KCNE1 enhances phosphatidylinositol 4,5-bisphosphate (PIP2) sensitivity of IKs to modulate channel activity

Abstract

Phosphatidylinositol 4,5-bisphosphate (PIP(2)) is necessary for the function of various ion channels. The potassium channel, I(Ks), is important for cardiac repolarization and requires PIP(2) to activate. Here we show that the auxiliary subunit of I(Ks), KCNE1, increases PIP(2) sensitivity 100-fold over channels formed by the pore-forming KCNQ1 subunits alone, which effectively amplifies current because native PIP(2) levels in the membrane are insufficient to activate all KCNQ1 channels. A juxtamembranous site in the KCNE1 C terminus is a key structural determinant of PIP(2) sensitivity. Long QT syndrome associated mutations of this site lower PIP(2) affinity, resulting in reduced current. Application of exogenous PIP(2) to these mutants restores wild-type channel activity. These results reveal a vital role of PIP(2) for KCNE1 modulation of I(Ks) channels that may represent a common mechanism of auxiliary subunit modulation of many ion channels.

Fig. 1.

KCNE1 slows the PIP₂-dependent channel rundown. (A) KCNQ1 and KCNQ1 + KCNE1 currents at various times after patch excision. Voltage was stepped from a holding potential of -80 to +80 mV and then back to the holding potential. (B) Time dependence of current amplitude after patch excision. Normalized tail current amplitude at various time, I_t/I₀, is plotted; I₀ is the tail current amplitude immediately following patch excision. KCNQ1 (filled circles), n = 9; KCNQ1 + KCNE1 (open circles), n = 12; KCNQ1 + KCNE1 + 10 μM PIP₂ (open squares), n = 6; KCNQ1 + KCNE1 coexpressed with Ci-VSP (open diamonds), n = 6. Solid lines are monoexponential fits to data. Voltage was stepped from a holding potential of -80 mV to +80 mV for 5 s and then back to the holding potential for 5 s. (C) Time constants of exponential fits and initial delay to current rundown. (D) Normalized tail current amplitude at various times, I_t/I₀, with KCNQ1: KCNE1 mRNA ratio 1∶1 (open circles), n = 12; 1∶0.05 (open diamonds), n = 6; 1∶0.01 (open squares), n = 6; 1∶0 (filled circles), n = 9. (E) Time constants of exponential fits and initial delay to current rundown. In this and other figures, the data are presented as mean ± SEM.

Fig. 2.

KCNE1 enhances PIP₂ sensitivity. (A) Normalized tail current amplitude at various times following patch excision and PIP₂ application. PIP₂ was applied at all times after the arrow. Voltage protocol to elicit the currents is the same as in Fig. 1. (B) PIP₂ dose response of WT KCNQ1 and WT and mutant KCNQ1 + KCNE1 channels. I_rec is tail current amplitude at steady state after recovery of channel activity following PIP₂ application. I₀ is the tail current amplitude immediately following patch excision. KCNQ1 (filled circles), KCNQ1 + KCNE1 (open circles), R539W + KCNE1 (open diamonds), and R555C + KCNE1 (open squares). Solid curves are fits to the Hill equation. Hill coefficients are 2.0, 1.5, 1.6, and 1.0 for WT KCNQ1 + KCNE1, R555C + KCNE1, R539W + KCNE1, and KCNQ1, respectively. For KCNQ1 + KCNE1, the responses of currents to diC8-PIP₂ (up triangles) and diC4-PIP₂ (down triangles) (5 and 500 μM) are also plotted. (C) EC₅₀ calculated by the Hill fit (n = 6–9).

Fig. 3.

Molecular determinants of PIP₂ sensitivity in KCNE1. (A) Normalized tail current amplitude at various times following patch excision. WT KCNQ1 (filled circles), n = 9; WT KCNQ1 + KCNE1 (⋈), n = 9; KCNQ1 + mutant KCNE1: R67Q (up triangles), n = 6; K69C (open diamonds), n = 6; K70C (open circles), n = 6; H73N (open squares), n = 6; R67E/K69E/K70E triple mutation (TM), (down triangles), n = 6. (B) Time constants of exponential fits and initial delay to current rundown. The axis is labeled with the KCNE1 construct that was coexpressed with KCNQ1. (-) indicates KCNQ1 alone. (C) The transmembrane segment and juxtamembraneous C-terminal region of the KCNE1 NMR structure (PDB ID code 2K21). The residues important for PIP₂ sensitivity are shown in stick presentation.

Fig. 4.

LQT mutations in KCNE1 reduce PIP₂ sensitivity. (A) Whole-cell currents and (B) conductance-voltage (G-V) relation in oocytes. WT KCNQ1 + KCNE1 (open squares), n = 6; KCNQ1 + mutant KCNE1: R67C (filled circles), n = 6; R67H (filled diamonds), n = 6; K70N (filled triangles), n = 6; K70M (filled squares), n = 6; Voltages were stepped from a holding potential of -80 mV to a test potential (-80 to +80 mV) and then repolarized to -40 mV. (C) Whole-cell current amplitude measured +80 mV normalized by that of WT channels (filled bars) and V_1/2 measured from G-V relation (open bars). (D) PIP₂ dose response of KCNQ1 + mutant KCNE1 channels: K70N (filled circles), R67C (open circles). Solid curves are fits to the Hill equation. Hill coefficients are 3 and 4, and EC50 are 114 μM and 99 μM for KCNQ1 + K70N and KCNQ1 + R67C, respectively. (E) Tail current amplitude in response to the same voltage protocol as described in Fig. 1 for WT and mutant channels. (filled bars) Indicate the tail current amplitude immediately following patch excision. (open bars) Indicate the tail current amplitude after steady-state recovery of channel activity in response to 300 uM exogenous PIP₂. (F) G-V relations of KCNQ1 + K70N (filled symbols) and WT KCNQ1 + KCNE1 (unfilled) before and after patch excision in PIP₂-containing solutions. KCNQ1 + K70N: on cell (filled circles), V_1/2 = 48.5 ± 2.2 mV; excised in 50 μM PIP₂ (filled diamonds), V_1/2 = 50.7 ± 0.8 mV; excised in 150 μM PIP₂, (filled triangles), V_1/2 = 29.9 ± 3.2 mV; excised in 300 μM PIP₂ (filled squares), V_1/2 = 8.9 ± 2.6 mV. KCNQ + KCNE1: on cell (open circles), V_1/2 = 21.6 ± 3.2 mV; excised in 300 μM PIP₂ (open squares), V_1/2 = 8.5 ± 1.2 mV. n = 2 for G-V relation of KCNQ1 + K70N in 50 μM PIP₂, n = 6–12 for all other experiments.

Fig. 5.

PIP₂ modulation is independent of phosphomimetic mutations of I_Ks channels. (A) Whole-cell currents of S27D/S92D channels. To measure deactivation time constants, voltages were stepped from a holding potential of -80 mV to a depolarization potential (+40 mV) and then repolarized to test potentials (-70 to 0 mV) (Left). To measure the G-V relation, voltages were stepped from a holding potential of -80 mV to a test potential (-80 to +80 mV) and then repolarized to -40 mV (Right). (B) Time constant of deactivation vs. voltage and (C) G-V relation. WT KCNQ1 + KCNE1 (open circles), n = 6; S27D/S92D + KCNE1 (filled circles), n = 7. (D) Time dependence of normalized tail current amplitude after patch excision. WT KCNQ1 + KCNE1 (open circles), n = 12; S27D/S92D + KCNE1 (filled circles), n = 6. Solid lines are monoexponential fits to data. (E) Time constant of tail currents in D. The deactivation time constant of the mutant channel is significantly larger than that of the WT (P < 0.05).

Fig. 6.

Partial sequence alignment of all members of the KCNE family. Numbering indicates the position on KCNE1. The filled box highlights the predicted transmembrane domain (TMD) (53). Open boxes indicate conserved residues that are key determinants of PIP₂ sensitivity.