PIP2-dependent coupling of voltage sensor and pore domains in Kv7.2 channel

Figures

• 

  Fig. 1. Simulation design for identifying PIP2 localization in open and closed Kv7.2 channels. a Left: A tetrameric Kv7.2 channel used in our simulations, where each subunit is highlighted with a different color in surface representation. Right: Detailed view of the important structural features. A tetrameric human Kv7.2 channel in a closed state was modeled from the cryo-EM structure of human Kv7.1 channel with a closed pore (PDB ID: 5VMS). For modeling the Kv7.2 channel in an open state, TMD (targeted MD) was used to drive the transition of the closed Kv7.2 state model towards the template based on the open state of Kv1.2/ Kv2.1 channel (PDB: 2R9R). b Top view of the simulation replicates (Runs) showing the initial placement of 8 PIP2 molecules in both upper and lower leaflets of the lipid bilayer surrounding Kv7.2 channel in the absence of calmodulin. For each state/conformation (open or closed), 3 independent simulation replicates were performed in a tetrameric Kv7.2 channel by randomly shuffling the initial positions of PIP2 lipids, as shown by different colors (Runs 1-3). c–d Volumetric maps of PIP2 headgroup occupancy extracted collectively from the simulation trajectories are…

• 

  Fig. 2. PIP2 coordination in 4 binding sites captured in the modeled structure of the open Kv7.2 channel. a–d PIP2 coordination in Site-O1 (a), Site-O2 (b), Site-O3 (c), and Site-O4 (d). Kv7.2 protein is shown in ribbon representation with S4 in red, helices A and B in brown, and the rest of the protein in ice blue. A PIP2 lipid (carbon atoms in yellow, oxygen in red, and phosphorus atoms in tan) and the residues of the binding pocket at each site at the end of simulations are shown in sticks (basic residues in blue, polar in green, acidic in red, and hydrophobic in white). e–h The contact probability for each chemical moiety of PIP2 headgroup with the key residues in the binding Site-O1 (e), Site-O2 (f), Site-O3 (g), and Site-O4 (h). The chemical moieties of PIP2 headgroup include P1-phosphate (black), P4-phosphate (red), P5-phosphate (orange), 2-hydroxyl (cyan), 3-hydroxyl (brown), and 6-hydroxyl (pink) group of the inositol ring. A heavy-atom distance cutoff of 4 Å was used to define a contact between a protein residue and a phosphate group of PIP2, whereas a 3.5 Å cutoff was used to define a contact between a residue and a hydroxyl group of PIP2. Analysis of the contact probabilities…

• 

  Fig. 3. Charge-neutralizing mutations disrupt PIP2 binding to the mutated residues in the open Kv7.2 channel. a, d, g, j Time evolution of the distance between the COM of PIP2 headgroup and the COM of the mutated residue upon introducing mutations including R214Q (a), K219N (d), R325Q (g), and R353Q (j) in PIP2-bound Kv7.2 channel in an open state from three independent simulations (different colors). Introduction of these mutations resulted in the dissociation of bound PIP2 lipid, as indicated by the distance increase. b, e, h, k The contact probability for each chemical moiety of PIP2 headgroup with the key residues in open Kv7.2 channels containing R214Q (b), K219N (e), R325Q (h), and R353Q (k). The chemical moieties of PIP2 headgroup include P1-phosphate (black), P4-phosphate (red), P5-phosphate (orange), 2-hydroxyl (cyan), 3-hydroxyl (brown), and 6-hydroxyl (pink) group of the inositol ring. A heavy-atom distance cutoff of 4 Å was used to define a contact between a residue and a phosphate group of PIP2, whereas a 3.5 Å cutoff was used to define a contact between a residue and a hydroxyl group of PIP2. Analysis of the contact probabilities was performed over the last 200 ns of the…

• 

  Fig. 4. Charge-neutralizing mutations of potential PIP2 binding residues decrease voltage- and PIP2-dependent activation of homomeric Kv7.2 channels. Whole cell voltage clamp recordings of macroscopic K+ currents through Kv7.2 WT or mutant channels were performed in CHO hm1 cells cotransfected with GFP and PIP5K. The DNA plasmid ratio was 0.2:0.45:0.45 (GFP: Kv7.2 WT or mutant: PIP5K). Cells were held at −80 mV. K+ currents were evoked by depolarizing voltage steps for 1.5 s from −100 mV to +20 mV in 10-mV increments, followed by a step to 0 mV for 300 ms. a Representative traces after subtraction of leak currents. Leak current was defined as non-voltage-dependent current from GFP-transfected cells. Note different Y-axis scales for systems with and without PIP5K. b Average peak current densities (pA/pF) of Kv7.2 WT or mutant channels with or without PIP5K coexpression at all voltage steps. c Average peak current densities of WT or mutant Kv7.2 channels at +20 mV. p values are computed from one-way ANOVA post-hoc Fisher’s test. d Normalized conductance (G/Gmax) at all voltage steps. e Average activation constant (τ) αt +20 mV. The number of GFP-positive cells that were recorded without…

• 

  Fig. 5. Charge-neutralizing mutations of potential PIP2 binding residues alter Kv7.2 current response to Dr-VSP activation. a Representative current traces showing Dr-VSP-mediated Kv7.2 current decay in CHO hm1 cells co-expressing Dr-VSP and Kv7.2 WT or mutants from −20 mV to +100 mV. CHO cells were held at –70 mV, and a brief voltage step to –60 mV was applied to calculate the linear leak. 10 s step depolarizations were applied in 20-mV steps from –20 to +100 mV with 2 min inter-step intervals to allow PIP2 regeneration. Red trace shows current decay curve when cells were held at +40 mV. b Ratio of current decay in Kv7.2 WT or mutant channels at each voltage step. c Kv7.2 current decay ratio at +40 mV, +60 mV, +80 mV and +100 mV. The number of cotransfected cells that were recorded with EGFP-tagged Dr-VSP: Kv7.2 WT (n = 27), R214Q (n = 14), K219N (n = 13), R325Q (n = 12), R353Q (n = 15), R214Q/ K219N (n = 13) or R214Q/K219N/R353Q (n = 11). The source data for Fig. 5b is available in Figshare93. Data represent the mean ± SEM. One-way ANOVA Fisher’s test results are shown (*p < 0.05, **p < 0.01 and ***p < 0.005).

• 

  Fig. 6. Charge-neutralizing mutations of potential PIP2 binding residues in Site-O4 disrupt the correlated motions of the VSD and the S6 gate of Kv7.2 channels. Network-based community analysis in WT (a) and mutant Kv7.2 channels containing R214Q (b), K219N (c), and R325Q (d) mutations in Site-O4. A community represents a set of residues that move in a correlated manner during the MD simulations. The thickness of the edges between the amino acids within each community corresponds to the strength of correlation between them. The network analysis was performed after combining all 3 independent simulation trajectories. a Binding of PIP2 at Site-O4 in the WT channel results in correlated motions of the VSD (the S4 and the S4-S5 linker) and the gate S6, highlighted by a single community in green. b Introduction of the R214Q mutation leads to uncorrelated motions of the VSD and the gate, highlighted by 3 separate communities colored in pink, green, and gray. c Introduction of the K219N mutation leads to uncorrelated motions of the VSD and the gate, highlighted by the smaller community in green and the emergence of an additional community colored in pink. d Kv7.2-R325Q channels displayed weaker…

• 

  Fig. 7. PIP2-mediated conformational changes of helices A and B in open Kv7.2 channel. a Snapshot of a representative Kv7.2 monomer in PIP2-free membranes. b Snapshot of a representative Kv7.2 monomer in PIP2-containing membranes. c Orientation angle of helices A and B with respect to the membrane normal. The distribution of the orientation angle of helices A and B in WT Kv7.2 channel over the last 200 ns of all the simulations in membranes with (w/) or without (w/o) PIP2 as well as that of mutant Kv7.2-R353Q channel in membranes with PIP2. In PIP2-free membranes, the helical pair of the WT channel adopts a largely solvent-exposed conformation. Addition of PIP2 induces a drastic conformational change in these helices from a solvent-exposed conformation to a bilayer-interacting conformation. However, the R353Q mutation in the AB linker moves the helices closer to a solvent-exposed conformation even in the presence of PIP2. d Large-scale conformational change in the cytoplasmic helices provides a pathway for PIP2 movement along Helix-B to its ultimate localization at Site-O4. All the phosphorus atoms of the bilayer are shown in vdW (van der Waals) representation. Kv7.2 protein is shown in…