Two-stage electro–mechanical coupling of a KV channel in voltage-dependent activation

Figures

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  Fig. 1. Classic interactions are necessary for E–M coupling when the VSD transitions into the intermediate state. a VCF recordings of pseudo-WT KV7.1* (KV7.1-C214A/G219C/C331A). KV7.1+KCNE1 currents are shown at the same scale. The F–V relationships (blue circles) are fitted with a double Boltzmann function. F1–V represents the VSD transition from resting to intermediate state; F2–V represents the VSD transition from the intermediate to activated state. G-V represents channel opening with VSD transition at both the intermediate (IO) and the activated (AO) states. b Cartoon scheme illustrating the gating mechanism of two-step VSD movements and distinct two open states. c VCF recordings of V254M in KV7.1*. V254M+KCNE1 currents are shown at the same scales. The F–V relationships of KV7.1* and V254M are shown in gray and blue, respectively. d Cartoon scheme illustrating that V254M disrupts E–M couplings for both IO and AO. e Summary of data for WT and mutant KV7.1. VSD activation (blue, percentage change in fluorescence) and pore opening (black, current amplitude) are normalized to the WT. n ≥ 3. Blank: cells not injected with channel mRNA. Data points are shown in small open circles. f V50…

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  Fig. 2. LQTS mutation W248R specifically eliminates the E–M coupling when the VSDs adopt the activated conformation. a, b VCF recordings of W248R in KV7.1*. W248R+KCNE1 currents are shown at the same scale. The F–V relationships of KV7.1 and W248R are shown in gray and blue, respectively. n ≥ 3. All averaged data are shown in mean±SEM. c Representative currents of W248R/E1R/R2E and W248R/E1R/R4E activated from −120 mV to 60 mV with 20 mV increments. Same scale for both currents. The inset shows western blot data for membrane expression of W248R/E1R/R4E. d VCF recordings of W248R/F351A. e Cartoon scheme illustrating that W248R specifically disrupts the AO E–M coupling. f Mapping the key residues at the S4-S5L/S6c interface (green, V254, H258, A341, P343, and G345) in Fig. 1, and W248 and S338 (blue) onto the KV7.1 cryoEM structure (PDB: 5VMS)2. Source data are provided as a Source Data file.

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  Fig. 3. Key residues involved in the E–M coupling when the VSD adopts the activated conformation. a A cartoon scheme to illustrate that ML277 specifically enhances AO state E–M coupling27. b Currents of WT KV7.1 and W248R before and after adding 1 µM ML277. Voltage: +40 mV then returned to −40 mV. c 1 µM ML277-induced current increase on KV7.1 WT and with mutations in the VSD, S4-S5L, S5, and S6. The residues were mutated to alanine (A), tryptophan (W), or to known LQTS mutations. The dotted line shows 2X the standard error below the average current increase of WT KV7.1. Mutations that show ML277-induced current increases (mean + SEM) below the dotted line are labeled as red. N.C. mutations show little or no current. Inact. mutations show obvious c-type like inactivation5. Data points are shown in small open circles. d Current ratios of IIKs/IKv7.1 (black) and IE1R/R4E/IE1R/R2E (blue) for WT and mutant KV7.1. Star indicates LQTS-associated mutations. e V50 values of G–V, F1–V, and F2–V relations for WT and mutant KV7.1 channels. Data points are shown in small open circles. f Current ratios of IIKs/IKv7.1 (black) and IE1R/R4E/IE1R/R2E (blue) for WT and the S4c mutant channels. g V50…

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  Fig. 4. E–M coupling interactions when the VSD adopts the activated conformation. a, b Double mutant cycle analysis of the interaction between L251 and I268. ∆∆G = ∆G – (∆G1 + ∆G2) = 1.8 kcal/mol, which indicates interaction between the two residues. n ≥ 3. All averaged data are shown in mean ± SEM. c Double mutant cycle analysis of interactions between W248 and I268, L251 and I268, L251 and F339, M238 and L271, and L239 and L271 (∆∆G = 1.4–4.4 kcal/mol). n ≥ 3. d Mapping the five pairs of interacting residues onto the KV7.1 cryoEM structure. Color codes are the same as in Fig. 3h. Source data are provided as a Source Data file.

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  Fig. 5. MD simulation correlates temporal sequence of VSD movement to engagement of the two-stage E–M coupling interactions. a Interaction frequencies of KV7.1 residue pairs as observed during the MD trajectories for the KV7.1 RC, IO, AO, and AC models. Frequency threshold of 0.4 or higher is defined as indicative of interaction between two residues (see Methods). b Representations of the IO state model of a KV7.1 subunit. Inset: enlarged structures of the boxed area. Blue, residues identified as important for E–M coupling in experiments (Fig. 1c–g); gray: V255 and T265, which were identified in MD simulations, but for which mutations still show functional currents (Fig. 3c). Residue V255 was found to be important for the AO state E–M coupling (Fig. 3). Each pair of residues is represented by a black dashed line connecting their respective sidechains. c Interaction frequencies of AO model-specific (top), IO model-specific (bottom left), and in both IO and AO models (bottom right) residue pairs during the MD trajectories. d Representations of the AO (top) and IO states (bottom) models of KV7.1. An entire subunit (in gray) as well as S5' and S6' segments of an adjacent subunit (in purple)…

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  Fig. 6. Statistical coupling analysis suggest conservation of the two-stage E–M coupling across domain-swapped KV superfamily. a Sample multiple sequence alignment (MSA) containing domain-swapped KV channels (KV1-KV9) used as input for statistical coupling analysis (SCA). The bottom histogram shows counts for pairwise sequence identity between all pairs of residues within the MSA. MSA uses the nomenclature established by the HUGO Gene Nomenclature Committee (HGNC) for voltage-gated potassium channels80. b SCA covariance matrix calculated by SCA (see Methods). X and Y axes indicate amino acid positions, while colors indicate the degree of covariance, with blue and red corresponding to lowest and highest degrees of covariation. c The independent component (IC) submatrix visualizes a subset of the SCA matrix (panel b) in which the residues are the highest covariant. Each IC represents a group of co-varying/co-evolving amino acids as calculated by SCA (see Methods). Axes are amino acid position numbers. Diagonal boxes indicate covariation within each IC, off-diagonal boxes indicate cross talk between ICs. Sector 1 and sector 2 were defined by combining the IC as shown (also see Methods and…