Long QT mutations at the interface between KCNQ1 helix C and KCNE1 disrupt I(KS) regulation by PKA and PIP₂

Abstract

KCNQ1 and KCNE1 co-assembly generates the I(KS) K(+) current, which is crucial to the cardiac action potential repolarization. Mutations in their corresponding genes cause long QT syndrome (LQT) and atrial fibrillation. The A-kinase anchor protein, yotiao (also known as AKAP9), brings the I(KS) channel complex together with signaling proteins to achieve regulation upon β1-adrenergic stimulation. Recently, we have shown that KCNQ1 helix C interacts with the KCNE1 distal C-terminus. We postulated that this interface is crucial for I(KS) channel modulation. Here, we examined the yet unknown molecular mechanisms of LQT mutations located at this intracellular intersubunit interface. All LQT mutations disrupted the internal KCNQ1-KCNE1 intersubunit interaction. LQT mutants in KCNQ1 helix C led to a decreased current density and a depolarizing shift of channel activation, mainly arising from impaired phosphatidylinositol-4,5-bisphosphate (PIP2) modulation. In the KCNE1 distal C-terminus, the LQT mutation P127T suppressed yotiao-dependent cAMP-mediated upregulation of the I(KS) current, which was caused by reduced KCNQ1 phosphorylation at S27. Thus, KCNQ1 helix C is important for channel modulation by PIP2, whereas the KCNE1 distal C-terminus appears essential for the regulation of IKS by yotiao-mediated PKA phosphorylation.

Keywords: Arrhythmia; IKS; KCNE; KCNQ; Long QT; Potassium channel.

Fig. 1.

Effects of KCNE1 C-terminus deletions and LQT5 mutations on I_KS currents. (A) A cartoon of KCNE1, indicating the location of the LQT5 mutations and the deletions in the C-terminus. Representative current traces of WT KCNQ1, co-expressed in CHO cells with WT KCNE1 (B), LQT5 mutant V109I (C), LQT5 mutant P127T (D) or with KCNE1 deletion mutants at the distal C-terminus Δ109–129 (E) or the proximal C-terminus Δ69–77 (F). Cells were held at −90 mV. Membrane voltage was stepped for 3 s from −50 mV to +60 mV (or to +100 mV for Δ69–77) in 10 mV increments and then repolarized for 1.5 s to −60 mV. Current–voltage (G) and conductance–voltage (H) relationships of WT KCNQ1+WT KCNE1 or LQT5 mutants (n = 11–17). Current–voltage (I) and conductance–voltage (J) relationships of WT KCNQ1+WT KCNE1 or deletion mutants (n = 7–10).

Fig. 2.

LQT1 and LQT5 mutations disrupt the interaction between KCNQ1 helix C and KCNE1 C-terminus. (A) Representative immunoblot of GST pulldown (GST PD) (upper row) of the His-MBP-tagged KCNE1 C-terminus (residues 67–129, E1CT in diagram) or control His-MBP peptide, by GST-tagged KCNQ1 helix C (residues 535–572, Q1 Helix C, in diagram), illustrating the direct interaction of KCNE1 C-terminus with KCNQ1 helix C (n = 4). Equal amounts of His-MBP and His-MBP-tagged KCNE1 were used, as seen in the input (lower row). (B) Representative immunoblot of GST pulldown (upper row) of the His-tagged WT KCNQ1 C-terminus (residues 352–622 Δ396–504, Q1CT in diagram) by GST-tagged KCNE1 C-terminus deletion mutants (E1CT in diagram). Inputs are shown in the lower row. (C) Quantification of the pulldowns normalized to input (n = 5). *P<0.05, **P<0.01. (D) Representative immunoblot of GST pulldown (upper row) of the His-tagged WT KCNQ1 C-terminus (residues 352–622 Δ396–504) or LQT1 mutants by GST-tagged KCNE1 C-terminus (77–129). Inputs are shown in the lower row. (E) Representative immunoblot of GST pull-down (upper row) of His-tagged WT KCNQ1 C-terminus (residues 352–622 Δ396–504) by GST-tagged WT KCNE1 C-terminus (residues 67–129) or LQT5 mutants. Inputs are shown in the lower row. (F,G) Quantification of the pulldown shown in D and E, respectively, and normalized to input (n = 3–5). *P<0.05; **P<0.01; ***P<0.001.

Fig. 3.

LQT1 and LQT5 mutations do not affect the channel trafficking to the plasma membrane. (A) TIRF fluorescence images of CHO cells co-expressing CFP-tagged WT KCNQ1 and GPI-citrine. (B) Epi-fluorescence (left panels) and TIRF fluorescence (right panels) images of CHO cells expressing CFP-tagged WT KCNQ1+KCNE1 (upper panels) or CFP-tagged G589D LQT1 mutant+KCNE1 (lower panels). (C) TIRF fluorescence images of CHO cells co-expressing CFP-tagged helix C LQT1 mutants with KCNE1. (D) YFP-tagged WT KCNE1 or LQT5 mutant co-expressed with WT KCNQ1. Quantification of the TIRF fluorescent signals of CFP-tagged WT KCNQ1 and LQT1 mutants (E) and of YFP-tagged WT and LQT5 mutant (F) as normalized to membrane signal area (n = 16–60). ***P<0.001. Scale bars: 10 µm.

Fig. 4.

P127T LQT5 mutation and Δ109–129 deletion at the KCNE1 distal C-terminus suppress the cAMP-mediated upregulation of I_KS current. Representative current traces recorded from CHO cells co-expressing WT KCNQ1+WT yotiao and WT KCNE1 (A), KCNE1 P127T (C), KCNE1 Δ109–129 (E) and KCNE1 V109I (G). Cells were held at −90 mV and stepped to +30 mV for 3 s and then repolarized to −40 mV for 1.5 s. Cells were recorded in the absence (black traces) or presence (red traces) of 200 µM cAMP+0.2 µM okadaic acid. Current–voltage relationships of WT KCNQ1+WT yotiao and WT KCNE1 (B) or KCNE1 P127T (D) or KCNE1 Δ109–129 (F) or KCNE1 V109I (H). Voltage was stepped for 3 s from −50 mV to +60 mV in 10 mV increments followed by repolarization to −40 mV for 1.5 s. Red and black curves represent recordings with or without cAMP+okadaic acid in the patch pipette, respectively. (n = 6–7). Above +30 mV, the WT I_KS and, above +20 mV, the KCNE1 V109I current upregulation induced by cAMP+okadaic acid were significant. *P<0.05, **P<0.01.

Fig. 5.

LQT5 P127T mutant and deletion mutant Δ109–129 impair phosphorylation of KCNQ1 at S27 during cAMP-dependent stimulation. (A) Representative immunoblots (IB) of HEK 293 cells lysates co-expressing yotiao, WT KCNQ1 and WT, mutated (V109I and P127T) or truncated (Δ109–129) KCNE1 from cells treated in the absence and presence of 250 µM 8CPT+0.2 µM okadaic acid and immunoblots of HEK 293 cells lysates co-expressing yotiao WT KCNE1 and WT KCNQ1 or LQT1 mutant K557E from cells treated in the absence and presence of 250 µM 8CPT+0.2 µM okadaic acid. Blots were probed with antibodies against phosphorylated KCNQ1 S27 (first row from top), KCNQ1 (second row from top), yotiao (third row from top) and KCNE1 (fourth row from top). (B) Quantification of phosphorylated KCNQ1 S27 was calculated by dividing phosphorylated signal to KCNQ1 input (anti-KCNQ1 blot) for both unstimulated and cAMP-stimulated cells (250 µM 8CPT+0.2 µM okadaic acid) and normalized to stimulated WT KCNQ1+WT KCNE1+yotiao (n = 3–5). *P<0.05, ** P<0.01, ***P<0.001; ns: not statistically significant. (C) An alternative quantification of phosphorylated KCNQ1 S27 was expressed as the relative cAMP+okadaic acid-stimulated S27 phosphorylation where the extent of induced phosphorylation of WT I_KS was equal to 1 (n = 3–5). *P<0.05; ns: not statistically significant.

Fig. 6.

Effects of KCNQ1 helix C LQT1 mutations on I_KS currents. A cartoon of one KCNQ1 subunit indicating the location of helix C in the C-terminus and its sequence, where LQT1 mutations are situated (A). Representative current traces of WT KCNE1 co-expressed in CHO cells with WT KCNQ1 (B), LQT1 mutants S546L (C), R555C (D), R555H (E), K557E (F) and R562M (G). The voltage-clamp protocol is the same as that described in Fig. 1, except that higher membrane voltages of up to +160 mV were applied for some LQT1 mutations, because of their depolarizing shift. Current–voltage (H) and conductance–voltage relationships of WT and KCNQ1 LQT1 mutants co-expressed with WT KCNE1 (n = 5–9) (I).

Fig. 7.

LQT1 mutations in KCNQ1 helix C exhibit greater sensitivity to the voltage-sensitive phosphatase Dr-VSP. (A) Representative current traces of WT I_KS (KCNQ1+KCNE1) sensitivity to the voltage sensitive phosphatase Dr-VSP recorded by means of a tri-pulse protocol where membrane potential is first stepped to +10 mV (3 s, from −90 mV holding potential) to open the channel, followed by a +100 mV voltage step (2 s) to activate Dr-VSP and then return to +10 mV (3 s). This protocol was repeatedly applied every 10 s for 2 min. Left panel shows WT I_KS sensitivity to protocol application in the absence of Dr-VSP at the first (black trace) and last sweeps (red trace). Right panel shows WT I_KS sensitivity to protocol application in the presence of Dr-VSP at the first (black trace) and last sweeps (red trace) (n = 5). (B,C,E,G,I,K) Representative current traces of WT I_KS, S546L, R555C, R555H, K557E and R562M sensitivity to Dr-VSP recorded by one pulse protocol, where membrane potential is stepped to +100 mV (2 s, from −90 mV holding potential) to both open the channels and activate Dr-VSP. Shown are current traces at time 0 (black trace) and at time 10 s (red trace). Kinetics of current decline for WT I_KS and mutant channels are shown in the absence or presence of Dr-VSP. The comparison between WT I_KS and the five LQT1 mutants was quantified at an early time point (10 s) before the current drop reached steady state and was expressed as a ratio of currents measured at time 10 s over that recorded at time 0. At 10 s, ratios of current decline were 0.75±0.05 (n = 5), 0.74±0.02 (n = 7), 0.32±0.01 (n = 3, P<0.01), 0.54±0.06 (n = 4, P<0.05), 0.55±0.04 (n = 7, P<0.05) and 0.48±0.04 (n = 7, P<0.01) for WT, S546L, R555C, R555H, K557E and R562M, respectively.

Fig. 8.

LQT1 mutations in KCNQ1 helix C show impaired binding to PIP₂. (A) Representative immunoblot of PIP₂ pulldown (PIP2 PD) by PIP₂-coated agarose beads of His-tagged WT KCNQ1 C-terminus or LQT1 mutants (upper row). Inputs are shown in the lower row. (B) Quantification of the pull-down normalized to input (n = 3). *P<0.05; **P<0.01; ***P<0.001. (C) Cartoon summarizing the two different pathways involved in the mechanisms of LQT mutations located at the interacting KCNE1 distal C-terminus and KCNQ1 helix C module: one involving LQT1 mutants in KCNQ1 helix C that impairs PIP2 interaction and one of the LQT5 mutant P127T at the distal KCNE1 C-terminus, which impedes I_KS regulation by yotiao-mediated PKA phosphorylation.