Pharmacological rescue of specific long QT variants of KCNQ1/KCNE1 channels

Figures

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  FIGURE 1. ML277 and R-L3 modulate wild type IKS channels in the absence of KCNE1. Individual current traces recorded in response to 2 s depolarizing steps to +60 mV from (A) 70 mV holding potential are shown as insets in panels (B,C). Currents are shown in the absence (black traces) and presence (red traces) of test drugs. (A). Control drug-free recording of wild type KCNQ1 channel activity. (B). KCNQ1 conductance-/voltage relationships recorded in the absence (black) and presence (red) of 1 μM ML277. (C). KCNQ1 conductance-/voltage relationships recorded in the absence (black) and presence (red) 1 μM R-L3. (D). Deactivation tail time constants measured at −40 mV in the absence (control) and presence of 1 μM ML277 and 1 μM R-L3. (E). KCNQ1 tail current amplitude (measured at −40 mV) in the absence (control) and presence of ML277 (1 μM) and R-L3 (1 μM). (F). Current density measured at +60 mV for the conditions indicated on the abscissa. Data from individual experiments in (E,F) are connected by straight lines.

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  FIGURE 2. ML277 and R-L3 do not modulate IKS channels in the presence of KCNE1. Individual current traces recorded in response to 2 s depolarizing steps to +60 mV from (A) 70 mV holding potential are shown as insets in panels (B,C) in the absence (black) and presence (red) of test drugs. (A). Control KCNQ1-KCNE1 channel activity recorded at +60 mV. (B). KCNQ1-KNCE1 conductance-voltage curves in the absence (black) and presence (red) of 1 μM ML277. (C). KCNQ1-KNCE1 conductance-voltage curves in the absence (black) and presence (red) of 1 μM R-L3. (D). Deactivation tail time constants measured at −40 mV in the absence (control) and presence of 1 μM ML277 and 1 μM R-L3. (E). Summary data for peak KCNQ1-KCNE1 current density measured at +60 mV in the absence (control) and presence of 1 μM ML277 and 1 μM R-L3. Data from individual experiments are connected by straight lines.

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  FIGURE 3. ML277 and R-L3 do not enhance channel activity carried by KCNQ1 expressed with the D76N mutant KCNE1 subunit. (A). Conductance voltage curves of KCNQ1-D76N channels in the absence (black) and presence (red) of 1 μM ML277. (B). Conductance voltage curves of KCNQ1-D76N channels in the absence (black) and presence (red) of 1 μMR-L3. (C). Tail current density measured at −40 mV in the absence (control) and presence of 1 μM ML277 and 1 μM R-L3. Data from individual experiments are connected by straight lines. (D). Deactivation time constants measured at −40 mV in the absence (control) and presence of 1 μM ML 277 and 1 μM R-L3.

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  FIGURE 4. Potentiation of KCNQ1-L51H channel activity by ML277 and R-L3. (A). Normalized conductance/voltage relationship for KCNQ1/KCNE1-L51H channels before (black traces) and after (red traces) application of 1 μM ML277. The inset shows representative currents recorded at +60 mV. (B). Normalized conductance/voltage relationship for KCNQ1/KCNE1-L51H channels before (black traces) and after (red traces) application of 1 μM R-L3. The inset shows representative currents recorded at +60 mV. (C). Tail current amplitudes measured at −40 mV in control and 1 μM ML277 and 1 μM R-L3 solutions. (D). Summary data for KCNQ1/KCNE1-L51H peak current density measured 2 s at +60 mV in the absence and presence of 1 μM ML277 and 1 μM R-L3. Data in each cell are connected by straight lines. (E). Summary of measurement of slow deactivation time constant (τ50) in the absence and presence of both drugs. ML277: n = 10; R-L3: n = 9.

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  FIGURE 5. Potentiation of KCNQ1- G52R channel activity by ML277 and R-L3. (A). Normalized conductance/voltage relationship for KCNQ1-KCNE1-G52R channels before (black traces) and after (red traces) application of 1 μM ML277. The inset shows representative currents recorded at +60 mV. (B). Normalized conductance/voltage relationship for KCNQ1-KCNE1-G52R channels before (black traces) and after (red traces) application of 1 μM R-L3. The inset shows representative currents recorded at +60 mV. (C). Deactivation of tail current amplitude measured at −40 mV in control and 1 μM ML277 and 1 μM R-L3 solutions. (D). Summary data for peak KCNQ1/KCNE1-G52R channel activity measured 2 s at +60 mV in control and 1 μM ML277 and 1 μM R-L3 solutions. The currents measured in each cell are connected by straight lines. (E). Summary of slow deactivation time constant (τ50) measured at −40 mV in the absence and presence of 1 μM solutions of both drugs: ML-277: n = 6; R-L3: n = 10, R-L3.

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  FIGURE 6. Heterozygous KCNQ1- L51H mutant channel activity is potentiated by ML277 and R-L3. Cells were transfected with KCNQ1/KCNE1/KCNE1-L51H and exposed to 1 μM ML277 (A) and 1 μM R-L3 (B). Transfections were carried out with KCNQ1 and KCNE1 WT and KCNE1 mutant ratios of 1:0.5:0.5 as described in the text. Currents recorded at +60 mV are shown before (black traces) and after (red traces) exposure to drugs. (C). Summary data, in which peak currents measured at + 60 mV at 2 s in the absence and presence of drugs are connected by straight lines. (D). Normalized conductance/voltage curves measured in the absence (black) and presence (red) of 1 μM ML 277. (E). Normalized conductance/voltage curves measured in the absence (black) and presence (red) of RL-3. (F). Summary of slow deactivation time constant (τ50) in the absence and presence of 1 μM of both drugs. ML277: n = 14; R-L3: n = 10.

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  FIGURE 7. Heterozygous KCNE1-G52R mutant channel activity is potentiated by KCNQ1 agonists. Cells were transfected with KCNQ1/KCNE1/KCNE1-G52R and exposed to 1 μM ML277 (A) and 1 μM R-L3 (B). Transfections were carried out with KCNQ1 and KCNE1 WT and KCNE1 mutant ratios of 1:0.5:0.5 as described in the text. Currents recorded at +60 mV are shown before (black traces) and after (red traces) exposure to drugs. (C). Summary data for current density measured at +60 mV at 2 s in which straight lines connect peak current in the absence and presence of drugs in individual cells. (D). Normalized KCNQ1/KCNE1/KCNE1-G52R conductance/voltage curves measured in the absence (black) and presence of 1 μM ML 277 (red). (E). Normalized KCNQ1/KCNE1/KCNE1-G52R conductance/voltage curves measured in the absence (black) and presence of 1 μM RL-3 (red). (F). Summary of slow deactivation time tail constant (τ50) measured at −40 mV in the absence and presence of 1 μM each drug. ML277: n = 14; R-L3: n = 20.

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  FIGURE 8. Select LQT-1 mutant channel currents are augmented by ML277 and R-L3. (A). Cells were transfected with KCNQ1-S546L without KCNE1. (B). KCNQ1-S546L-KCNE1 channel activity measured at +60 mV in the absence (boack) and presence (red) of 1 µM ML277. (C). KCNQ1-S546L-KCNE1 channel activity measured at +60 mV in the absence (boack) and presence (red) of 1 μM R-L3. (D). Normalized conductance/voltage curves measured in the absence (black curves) and presence (red curves) of 1 μM ML 277 (left panel) or RL-3 (right panel). (E) Summary data for peak currents measured after 2 s depolarization to +60 mV in the absence (control) and presence of 1 µM ML277 or 1 μM R-L3 with straight lines joining recordings for individual cells.

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  FIGURE 9. Select LQT-1 mutant channel currents are not augmented by ML277 and R-L3. (A). Expression of KCNQ1-T265I without KCNE1 recorded at +60 mV. (B). KCNQ1-T265I-KCNE1 channel activity at +60 mV in the absence (black trace) and presence (red trace) of 1 µM ML277. (C). KCNQ1-T265I-KCNE1 channel activity at +60 mV in the absence (black trace) and presence (red trace) of 1 μM R-L3. (D). Normalized KCNQ1-T265I-KCNE1 channel conductance/voltage curves measured in the absence (black) and presence of 1 μM ML277 (red curve, left panel) or RL-3 (red curve right panel). (E). Summary data for peak current density measured after 2 s depolarization to +60 mV in the absence (control) and presence of 1 μM of each drug with straight lines joining recordings for individual cells.

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  FIGURE 10. Cd2+ sensitivity of KCNQ1 and KCNE1 mutants. (A) Currents in response to voltage step to +40 mV from −80 mV with a tail voltage of −40 mV in the absence (control) and presence of 0.5 mM cadmium. (B) Percent block of 0.5 mM cadmium for each mutation (n > 3). Significance tested by ANOVA with the Dunnett’s test for multiple comparisons with Control (Q1+E1-G55C). Ns = non-significant, *p < 0.05.