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Figure 1. Measurement of inactivation kinetics for WT and V535S Kv11.1 channels. (A) Voltage protocols used to measure the rates of onset of inactivation (i) and the rates of recovery from inactivation (ii), with key regions for the measurement of current highlighted in black. (B and C) Representative families of current traces measuring the rates of onset of inactivation (i) and rates of recovery from inactivation (ii) for WT (B) and V535S (C) Kv11.1 channels. Highlighted in bold are current traces at 0 mV (i) and −130 mV (ii) to aid comparison. (D) Chevron plots of the logarithm of the observed rates for onset of (open squares) and recovery from (closed squares) inactivation for WT (i) and V535S (ii) channels, plotted against voltage. Solid black lines are a fit of Eq. 1 (see Materials and methods), whereas the solid gray line in (ii) indicates kobs,V for WT channels to aid comparison. Dashed lines indicate the derived unidirectional rate constants for the onset of (kinact,V) and recovery from (krec,V) inactivation, with values at 0 mV indicated by arrows. The equilibrium constant for inactivation (Keq) at 0 mV was calculated by: Keq,0 = kinact,0/krec,0 (Eq. 2 in Materials and methods).
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Figure 2. Scanning mutagenesis of the Kv11.1 channel S4 helix. (A–C) Shifts, relative to WT, in log(Keq,0) values for alanine (A), serine (B), and tryptophan (C) mutagenesis scans of S4 residues spanning from Gly522 to Lys538. Data are presented as means ± SEM for n = 3–20 cells (see Table S1). Dashed lines indicate Δlog(Keq,0) > ±0.5 log units, which has been shown previously to be the minimum perturbation required to derive an accurate Φ-value (Cymes et al., 2002; Wang et al., 2011). Mutations that fulfill this minimum requirement are indicated by closed bars, whereas mutations with Δlog(Keq,0) < 0.5 log units are indicated by open bars. *, mutant channels that failed to express or expressed poorly.
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Figure 3. Families of mutations at important Kv11.1 channel S4 residues. (A and B) Shifts in log(Keq,0), relative to WT, for families of mutations at charged (A) or hydrophobic (B) residues that were identified as important determinants for inactivation gating from our mutagenesis scans of the S4 helix. Mutations are indicated by single–amino acid code on the y axis. Data are presented as means ± SEM for n = 4–14 cells (see Table S1). Mutations that cause a significant perturbation to inactivation, measured as Δlog(Keq,0) > ±0.5 log units relative to WT, are indicated by closed bars, whereas mutations with Δlog(Keq,0) < ±0.5 log units are indicated by open bars. *, mutant channels that failed to express or expressed poorly.
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Figure 4. Mutation of S4 residues affects inactivation at a midway point during the transition pathway. (A) REFER plots of the log forward unidirectional rate constant, log(kinact,0), against the log equilibrium constant, log(Keq,0), for inactivation, in families of mutations at individual residues: i, Leu529; ii, Leu530; iii, Leu532; iv, Val535. (B and C) REFER plots for all mutations at Leu529, Leu530, Leu532, and Val535 combined (B) or all S4 residue mutations (C). Data are presented as means ± SEM for 3–14 cells. The slope of the linear regression analysis (solid black lines) for each family of mutations represents the Φ-value, as indicated, and can be compared with the slope for all other S4 mutations (solid gray lines). ANCOVA was used to determine statistical significance; *, P < 0.05.
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Figure 5. Combinations of S4 mutants do not have additive effects on the energetics of Kv11.1 channel inactivation. (A) Bar graph comparing shifts in log(Keq,0), relative to WT, for triple S4 mutant (L529S + L532S + V535S), double mutants L532S + V535S and L529S + V535S, and single mutants V535S, L532S, and L529S. Data are presented as means + SEM for 5–18 cells. (B) REFER plot of triple S4 mutant (L529S + L532S + V535S), double mutants L532S + V535S and L529S + V535S, and single mutants V535S, L532S, and L529S. Data are presented as means ± SEM for 5–18 cells. The linear slope (solid black line) indicates a Φ-value of 0.42 and was not significantly different to that derived from all of the individual S4 mutants (Φ = 0.50; P > 0.05 using ANCOVA).
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Figure 6. Perturbations to inactivation gating and activation gating show poor correlation for S4 residue mutations. Plot comparing perturbations to ΔG0, versus WT, of inactivation gating (ΔΔG0Inact, represented by Δlog(Keq,0)) compared with perturbations to ΔG0, versus WT, of activation gating (ΔΔG0Activ; see Materials and methods) for each S4 residue mutation. Solid black line represents linear regression analysis constrained to go through WT, whereas dashed gray line is an unconstrained fit (R2 = 0.38).
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Figure 7. Only combinations of S4 mutants alter the voltage sensitivity of Kv11.1 channel inactivation. (A and B) Dot plots showing the linear slope values calculated from the derived unidirectional rate constants (shown as black lines in Fig. 1 D) for the onset of (kinact; A) and recovery from (krec; B) inactivation for groups of mutants (to Ala, Ser, and Trp) at each residue in the S4 domain, as well as the double and triple mutants. Slope values indicate the voltage dependence of the forward (inactivation; A) and reverse (recovery from inactivation; B) gating transitions. The mean of all mutations at each individual residue falls within ±2 standard deviations (gray box) of the mean calculated from all single S4 mutants.
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Figure 8. Multi-domain model of inactivation gating in Kv11.1 channels. (A–D) REFER plots to compare S4 residue mutations (shown in black) against S4S5 linker (A), S5 (B), S5P linker (C), and S6 (D) residue mutations (shown in gray). ANCOVA shows that the linear regression slope, which represents the Φ-value, was significantly different for the S4 helix compared with the S5, S5P, and S6 helices (P < 0.05), but not the S4–S5 linker. (E) Cartoon showing two opposing subunits of Kv11.1. Segments in the right-hand subunit are labeled S1–S6 and pore helix (PH). Segments in the left-hand subunit are color and number coded, according to derived Φ-values (present study and Wang et al., 2011; Perry et al., 2013), to show the relative sequence of events that occur during the open-to-inactivated gating transition, where red (1) indicates the first step and purple (8) indicates the putative final step. The S4 helix (green, 5), as well as the S4–S5 linker, experience a change in environment shortly after the S5 helix and S5P linker but before the S6 helix. We propose that hydrophobic interactions between the S4 helix and the S5 helix help couple the voltage-sensor domain to the pore domain during the open-to-inactivated gating transition of Kv11.1 channels.
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Figure 9. Homology model of the Kv11.1 channel. View parallel to the membrane of an amplified region of a Kv11.1 channel homology model created using the Kv1.2/2.1 chimera crystal structure (Long et al., 2007) as a template, according to the alignment shown in Fig. S3. The amplified region is indicated by the boxed region of the entire four-subunit homology model shown in the inset. Hydrophobic residues on the S4 helix (Leu529, Leu532, and Val535) of one subunit (sub1; shown in green, with residues colored orange) face toward hydrophobic residues on the S5 helix (Ile560, Leu564, and Ile567) of the neighboring subunit (sub2; shown in blue, with residues colored purple). The arrow represents a cavity filled by lipid in the Kv1.2/2.1 crystal structure.
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Figure 10. Double mutant cycle analysis of S4 and S5 helix residues. (A) Schematic showing the principle of double mutant cycle analysis to test for an energetic coupling between Leu529 on the S4 helix and Ile560 on the S5 helix. Perturbations to inactivation, measured by changes in log(Keq,0) relative to WT, caused by individual mutations, L529S (ΔΔGmut1) and I560A (ΔΔGmut2), are compared with the perturbation caused by the double mutant L529S + I560A (ΔΔGmut1+mut2). Measured Δlog(Keq,0) values representing ΔΔGmut1, ΔΔGmut2, and ΔΔGmut1+mut2 are shown in parentheses. An energetic coupling between Leu529 (S4) and Ile560 (S5) residues is indicated by ΔΔGmut1+mut2 being significantly different from ΔΔGmut1 + ΔΔGmut2 (P < 0.05; see Materials and methods). (B–E) Double mutant cycle analysis for S4 helix mutations L529S (B), L530S (C), L532S (D), and V535S (E), paired with mutations on the S5 helix (I560A, L564A, or I567A) or S5P linker (D591K). Measured values of Δlog(Keq,0) for individual or double mutants are shown as closed bars, whereas the theoretical additive values, ΔΔGmut1 + ΔΔGmut2, are shown by open bars. Data presented as means ± SEM for 6–14 cells (see Table S1).
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