ML277 regulates KCNQ1 single-channel amplitudes and kinetics, modified by voltage sensor state

Abstract

KCNQ1 is a pore-forming K+ channel subunit critically important to cardiac repolarization at high heart rates. (2R)-N-[4-(4-methoxyphenyl)-2-thiazolyl]-1-[(4-methylphenyl)sulfonyl]-2 piperidinecarboxamide, or ML277, is an activator of this channel that rescues function of pathophysiologically important mutant channel complexes in human induced pluripotent stem cell-derived cardiomyocytes, and that therefore may have therapeutic potential. Here we extend our understanding of ML277 actions through cell-attached single-channel recordings of wild-type and mutant KCNQ1 channels with voltage sensor domains fixed in resting, intermediate, and activated states. ML277 has profound effects on KCNQ1 single-channel kinetics, eliminating the flickering nature of the openings, converting them to discrete opening bursts, and increasing their amplitudes approximately threefold. KCNQ1 single-channel behavior after ML277 treatment most resembles IO state-locked channels (E160R/R231E) rather than AO state channels (E160R/R237E), suggesting that at least during ML277 treatment, KCNQ1 does not frequently visit the AO state. Introduction of KCNE1 subunits reduces the effectiveness of ML277, but some enhancement of single-channel openings is still observed.

Figure 1.

KCNQ1 responds to ML277 with a large increase in current. (A) Top: Representative KCNQ1 current traces (t) just as (t5) and after 1 µM ML277 was added to the bath. Cell was pulsed from a holding potential of −80 mV to +60 mV for 2 s, then for 1 s to −40 mV. The interpulse interval was 15 s. Bottom: Effect of increasing interpulse intervals at a holding potential of −80 mV on current waveform at +60 mV for 2 s in the presence of 1 µM ML277. Trace t1 obtained in control solution, others after rest intervals as indicated in the presence of ML277. (B) Representative KCNQ1 currents in control (top) and in 1 µM ML277 conditions (bottom), obtained using 4 s voltage-clamp pulses from −90 to +80 mV in 10 mV steps. Interpulse interval was 15 s, and tail currents were measured at −50 mV for 1 s. (C) Normalized G-V plots for KCNQ1 obtained from peak tail currents at different concentrations of ML277 as indicated (n = 5–11). The V_1/2 for KCNQ1 in control conditions was −20.9 ± 2.3 mV. (D) ML277 concentration-response curves at +60 mV using tail current data normalized to same cell control values and fit with drug versus response, three parameter curves to obtain EC₅₀s using GraphPad software, n = 2–6. (E) Representative single-channel recordings of GFP-tagged KCNQ1 in control conditions above (blue) and in 1 µM ML277 below (red) from the same patch. The voltage protocol is shown above the control sweeps, and the interpulse interval was 10 s. (F) Ensemble averages of 17 active sweeps of control KCNQ1 (blue trace) and after 1 µM ML277 treatment (red trace). Control traces were combined from two different patches including patch shown in A and B. ML277 traces were only from the patch shown. (G) All-points histograms of 40 KCNQ1 single-channel sweeps in control (blue bars) and 40 sweeps in 1 µM ML277 (red bars).

Figure S1.

Peak KCNQ1 currents after ML277 addition and response to HMR. (A) Diary plots of peak KCNQ1 current over time after 1 µM ML277 addition, normalized to last control level for eight different cells. Cells were held at −80 mV and pulsed to +60 mV for 2 s, then to −40 mV for 1 s repeatedly with an interpulse interval of 15 s. In a few examples (red triangles, yellow circles), after ML277 has its effect, the current begins to decrease again before stabilizing, and while the decrease did not always happen in the recording time frame, it suggests that ML277 might not act as a PIP2 mimetic given that PIP2 has previously been shown to prevent rundown of whole-cell currents (Li et al., 2011; Loussouarn et al., 2003). (B) Concentration-response curves for HMR inhibition of KCNQ1 currents under different experimental conditions; KCNQ1 pulsed to +60 mV and HMR added after washout of ML277 (red circles, IC₅₀ = 3.44 µM); KCNQ1 pulsed to +60 mV and HMR added with ML277 remaining in solution (magenta triangles, IC₅₀ = 5.88 µM); KCNQ1 pulsed to −20 mV and HMR added with ML277 remaining in solution (green triangles, IC₅₀ = 4.86 µM); and KCNQ1 in control bath solution pulsed to +60 mV (blue circles, IC₅₀ = 2.54 µM), n = 2–6. (C) Paired peak tail current amplitudes before and after exposure to 1 µM ML277. Protocol was as in A. (D) Chart of peak KCNQ1 tail current amplitudes in ML277 divided by peak current in control conditions with four different test protocols as labeled. (E) Diary plots of KCNQ1 peak currents showing response to increasing concentrations of HMR under the different experimental conditions detailed in B. A pause in the diary plot is shown where a voltage-clamp protocol was run before beginning the 10-min washout. (F) Top: G-V plot of KCNQ1 before (blue circles) and after exposure to 0.1% DMSO (cyan circles). V_1/2 of activation and slope values was −22.5 ± 1.5 mV and 12.8 ± 2.0 for control, −23.7 ± 1.8 mV and 14.5 ± 1.8 in DMSO, n = 4 for each. Cells were held at −90 mV and pulsed from −90 to +80 for 4 s, then to −40 mV for 0.9 s, with an interpulse interval of 15 s. Bottom: Plot of peak tail currents in 0.1% DMSO divided by control values after a 4-s, +60 mV pulse. (G) Ensemble averages of 53 sweeps of KCNQ1 treated with 1 µM ML277 (red) and 53 sweeps of the same cell after treatment with 20 µM HMR. Protocol is shown above. The interpulse interval was 10 s. All error bars in the figure denote mean ± SEM.

Figure 2.

ML277 increases amplitude of EQQQQ openings. (A) Representative whole-cell current traces from cells before (blue trace) and after exposure to 1 µM ML277 (red trace) for channel complexes containing increasing numbers of KCNE1. The example for EQ shows an extra sweep in orange to highlight the current rundown that took place after 8 min recording. (B) Bar chart showing the tail current amplitude in ML277 divided by control amplitude for Q1 and WT I_Ks of different stoichiometries. ****, P < 0.0001. (C) V_1/2 of activation before and after 1 µM ML277 treatment for EQ, EQQ, and EQQQQ (n = 6–12). See Table 1 for slope factors. All error bars denote mean ± SEM. (D and E) Representative single-channel recordings of EQQQQ in control (D) and in 1 µM ML277 (E) from the same patch. The voltage protocol is shown above the control sweeps, and the interpulse interval was 10 s. (F) Ensemble average currents of 16 active sweeps in control EQQQQ (blue trace) and after 1 µM ML277 treatment (red trace) from the same patch. (G) All-points histograms of the same data used in F. (H) First latencies for EQQQQ in control and ML277 conditions. Mean first latencies were 0.91 ± 0.07 s for control (108/287 active, 38%) and 0.64 ± 0.04 s in the presence of ML277 (223/703 active, 32%), n = 3.

Figure S2.

G-V plots and some representative currents for EQQQQ, EQQ, EQ, and KCNQ1 E160R mutants before and after ML277 treatment. Cartoons below construct labels denote the number and location of subunits in the channel tetramer containing WT (blue circle) or E160R containing KCNQ1 subunits (red circle) or KCNE1 (green circle). (A) Representative whole-cell currents of EQQQQ obtained using an activation protocol before (top) and after 1 µM ML277 (bottom). Cell was pulsed from a holding potential of −80 mV to +60 mV for 4 s, then for 1 s to −40 mV. The interpulse interval was 15 s. Right: G-V plots of EQQQQ before (blue circles) and after (red circles) 1 µM ML277 exposure. (B–D) Representative whole-cell currents of E160R-Q*Q (B), E160R-QQQ*Q* (C), and E160R-Q*QQ*Q* (D) obtained using an activation protocol before (top) and after 1 µM ML277 (bottom). Cells were held at −90 and pulsed from −90 to +80 or +100 in 10 mV steps for 4 s, then to −40 mV for 0.9 s. The interpulse interval was 15 s. G-V plots are shown in the graph to the right. The E160R-Q*Q record in red triangles in A represents only one cell responding as such at the 1 µM ML277 dose, red circles in A represent 2 cells, for all others in the figure n = 3 or 4. (E–G) G-V plots for E160R-Q*QQQ (E), EQQ (F), and EQ (G). Protocol as in A. (H) Plot of deactivation time constants (τ) versus voltage for EQ, EQQ, and EQQQQ before and after 1 µM ML277. See Table 2 for n values and statistics. Error bars throughout figure denote mean ± SEM.

Figure 3.

ML277 increases the amplitude of EQQ and EQ openings. (A) Single-channel recordings of EQQ (top) and EQ (bottom) before (blue traces) and after addition of 1 µM ML277 (red traces) from the same patch as control data. The voltage protocol is shown above the data. (B) All-points amplitude histograms of three active sweeps of EQQ before (blue) and after 1 µM ML277 addition (red; top) and 27 active sweeps combined from three different cells of EQ before (blue) and after 1 µM ML277 addition (bottom). (C) Scatter plot of first latencies to opening of the active sweeps for EQ, EQQ, and EQQQQ before and after ML277 exposure. See Table 3 for mean values.

Figure 4.

R2 and R4 mutants also respond to ML277 with an increase in current amplitude. (A and B) Cartoons representing the putative interactions between the charges in the S2 and S4 TMDs of the VSDs that hold them in the IO state (R2; A) and the AO state (R4; B). Next to the cartoons are representative whole-cell current traces from cells before (blue trace) and after exposure to 1 µM ML277 (red trace). Cells were held at −90 mV and pulsed for 4 s to +60 mV and then −40 mV for 0.8 s. Interpulse interval was 15 s. (C) Bar chart of peak tail currents measured in ML277 divided by control peak current measurement for R2 (n = 4) and R4 (n = 5). The mean ratio ± SEM for R2 is 3.75 ± 0.59, and for R4 it is 5.86 ± 1.30. (D) Normalized G-V plots for R2 (top) and R4 (bottom) obtained from peak tail currents at different concentrations of ML277 as indicated (n = 3–11). Voltage protocol was the same as for A and B. (E) ML277 concentration-response curves at +60 mV for R2 (n = 3–8) and R4 (n = 2–6), using tail current data normalized to same cell control values and fit with drug versus response. Three parameter curves were used to obtain EC₅₀s using GraphPad software.

Figure 5.

Increased single-channel current amplitudes in R2 and R4 mutants with ML277. (A and B) Representative single-channel recordings of R2 in control (A) and in 1 µM ML277 (B) from the same patch. The voltage protocol is shown above the control sweeps, and the interpulse interval was 10 s. (C) All-points histograms of 10 R2 single-channel recording sweeps in control (blue) and 10 sweeps in 1 µM ML277 (red) from the same patch. All-points histograms of R4 single-channel sweeps (right). (D) Representative sweeps of single-channel recordings of R4 in control conditions (upper sweep) and in 1 µM ML277 (lower sweep) from the same patch. The voltage protocol is shown above the control sweeps, and the interpulse interval was 10 s. (E) Amplitudes of openings for R2, R4, and KCNQ1 after exposure to ML277 as determined by Gaussian fits of all-points histograms. Error bars denote mean ± SEM.

Figure 6.

KCNQ1 mutants with 1–4 VSD restrained in the closed state respond to ML277 with an increase in current. (A) Cartoon shows the putative interactions between charges in S2 and S4 in the E160R mutant. Cartoons above each representative whole-cell recording denote the number and location of subunits in the channel tetramer containing WT (blue circle) or E160R subunits (red circle). Protocol is as in Fig. 4, A and B. Control traces are blue, and those in 1 µM ML277 are red. A sample from a GFP-only transfected cell is also shown. (B) Graph of the ratio of peak tail currents in ML277 to control for different channel complexes. Subunits containing E160R are denoted by an *. (C) Bar chart showing the median and range of tail current amplitudes for each channel type before (blue bars) and after ML277 (red bars). (D) G-V plots of Q*Q from peak tail currents at −40 mV, shown in control (blue circles), and at different concentrations of ML277 as indicated. All error bars denote mean ± SEM (n = 3–15). (E) ML277 concentration-response curves at +60 mV using tail current data normalized to same cell control values and fit with drug versus response. Three parameter curves to obtain EC₅₀s using GraphPad software, n = 3–7.

Figure S3.

HMR blocks ML277-enhanced Q* currents, and ML277 blocks the S338F mutant KCNQ1 channel. (A and B) Tail currents from channels made up of (A) E160R-Q (Q*) and (B) GFP transfected cells, under control conditions (blue), after 1 µM ML277 (red), after 10 µM HMR + ML277 (green), and after washout (cyan). Cells were held at −80 mV, pulsed to +60 mV for 4 s, and then pulsed to −40 mV for 0.75 s. (C) Representative whole-cell current traces from cells before (blue trace) and after exposure to 1 µM ML277 (red trace) for KCNQ1 channel containing the S338F mutation. (D) Paired peak tail current measurements before and after ML277 treatment, measured after a 4-s pulse to +60 mV. (E) Bar chart of peak tail currents measured in ML277 divided by control peak current measurement for S338F mutant. The mean was 0.42 ± 0.10 (n = 4).

Figure 7.

Open probability of ML277 activated KCNQ1 and R2 channels decrease with voltage. (A) Representative whole-cell currents of KCNQ1 obtained during a voltage-clamp protocol after 1 µM ML277 treatment. Cells were held at −90 and pulsed from −90 to +60 or +100 in 10-mV steps for 4 s, then to −40 mV for 0.9 s. The interpulse interval was 15 s. Only the −20 and 60 mV traces are shown for clarity. (B) Representative portions of cell-attached recordings of a three-channel patch of KCNQ1 + ML277 at +60 mV (upper trace) and −20 mV (lower trace). (C) All-points amplitude histograms from the tail currents of the same cell-attached patch as in B. Histograms are for 10 traces at each test pulse. (D) Representative whole-cell currents of R2 after treatment with 2 µM ML277. Protocol as in A. Only −20 and +60 mV are shown for clarity. (E) Representative portions of cell-attached recordings of a two-channel patch of R2 + 1 µM ML277 at +60 mV (upper trace) and −20 mV (lower trace). (F) Percent closed time at each voltage for an approximately two-channel patch of R2 or KCNQ1. Same protocol as in A except HP was −80 mV. Only sweeps with at least one recognizable closing could be analyzed. (G) G-V plots of R2 before (n = 3) and after treatment with 2 µM ML277 (n = 3) and R4 after treatment with 2 µM ML277 (n = 5). Protocol as in A. (H) Bar chart showing the tail current amplitude in ML277 divided by control amplitude for KCNQ1 in different concentrations of ML277. All error bars denote mean ± SEM.

Figure 8.

The K41C mutation in KCNE1 allows ML277 to activate I_Ks as long as the complexes are not fully saturated. (A) Representative whole-cell currents of K41C-E’QQQQ obtained using a voltage-clamp protocol before (top) and after 1 µM ML277 (bottom). Cells were held at −90 and pulsed from −90 to +100 in 10-mV steps for 4 s, then to −40 mV for 0.9 s. The interpulse interval was 15 s. Only even voltages are shown for clarity. The graph on the right shows the G-V plots (n = 3). (B) Representative whole-cell currents of K41C-E’QQ obtained using the same activation protocol as described in A. The upper record is before ML277, and the lower trace is after 1 µM ML277 was added to the bath. The graph on the right shows the G-V plots (n = 4 or 5). (C) G-V plots for K41C-E’Q in control conditions and in 1 µM ML277 (n = 4 or 5). (D) Bar chart shows the tail current amplitude in ML277 divided by control amplitude for WT channel complexes and those containing the K41C-KCNE1 mutant with different stoichiometries of E1:Q1. (E) Bar chart showing the tail current amplitude in ML277 divided by control amplitude for WT I_Ks and I_Ks assembled with various mutants of KCNE1. EQ and K41C-E’Q are linked constructs, and the others are cotransfected. (F) Representative whole-cell currents of KCNE5-Q obtained using the same activation protocol as described in A. The upper record is before ML277, and the middle record is after 1 µM ML277 was added to the bath. The lower panel shows a sample control trace (blue) and a trace collected in the presence of ML277. The cell was held at −90 mV, pulsed to +60 mV for 4 s, and then −40 mV for 0.8 s. The interpulse interval was 15 s. The graph on the right shows the G-V plots (n = 4). (G) Bar chart showing ratio of the tail current for KCNE5-Q in ML277 divided by control after a test pulse to 60 mV for 4 s. (H) Sequence alignment of human KCNE proteins for the TMD region and that just N-terminal to it. Arrow indicates K41 in KCNE1. All error bars denote mean ± SEM.