A PIP2 substitute mediates voltage sensor-pore coupling in KCNQ activation

Abstract

KCNQ family K⁺ channels (KCNQ1-5) in the heart, nerve, epithelium and ear require phosphatidylinositol 4,5-bisphosphate (PIP₂) for voltage dependent activation. While membrane lipids are known to regulate voltage sensor domain (VSD) activation and pore opening in voltage dependent gating, PIP₂ was found to interact with KCNQ1 and mediate VSD-pore coupling. Here, we show that a compound CP1, identified in silico based on the structures of both KCNQ1 and PIP₂, can substitute for PIP₂ to mediate VSD-pore coupling. Both PIP₂ and CP1 interact with residues amongst a cluster of amino acids critical for VSD-pore coupling. CP1 alters KCNQ channel function due to different interactions with KCNQ compared with PIP₂. We also found that CP1 returned drug-induced action potential prolongation in ventricular myocytes to normal durations. These results reveal the structural basis of PIP₂ regulation of KCNQ channels and indicate a potential approach for the development of anti-arrhythmic therapy.

Fig. 1. A site in KCNQ1 for CP1 interaction.

a PIP₂ (upper) and CP1 (lower) molecules. The head of PIP₂ that was used for molecular similarity calculations is marked with a rectangle. The negatively charged groups of the two molecules are marked with circles. b Left, PIP₂ docked on the KCNQ1 and calmodulin complex. Right, magnified view of PIP₂ (upper) or CP1 (lower) docked on KCNQ1 at the VSD-PD interface, and the residues interacting with PIP₂ or CP1 are indicated. Two neighboring subunits of KCNQ1 are colored sky blue and pink, respectively. The bound calmodulins are colored light gray and tan, respectively. c Mutations alter CP1 effects on KCNQ1 conductance–voltage (G–V) relations. The mutated residues are predicted to interact with CP1 in docking. G–V relations of the wild type (WT) and mutant KCNQ1 in the absence (open symbols) or presence (solid symbols) of 10 µM CP1 are shown. d The effect of 10 µM CP1 on G–V shift in voltage range of WT and mutant KCNQ1 (WT n = 11 and mutant n = 3–7). V_1/2 is the voltage where conductance G is half-maximum, and ΔV_1/2 = V_1/2 (CP1) − V_1/2 (control). *Residues predicted to interact with CP1 in docking. N.C. no current expression. ^#Significantly different from the WT (p ≤ 0.05, Tukey–Kramer ANOVA test). For this and subsequent figures, error bar represents standard error of mean (sem), n ≥ 3 unless specified otherwise. In this and other experiments, except for those indicated otherwise, CP1 was applied to the bath solution.

Fig. 2. CP1 rescues KCNQ1 currents after PIP₂ depletion.

a, b KCNQ1 currents recorded from a Xenopus oocyte co-expressed with CiVSP in response to voltage pulses to +60 mV (the voltage protocol is depicted in the inset in (a). Currents of the first trace control (black), after rundown (gray), and after injection of ~10 µM CP1 into oocytes (red) are shown (a). Averaged time course of normalized current amplitude of KCNQ1 with rundown (black) and after CP1 injection (red) (b) (n = 3). c, d I_Ks (KCNQ1 + KCNE1) co-expressed with CiVSP in response to voltage pulses to +60 mV. Currents of the first trace control (black), after rundown (gray), after bath application of 10 µM CP1 (red), and after bath application of 100 µM chromanol 293B (blue) are shown (c). Averaged time course of normalized current amplitude of I_Ks with rundown (black), CP1 application (red), and chromanol 293B (d) (n = 7). e, f I_Ks currents recorded in the inside-out patch in response to voltage pulses to +80 mV (the voltage protocol is depicted in the inset in (e). Representative I_Ks current traces ran down after patch excision due to PIP₂ depletion (e, upper), and rescued by 10 µM CP1 application (e, lower). The changing color of the current traces and arrows indicates the time sequence of rundown and rescue (e). Normalized current amplitude following patch excision and CP1 application (n = 3) (f).

Fig. 3. CP1 modulates voltage-dependent activation of the KCNQ1 channel.

a KCNQ1 currents elicited in the absence and presence of 10 µM CP1. From a holding potential of −80 mV, test pulses were applied once every 20 s to potentials ranging from −120 mV to +80 mV with 10-mV increments (the voltage protocol is depicted in the inset). The tail currents were elicited at −40 mV. b Current–voltage relations of KCNQ1 in the absence or presence of 10 µM CP1. c Voltage-dependent activation curves (G–V) of KCNQ1 in the absence or presence of 10 µM CP1. d Time constants of deactivation, obtained by fitting an exponential function to the currents, in the absence or presence of 10 µM CP1. e Dose response for mean ΔV_1/2 of activation induced by CP1. f Current traces of pseudo WT KCNQ1 elicited in the absence and presence of 10 µM CP1. g G–V curves of pseudo WT KCNQ1 in the absence or presence of 10 µM CP1 (n = 11). h Fluorescence traces of pseudo WT KCNQ1 in the absence and presence of 10 µM CP1. i Fluorescence at steady-state voltage relations of pseudo WT KCNQ1 in the absence or presence of 10 µM CP1 (n = 7).

Fig. 4. Effects of CP1 on I_Ks channels.

a, b I_Ks currents in the absence (left) and presence of 2 µM CP1 (right) recorded with 100-s and −120-mV interval pulses between testing voltages. Holding potential: −80 mV; testing potential: −120 to +80 mV; returning potential: −40 mV (a). G–V relations (b). c, d I_Ks recorded at the same conditions as in (a), except for the interval pulses, was 20 s at −80 mV. e, f I_Ks in the absence or presence of 10 µM CP1. The voltage protocol was the same as in (b). g, h Current (g) and relative conductance (h) increases of I_Ks channels by 10 µM CP1. In h, the measured conductance in CP1 at all voltages was divided by the maximum measured conductance in control, and then normalized with the maximum measured conductance in control being 1. i Dose response for ΔV_1/2 of G–V relations to different CP1 concentrations as compared with those of KCNQ1 (dashed line).

Fig. 5. Subtype selectivity of CP1 on KCNQ channels.

a Current traces of KCNQ channels as indicated in the absence and the presence of 10 µM CP1. Holding potential: −80 mV, test pulses: −120 to +80 mV with 10-mV step, and returning potential: −40 mV. b–d Effects of 10 µM CP1 on the outward current at −10 mV, (KCNQ1/E1 at +40 mV) (b), ΔV_1/2 (c), and deactivation time constant from an exponential fit to the current at −120 mV (d) for different KCNQ channels. In b, # indicates significant change of current by CP1.

Fig. 6. Effects of CP1 on other ion channels expressed in Xenopus oocytes.

a Currents of Kir1.1, Ca_V1.2, Na_V1.5, hERG, and K_V4.2 before and after application of 10 µM CP1, respectively. The voltages for holding, test, and returning pulses were Kir1.1, 0 mV, −100 to +60 mV, 0; Ca_V1.2, −100 mV, −100 to +60, −100 mV; Na_V1.5, −100, −120 to +40 mV, −120 mV; hERG, −80 mV, −90 to +60 mV, −60 mV; K_V4.2, −80 mV, −100 mV to +80 mV, −40 mV; HCN4, −30 mV, −30 to −140 mV, −120 mV. b Current–voltage relations of Kir1.1 channel in the absence or presence of 10 µM CP1 (n = 7). c–g G–V relations of indicated channels with and without 10 µM CP1. The ΔV_1/2 of G–V relations (mV) are Ca_V1.2, 3.38 ± 0.71 mV (c); Na_V1.5, −2.26 ± 0.95 mV (d); hERG, 6.85 ± 1.81 mV (e); K_V 4.2, −2.26 ± 2.88 mV (f); HCN4, 17.13 ± 1.59 mV (g).

Fig. 7. Effects of CP1 on I_Ks and action potentials in cardiomyocytes.

a I_Ks currents in guinea pig ventricular myocytes in the absence and presence of 30 µM CP1 in the whole-cell patch-clamp configuration. Holding potential: −40 mV; testing potentials: −20 to +60 mV with 10-mV increment; returning potential: −20 mV. The I_Ks and its tail currents at the returning potential in control were obtained by subtracting the currents in the presence of chromanol 293B (10 µM) from those in the control only, and the I_Ks and its tail currents in the presence of CP1 were obtained by subtracting the currents in the presence of CPI (30 µM) plus 293B (10 µM) from those in the CPI only. b Averaged I_Ks currents in control and different CP1 concentrations [CP1] at 60 mV. c Dose response of I_Ks channels at +60 mV for CP1, EC₅₀ = 7.54 µM. d Averaged I_Ks tail currents in control and different [CP1] at −20 mV. e Dose response for V_1/2 of activation induced by CP1 for I_Ks channels, EC₅₀ = 7.86 µM (n = 6). f Effects of CP1 on normal actional potential duration (APD). Guinea pig ventricular myocytes were first perfused with 10 µM CP1 and 10 µM chromanol. After APD reached steady state, 10 µM CP1 was constitutively perfused alone. Last, CP1 was washed out for near-full reversal of APD shortening. g Effects of 0.2 µM CP1 on LQT action potentials. To mimic the LQT, 100 µM moxifloxacin was applied 2 h before the treatment of CP1. h Change of action potential duration after application of different [CP1]s (n = 5–7). Tukey–Kramer ANOVA test was used to compare control cells in different CP1 concentration, # is significant at P < 0.05. Unpaired two-tailed Student t tests were used to compare control and moxifloxacin cells at different CP1 concentration: * is significant at P < 0.05.

Fig. 8. Residues important for VSD-pore coupling in KCNQ1.

The residues are shown as colored sticks in the cryo-EM structure of hKCNQ1 (PDB entry: 6uzz). The colors indicate residues in the classic interactions (blue, including V254, H258, A341, P343, and G345), the interactions specifically when the VSD is at the activated state (cyan, including M238, L239, D242, R243, W248, L250, L251, V255, F256, Y267, I268, L271, G272, F335, S338, F339, and L342), specific interactions with PIP₂ (orange, T247, R259, Q260, and T264), specific interactions with CP1 (magenta, R249 and S253), and interactions with both PIP₂ and CP1 (red, K354 and K358). a One KCNQ1 subunit. b Enlarged frame. c Enlarged frame from different views.