Calmodulin acts as a state-dependent switch to control a cardiac potassium channel opening

Figures

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  Fig. 1. The S2-S3 linker within the KCNQ1 VSD modulates KCNQ1 currents. (A and B) Structural depiction of one monomer of the tetrameric KCNQ1/CaM complex when determined in the absence (A) and presence (B) of PIP2 [i.e., CTD-bent (A) and CTD-straight (B) conformations]. (C) Left: A simplified KCNQ1 gating model (16, 18–21) with two open states: IO and AO. Horizontal transitions correspond to VSD activation, vertical transitions correspond to pore opening. RC, resting closed; IC, intermediate closed; AC, activated closed. Right: Exemplar biexponential activation kinetics fitting of a Q1-WT normalized current. Voltage was stepped from −80 to 0 mV. (D) Sequence alignment of the KCNQ family and KCNA2 at the S2-S3 linker region predicted to interact with CaM. (E) Mutagenesis screen of the KCNQ1 S2-S3 linker region shown in (D). Y axis: ΔV1/2 from WT channel [see (F) for example]. X axis: residue position. Stars indicate significant ΔV1/2 from WT (table S1). Error bars are SEM. (F) Left: Exemplar recordings from KCNQ1 WT and S2-S3 linker mutant channels. Voltage protocol is shown in inset. Currents were collected with 10-mV increments at the test pulse but shown in 20-mV increments to avoid…

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  Fig. 2. CaM interacts with the KCNQ1 S2-S3 linker during voltage-dependent gating. (A) Structural depiction of CaM residues predicted to interact with Q1-S182 in the CTD-bent structure. Residues depicted with sticks are colored according to residue type: positively (negatively) charged, blue (red); polar, green. Ca2+-ions are shown as yellow spheres. (B) Summary ΔV1/2 of human Q1-WT coexpressed with mutant CaM from Q1-WT coexpressed with CaM-WT (table S2). CaM-N98S and CaM-F142L are LQTS-associated CaM mutants. Purple bars and stars indicate significant ΔV1/2. Error bars are SEM. (C) Exemplar currents from coexpression of CaM-Y100D with Q1-WT (left) or Q1-S182K (right) channels. Voltage protocol in inset. (D) Average V1/2 for Q1-WT and -S182K channels associated with either CaM-Y100D (YD) or endogenously supplied CaM-WT (endo). Long bar, mean; short bars, SEM. Star, significantly different. P values, calculated by t test, are 2.18 × 10−8 (Q1-WT/CaM-WT to Q1-WT/CaM-Y100D), 5.25 × 10−17 (Q1-WT/CaM-WT to Q1-S182K/CaM-WT), and 0.73 (Q1-S182K/CaM-WT to Q1-S182K/CaM-Y100D). n.s., not significant. (E) Exemplar biexponential fits of activation kinetics for Q1-WT and -S182K channels associated…

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  Fig. 3. Network analysis of MD simulations demonstrates that the CaM-VSD interactions modulate KCNQ1 VSD-pore allosteric pathways. (A) Flowchart of MD simulation analysis pipeline. The KCNQ1/CaM complex cryo-EM structure was inserted into a membrane and simulated with MD in explicit solvent. The protein was transformed into a network; each node is a residue while each edge weight corresponds to interaction strength between residues. Network analysis was applied to track information flow from defined source residues (nodes) to sink residues. High–information flow nodes carry key allosteric pathways from the source to sink residues. (B) CTD-bent information flow (VSD gating charges to the pore) through each residue projected with color intensity onto the KCNQ1/CaM CTD-bent structure. Dark color, high information flow (capped at 0.02). Source residues (gating charges), orange spheres; sink residues (pore), magenta spheres. Two full subunits out of four are shown for clarity; the VSDs, HA/HB, and CaMs in the front and back subunits are omitted. (C) Information flow profiles of human KCNQ1 and CaM along residue sequence. Light purple shadings, rough position of the S2-S3 linker/CaM interface.…

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  Fig. 4. CaM-VSD interactions control AO state occupancy. (A) VCF experiments using KCNQ1* psWT. Left and right columns: Exemplar ionic current/VSD fluorescence signal measurements (voltage protocols in inset) and average G-V/F-V relationships, respectively. The F-V relationship (solid black curve) is fitted with a double Boltzmann equation (F1 and F2, dotted black curves), see Materials and Methods. (B) Same as (A), but for KCNQ1* S182K mutant channel. In G-V and F-V plots: black, psWT; red, S182K. (C) KCNQ1 gating model with two open states, IO and AO. The KCNQ1 VSD transitions into the intermediate and activated conformations engage distinct sets of VSD-pore coupling interactions (green and violet boxes) to trigger pore opening. (D) Left: Cartoon schematic illustrating the effect of KCNE1 coexpression (top) and Q1-S338F mutation (bottom) to ablate the IO and AO states respectively to leave KCNQ1 to conduct only at one open state. Right: Current exemplars for Q1-WT and mutant channels designed to probe whether Q1-S182K mutation specifically affects IO or AO state. Each box shows enlarged tail currents, with the red trace indicating the tail current associated with the labeled test…

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  Fig. 5. A hub in the KCNQ1 CTD cooperates with the CaM-VSD interactions to control AO state occupancy. (A) Left: Information flow projection onto the CTD-bent structure, capped at 0.02. For clarity, VSDs, HA/HB, and CaMs in the front and back subunits are omitted. Right: Orange, information flow profile along KCNQ1 sequence (CaM C-lobe used as the source) in the CTD-bent structure; dark blue, information flow profile with gating charges as source. Purple shading, information flow peak in the S2-S3 linker/CaM interface. Green shading, peak between the HB/HC and HA/S6 loops. (B) Δinformation flow projected on the CTD-bent structure (CaM C-lobe versus the gating charges as the source). Dark violet residues, decreased information flow as a result of using CaM C-lobe as source; brown, increased information flow. (C) Structural depiction of residues in the CTD hub. Green ribbons, HA; blue ribbons, HB; pink ribbons, HC; gray ribbons, S6. S6′ is from a neighboring subunit. Residues depicted with sticks are colored according to residue type: positively (negatively) charged, blue (red); hydrophobic, white. (D) Scaled distance density of interacting residues in the CTD hub.

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  Fig. 6. Functional validation that the CTD cooperates with CaM-VSD interactions in KCNQ1 gating. (A) Cartoon depiction of KCNQ1 channel with key CTD residues (blue and green circles) predicted to be allosterically downstream of the CaM-VSD interaction (red circle). (B) Current exemplars for Q1-R360E and Q1-S182K/R360E mutants. Voltage protocol in inset. (C and D) Top and middle: Current exemplars for mutants involving the S2-S3 linker (S182K), the proximal HA (P369R), and the HB-HC loop (P535A). Bottom: Average G-V relationships calculated for the respective mutants. Dashed lines are G-V fits for Q1-WT (gray) and Q1-S182K (red). N = 7 (Q1-P369R), 5 (Q1-S182K/P369R), 8 (Q1-P535A), and 3 (Q1-S182K/P535A). Q1-P369R versus WT ΔV1/2 = −4.77 ± 1.24 mV, P < 0.01. Q1-P535A versus WT ΔV1/2 = 0.75 ± 0.79 mV, P = 0.35. P values are calculated with t test assuming unequal variance. (E) KCNQ1 voltage-dependent gating model integrating CaM and PIP2. Color designations: KCNQ1 channel (dark gray), KCNQ1 S2-S3 linker (light blue star), KCNQ1 CTD hub (green diamond), CaM (orange), PIP2 (violet), and S4 in the VSD is colored red, yellow, and green for resting, intermediate, and activated states,…