Inactivation of the Kv2.1 channel through electromechanical coupling

Fig. 1. Structure of the Kv2.1 channel. a, Current traces for the structural construct of Kv2.1 (1–598) recorded in 2 mM external K+ from −100 mV to +100 mV (40 mV increments) using a holding voltage of −90 mV and a tail voltage of −50 mV. Red dotted line denotes zero current. b, Normalized conductance–voltage (G–V) relations obtained using tail currents from traces like those in a and voltage–steady-state inactivation relations curves (I–V) obtained from a three-pulse protocol (Fig. 3d) comparing the structural construct (G–V, V1/2 = 1.3 ± 1.2 mV, z =2.7 ± 0.2, n = 6 cells in two independent experiments; I–V (inactivation), V1/2 = −27.8 ± 0.9, z = 3.2 ± 0.1, n = 6 cells in two independent experiments) with the full-length Kv2.1 channel (G–V, V1/2 = 0.8 ± 1.8 mV, z = 2.8 ± 0.4, n = 10 cells in eight independent experiments; I–V (inactivation), V1/2 = −23.3 ± 1.0, z = 3.9 ± 0.3, n = 3 cells in independent experiments). Solid symbols represent mean and solid lines corresponds to fits of the Boltzmann Equations. Error bars denote the standard error of the mean (s.e.m.). c,d, Side and external views of the Kv2.1 EM map (c) and model (d), with each subunit shown in different colours. EM…

Fig. 2. Epileptic encephalopathy mutations in Kv2.1. a, Mutations causing epileptic encephalopathy in humans mapped onto one monomer of Kv2.1, shown as a side view. b, G–V relations recorded for epileptic encephalopathy mutations highlighted in a and obtained from a family of voltage steps ranging from −150 to +200 mV in 100 mM external K+. Symbols represent mean and solid curves correspond to a fit of the Boltzmann equation. See Extended Data Fig. 2e for traces and Supplementary Table 1 for parameters of the fits and n values. c, Location of F412 in the Kv2.1 structure with adjacent S6 helices highlighted with different colours. d, Gating currents recorded for the F412L epileptic encephalopathy mutation from −90 mV to +50 mV in 2 mM external K+ (10 mV increments) from a holding voltage of −90 mV using a P/−4 protocol to subtract leak and capacitive currents. Red dotted line denotes zero current. e, Normalized Q–V relation obtained for F412L by integrating the OFF gating currents. Symbols represent mean and green solid curve corresponds to a fit of the Boltzmann Equation with V1/2 = −25.2 ± 0.4 mV, z = 4.4 ± 0.2 (n = 4 cells in two independent experiments). For all panels error bars are…

Fig. 3. A nexus of hydrophobic residues around F412 that are critical for inactivation. a, A nexus of hydrophobic residues around F412 in the Kv2.1 structure with cryo-EM density for hydrophobic side chains. b, Current traces obtained for Kv2.1, L316A, L403A and L329A using 2 mM external K+ and P/−4 subtraction. Holding voltage was −90 mV, steps were from −90 mV to +60 mV (+50 mV for L329A) in 20 mV increments and tail voltage was −50 mV (−90 mV for L329A). c, G–V relations obtained from tail currents for Kv2.1 (n = 13 cells in 10 independent experiments), L316A (n = 3 cells in two independent experiments) and L403A (n = 5 cells in two independent experiments) using 2 mM external K+. Holding voltage was −90 mV, voltage steps were 200 ms and tail voltage was −50 and −60 mV for Kv2.1 and L316A, respectively. For L403A, voltage steps were 20 ms and tail voltage was 0 mV. Symbols represent mean and smooth curves are fits of a Boltzmann equation (Kv2.1, V1/2 = −1.7 ± 0.8 mV, z = 2.6 ± 0.1; L316A, V1/2 = −2.9 ± 0.8, z = 2.2 ± 0.1; L403A, V1/2 = 55.9 ± 1.3 mV, z = 1.5 ± 0.1). d, Effect of Kv2.1 mutations on the rate and extent of inactivation assessed using a three-pulse (P1–P3) protocol with 2…

Fig. 4. Status of the internal gate in the F412L mutant of Kv2.1. a, Current traces for Kv2.1 (left) and W365F (right). The holding voltage was −90 mV, depolarizations were from −100 to +100 mV (20 mV increments) and tail voltage was −50 mV. External K+ was 100 mM. Red dotted line denotes zero current. b, OFF gating currents recorded for Shaker W434F and Kv2.1 F412L in control (black) or after application of internal TEA (blue) using P/−4 subtraction and 2 mM external K+. The holding voltage for Shaker W434F was −100 mV, test depolarizations were from −100 to 0 mV (10 mV increments). For Kv2.1 F412L the holding voltage was −90 mV, test depolarizations were from −90 to +50 mV (10 mV increments). c, Time constants (τ) for single exponential fits of the decay of OFF gating current in the absence or presence of internal TEA for Shaker W434F (n = 5 cells in five independent experiments) and Kv2.1 F412L (n = 5 cells in five independent experiments). d, Normalized ionic currents recorded for co-expression of Kv2.1 and the Kv2.1-F412L mutant with 2 mM external K+ at 60 mV. e, Plot of time constants (τi) of inactivation against test voltage. τ values were obtained by fitting single (Kv2.1) or…

Fig. 5. Structural basis of inactivation in Kv2.1 channels. a, Current traces for the structural L403A construct of Kv2.1 (1–598) recorded in 2 mM external K+ from −100 mV to +60 mV (20 mV increments) using a holding voltage of −100 mV. Red dotted line denotes zero current. b, Conductance–voltage (G–V) relations and voltage–steady-state inactivation relations (I–V) obtained from a three-pulse protocol (Fig. 3d) comparing the L403A Kv2.1 (1–598) (G–V, V1/2 = 56.7 ± 1.2 mV, z = 1.5 ± 0.1, n = 5 cells in two independent experiments; I–V (inactivation), V1/2 = −6.9 ± 0.7, z = 4.6 ± 0.1, n = 7 cells in two independent experiments) with the L403A mutant in the full-length Kv2.1 channel (Fig. 3c,f). G–V relations were obtained from tail currents using a holding voltage of −90 mV, 20 ms voltage steps to between −50 mV and +120 mV (10 mV increments) and a tail voltage of 0 mV. Solid symbols represent mean and solid lines corresponds to fits of the Boltzmann equation. Error bars are s.e.m. c, Side (left) and external (right) views of the Kv2.1-L403A EM map, with each subunit shown in different colours. EM densities that could correspond to lipids are in yellow. d, Model of the ion selectivity…

Extended Data Fig. 1. Biochemistry and cryo-EM imaging for the Kv2.1 channel. a) Cartoon illustrating the Kv2.1 construct used for structure determination. b) Gel filtration chromatograms for the full-length Kv2.1 channel (1-853) and the 1-598 C-terminal truncated construct in detergent solutions using a superose6 increase (10/300) column. The full-length construct showed a broader peak, suggesting heterogeneity of the protein. c) Time-course for expression of the Kv2.1 (1-598) construct in tsA201 transduced with different baculoviruses titers (indicated as %) using mVenus fluorescence in gel filtration chromatograms (superose6 increase 5/150 column) to measure expression. d) Gel filtration profile (Superose6 increase; 10/300 column) for the Kv2.1 (1-598) construct after nanodisc reconstitution. Similar profiles were observed in over 7 independent experiments. The peak in the green box was collected and concentrated for preparing cryo-EM samples. SDS-PAGE analysis of the Kv2.1 (1-598) sample used for cryo-EM data collection. See Supplementary Information Fig. 4 for uncropped gel. e) Direction distribution plots of the 3D reconstruction illustrating the distribution of particles in different…

Extended Data Fig. 2. Conformation of the Kv2.1 structure and functional properties of epileptic encephalopathy mutations. a) Side view of the model and EM maps for the S2, S3 and S4 helices within the VSD of Kv2.1. Basic residues in S4 and residues in the charge transfer center (F236, E239 and D262) are shown as sticks. b) Hole representation of the ion permeation pathway of the Kv2.1 structure and plot of pore radius along the length of the pore. c) View of the electrostatic surface of the extracellular PD of the Shaker channel (PDB 7SIP) and Kv2.1 (1-598). d) Side view of a single monomer of Kv2.1 with epileptic encephalopathy mutations shown in stick representation. Regions contributing to the PD and VSD are indicated and an expanded view of the PD is also shown. e) Current traces obtained with 100 mM extracellular K+ from cells expressing mutant Kv2.1 channels. For WT Kv2.1, S198F, T206K, T206M, L207P and K387N mutants, steps were from −100 mV to +100 mV in 40 mV increments, holding voltage was −90 mV and tail voltage was −50 mV. R302C currents were recorded using a protocol with a holding voltage of −120 mV, using a pre-pulse to −140 mV followed by a family of voltage steps from −140 mV to +80…

Extended Data Fig. 3. Gauging the movements of S4 in Kv2.1 (1-598) with disulfide and metal bridges. a) Side view of the VSD of one subunit (blue) and the PD of the adjacent subunit (red). Q293 in the R1 position of S4, T199 in the S1 helix and C232 in the S2 helix are shown as sticks. Sequence alignment shows the TM segments within the VSDs of Shaker and Kv2.1. b) Superimposed traces obtained from the same oocyte expressing the T199C/Q293C double mutant of Kv2.1 treated as indicated. Holding voltage was −90 mV, test voltage was +100 mV, tail voltage was −50 mV and external K+ was 50 mM. The control (gray) trace was obtained before any treatment, the Cu-Phe trace (dark yellow) in presence of Cu-Phe (1.5 µM–5 µM) and the DTT trace (black) after incubation with DTT (10 mM) for 10 min. c) G-V relations obtained for cells treated as in b using tail current measurements (-50 mV) and normalizing to the maximal current amplitude after DTT treatment (n = 7 cells in 2 independent experiments). d) Superimposed traces obtained from the same oocyte expressing the T199C/Q293C double mutant of Kv2.1 treated as indicated. Holding voltage was −90 mV, test voltage was +100 mV, tail voltage was −50 mV and external K+…

Extended Data Fig. 4. Exploring the non-conducting mechanism for Kv2.1 F412L. a) Close up view of the model and EM map for D378 in the P-loop and W365 in the pore helix. Dashed line represents a hydrogen bond. b) Fraction of non-inactivated channels (P3/P1) in response to the P2 family of voltage steps from −100 to +60 mV for Kv2.1 (black squares; n = 3 in 2 independent experiments) and the W365F mutation (orange circles; n = 5 in 2 independent experiments). Similar protocol to that illustrated in Fig. 3d. External K+ was 2 mM. c) Fraction of non-inactivated channels for Kv2.1 W365F recorded in 2 or 100 mM external K+ using P/−4 subtraction. Coloured diamonds denote individual experiments (n = 8 cells in 2 independent experiments for 2 mM K+ and n = 6 in 2 independent experiments for 100 mM K+) and solid black diamonds represent mean. d) Family of current traces for Kv2.1 with 2 mM external K+ in the absence and presence of internal TEA. Test depolarizations were from −100 to +50 mV in 10 mV increments from a holding voltage of −90 mV using P/−4 subtraction. Insets show tail currents recorded at –50 mV. Red line indicates zero current. e) Plot of time constant of deactivation for Kv2.1 in the absence…

Extended Data Fig. 5. 4-AP interferes with inactivation in Kv2.1. a) Current traces for Kv2.1 with 2 mM external K+ in the absence and presence of 4-AP (10 mM). Test depolarizations were from −90 to +60 mV in 20 mV increments from a holding voltage of −90 mV using a tail voltage of −60 mV and P/−4 subtraction. Red and black dashed lines indicates zero current. b) G-V relations obtained measuring the peak tail currents from traces as shown in panel a for Kv2.1 (n = 3 in 2 independent experiments) before and after 4-AP addition. Tail currents were normalized to the maximum value in control solution. c) G-V relations independently normalized to the maximum tail current amplitude recorded in the absence or presence of 4-AP. Fits of the Boltzmann equation to the data are shown as solid curves. For control, V1/2 = −1.1 ± 0.9 mV and z = 2.4 ± 0.1 For 4-AP, V1/2 = 18.1 ± 0.9 mV and z = 2.1 ± 0.1. Same cells as in panel b. d) Current families for Kv2.1 obtained using a three-pulse protocol with 2 mM external K+ in the presence of 4-AP (10 mM). Holding voltage of −100 mV, the first pulse (P1) was to +60 mV, followed by a brief closure to −100 mV, the test pulse (P2) was from −100 to +60 mV for 20 s to allow the…

Extended Data Fig. 6. Structural comparison of Kv2.1 and the L403A mutant. a) Superimposition of Kv2.1 (gray) with each of the four protomers of the L403A mutant aligned using the VSDs. b) Cryo-EM map of a single subunit of Kv2.1 (gray) with the map for protomer D in the L403A mutant (red). c) Conformational changes at the interface between S5 and S6 helices between Kv2.1 (gray) and protomer D of the L403A mutant (red). d) Conformational changes in the S6 helix between Kv2.1 (gray) and protomer D of the L403A mutant (red). Cryo-EM maps for S6 helices from protomer D in the L403A mutant (red) and Kv2.1 (gray) are shown to the right. e,f) A symmetrical L403A tetramer generated by aligning four D protomers to Kv2.1 based on selectivity filter. Dash lines indicate clashes between neighboring I405 side chains (e), between the sidechain of L329 and backbone of G312 (f) and between the sidechain of F333 and that of L313 (f). g,h) A symmetrical L403A tetramer modeled by aligning four D protomers to Kv2.1 based on the VSDs. Dash lines show clashes between neighboring I405 side chains (g) and distances between backbone carbonyls within the selectivity filter that are shorter when compared to those of Kv2.1 and…

Extended Data Fig. 7. A selectivity filter mutant interferes with inactivation in Kv2.1 L403A. a) Current traces for the Kv2.1-L403A-T373A double mutant and Kv2.1-T373A with 2 mM external K+. Test depolarizations for Kv2.1-L403A-T373A were from −90 to +60 mV in 10 mV increments from a holding voltage of −90 mV using a tail voltage of −60 mV with P/−4 subtraction. Kv2.1-T373A test depolarizations were from −90 to +50 mV in 10 mV increments from a holding voltage of −90 mV using a tail voltage of −70 mV. Red line indicates zero current. b) G-V relations obtained by measuring the peak tail currents from traces as shown in panel a for Kv2.1- T373A (blue circles, n = 6 in 4 independent experiments) and Kv2.1- L403A-T373A (inverted maroon triangles, n = 6 in 4 independent experiments). For comparison purposes, data for L403A and Kv2.1 are also shown from Fig. 3c. Symbols represent mean and solid line is a fit of a Boltzmann equation to the data for Kv2.1-T373A (V1/2 = −38.4 ± 1.5 mV and z = 3.4 ± 0.4), Kv2.1-T373A-L403A (V1/2 = −25.1 ± 1.8 mV and z = 1.9 ± 0.1). c) Plot of fraction of non-inactivated during the P2 voltage step (P3/P1) using the same protocol as in Fig. 3d for Kv2.1-T373A-L403A (inverted…