shown are representative traces and current-voltage relations were determined as indicated.
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Figure 3. Conductance-voltage relations of WT Kv7.1 and mutants Q220C, V221C, F222C, S225C, A226C, I227C and R228C.Curves were fitted to one Boltzmann function. The following values were obtained: V50 = −24.1±2.3 mV, s = 16.3±2.1 mV (WT); V50 = −18.7±1.5 mV, s = 19.9±2.3 mV (Q220C); V50 = −20.5±3.5 mV, s = 33.8±7.1 mV (V221C); V50 = −8.3±1.3 mV, s = 20.5±2.2 mV (F222C); V50 = −1.9±1.5 mV, s = 13.8±1.7 mV (S225C); V50 = −18.0±1.1 mV, s = 13.4±1.0 mV (A226C); V50 = −13.6±1.7 mV, s = 23.7±3.2 mV (I227C); V50 = +3.9±4.1 mV, s = 18.5±3.5 mV (R228C). n = 6–20.
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Figure 4. Effects of external Cd2+ and Cu-Phen on mutant I227C.(A) Representative traces recorded from an oocyte expressing I227C and bathed in ND96 without (w/o) prior incubation with 100 µM DTT (1 hour at 20°C) and without Cd2+ or Cu-Phen; Currents were evoked as in Figure 2. (B) Oocytes expressing I227C channels were first pre-incubated with 100 µM DTT (1 hour at 20°C) and subsequently washed with ND96 in the absence or presence of 100 µM CdCl2. (C) Same as in B but with 100 µM Cu-Phen. Shown are representative traces and current-voltage relations that were determined as indicated. (D) Representative traces of an oocyte expressing mutant I227C, bathed in ND96 containing 100 µM Cu-Phen and subsequently washed with either ND96 (left panel) or ND96 containing 1 mM DTT (right panel). Currents were evoked by a step depolarization from −80 mV to +30 mV. Similar results have been obtained in 6 other cells. (E) Representative traces of an oocyte expressing I227C, preincubated for 10 min with 100 µM Cu-Phen at −80 mV and then washed out for 5 min with ND96 at −80 mV. Currents were then evoked either by a depolarizing step to 0 mV (red trace) or by a hyperpolarizing step to −140 mV (black trace), after which a fast re-application of Cu-Phen was applied (red or black trace after the arrow). Similar results have been obtained in 5 other cells.
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Figure 5. Effects of external Cd2+ on the double mutants I227C-C136A (A), I227C-C214A (B) and I227C-C331A (C).Oocytes were bathed in ND96 in the absence and presence of 100 µM CdCl2. Currents were evoked as in Figure 2. Shown are representative traces and current-voltage relations.
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Figure 6. Effects of external Cd2+ and Cu-Phen on mutant R228C.Oocytes were bathed in ND96 in the absence and presence of 100 µM CdCl2 (A) or 100 µM Cu-Phen (B). Shown are representative traces and current-voltage relations were determined as indicated. (C) R228C channels were opened by depolarization to +30 mV in a train of 60 sweeps at 0.2 Hz, in the absence (black traces) or presence of 100 µM Cu-Phen (red traces). The increase in current was calculated by the ratio of the amplitude of the 60th sweep to that of the 1st sweep. (D) Representative traces of an oocyte expressing R228C, preincubated for 10 min with 100 µM Cu-Phen at −80 mV and then washed out for 5 min with ND96 at −80 mV. Currents were evoked either by a depolarizing step to +30 mV (red trace) or by a hyperpolarizing step to −140 mV (black trace), after which a fast reapplication of Cu-Phen was applied (red or black trace after the arrow). Also shown, is the reversal by DTT of the current increase produced by the fast reapplication of Cu-Phen at +30 mV. Similar results have been obtained in 5 other cells.
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Figure 7. Effects of external Cd2+ (A) or Cu-Phen (B) on the double mutant R228C-C136A and of Cd2+ on R228C-C214A (C) and R228C-C331A (D).Oocytes were bathed in ND96 in the absence and presence of 100 µM CdCl2 or 100 µM Cu-Phen. Shown are representative traces and current-voltage relations were determined as indicated.
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Figure 8. Effects of external Cd2+ (A) or Cu-Phen (B) on mutant S225C.Oocytes were bathed in ND96 in the absence and presence of 100 µM CdCl2 or 100 µM Cu-Phen. Shown are representative traces and current-voltage relations were determined as indicated. (C) Shown are representative traces of an oocyte expressing mutant S225C before (red trace) and after (black trace) 10 min preincubation with Cu-Phen (100 µM) at −80 mV plus 5 min wash at −80 mV with ND96. Currents were evoked by a step depolarization to +30 mV. Similar results have been obtained in 6 other cells. Effects of Cu-Phen on the double mutants S225C-C136A (D), S225C-C214A (E) and S225C-C331A (F). Currents were evoked as in Figure 2. Current-voltage relations were determined as indicated.
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Figure 9. Plausible structural models accounting for the gating conformations of the voltage sensor domains of Kv7.1.(A) The closed (left) and open (right) conformations of Kv7.1 in side view (upper part) or top view (lower part). Each subunit is colored differently while alpha helices are shown as cylinders. (B) Top view of a subunit that reflects the closed state, where R228 becomes close to Glu160 (2.7 Å) and S255 to Cys136 (5.1 Å) within the same subunit. The indicated side chains are colored in CPK (grey, blue, red and yellow colors correspond to carbon, nitrogen, oxygen and sulfur atoms, respectively). (C) Side view of two adjacent subunits, which represent an intermediate open state (salmon) and an intermediate closed state (cyan). The two subunits are viewed from the inner part of the pore in horizontal outward direction. The inset shows the distance between Ile227 of two neighboring VSDs (5.2 Å) as well as the distance between R228 and Cys136 of the adjacent VSD (4.6 Å). (D) Adjacent VSDs in the open state (green), the intermediate open state (salmon), the intermediate closed state (cyan) and the closed state (yellow). Note that the VSD of the intermediate open state (salmon) tilts towards the neighboring VSD to interact with the S4 of the intermediate closed state (cyan), which undergoes axial rotation and translation from the closed state (yellow).
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