Zou X et al. (2022), Pharmacological rescue of specific long QT vari...

XB-ART-59504

Front Physiol 2022 Jan 01;13:902224. doi: 10.3389/fphys.2022.902224.

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

Zou X , Wu X , Sampson KJ , Colecraft HM , Larsson HP , Kass RS .

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The congenital Long QT Syndrome (LQTS) is an inherited disorder in which cardiac ventricular repolarization is delayed and predisposes patients to cardiac arrhythmias and sudden cardiac death. LQT1 and LQT5 are LQTS variants caused by mutations in KCNQ1 or KCNE1 genes respectively. KCNQ1 and KCNE1 co-assemble to form critical IKS potassium channels. Beta-blockers are the standard of care for the treatment of LQT1, however, doing so based on mechanisms other than correcting the loss-of-function of K+ channels. ML277 and R-L3 are compounds that enhance IKS channels and slow channel deactivation in a manner that is dependent on the stoichiometry of KCNE1 subunits in the assembled channels. In this paper, we used expression of IKS channels in Chinese hamster ovary (CHO) cells and Xenopus oocytes to study the potential of these two drugs (ML277 and R-L3) for the rescue of LQT1 and LQT5 mutant channels. We focused on the LQT1 mutation KCNQ1-S546L, and two LQT5 mutations, KCNE1-L51H and KCNE1-G52R. We found ML277 and R-L3 potentiated homozygote LQTS mutations in the IKS complexes-KCNE1-G52R and KCNE1-L51H and in heterogeneous IKS channel complexes which mimic heterogeneous expression of mutations in patients. ML277 and R-L3 increased the mutant IKS current amplitude and slowed current deactivation, but not in wild type (WT) IKS. We obtained similar results in the LQT1 mutant (KCNQ1 S546L/KCNE1) with ML277 and R-L3. ML277 and R-L3 had a similar effect on the LQT1 and LQT5 mutants, however, ML277 was more effective than R-L3 in this modulation. Importantly we found that not all LQT5 mutants expressed with KCNQ1 resulted in channels that are potentiated by these drugs as the KCNE1 mutant D76N inhibited drug action when expressed with KCNQ1. Thus, our work shows that by directly studying the treatment of LQT1 and LQT5 mutations with ML277 and R-L3, we will understand the potential utility of these activators as options in specific LQTS therapeutics.

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Species referenced: Xenopus laevis
Genes referenced: arfgap1 kcne1 kcnq1
GO keywords: potassium channel activity

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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 Î¼M ML277. (C). KCNQ1 conductance-/voltage relationships recorded in the absence (black) and presence (red) 1 Î¼M R-L3. (D). Deactivation tail time constants measured at â40 mV in the absence (control) and presence of 1 Î¼M ML277 and 1 Î¼M R-L3. (E). KCNQ1 tail current amplitude (measured at â40 mV) in the absence (control) and presence of ML277 (1 Î¼M) and R-L3 (1 Î¼M). (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.
	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 Î¼M ML277. (C). KCNQ1-KNCE1 conductance-voltage curves in the absence (black) and presence (red) of 1 Î¼M R-L3. (D). Deactivation tail time constants measured at â40 mV in the absence (control) and presence of 1 Î¼M ML277 and 1 Î¼M R-L3. (E). Summary data for peak KCNQ1-KCNE1 current density measured at +60 mV in the absence (control) and presence of 1 Î¼M ML277 and 1 Î¼M R-L3. Data from individual experiments are connected by straight lines.
	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 Î¼M ML277. (B). Conductance voltage curves of KCNQ1-D76N channels in the absence (black) and presence (red) of 1 Î¼MR-L3. (C). Tail current density measured at â40 mV in the absence (control) and presence of 1 Î¼M ML277 and 1 Î¼M R-L3. Data from individual experiments are connected by straight lines. (D). Deactivation time constants measured at â40 mV in the absence (control) and presence of 1 Î¼M ML 277 and 1 Î¼M R-L3.
	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 Î¼M 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 Î¼M R-L3. The inset shows representative currents recorded at +60 mV. (C). Tail current amplitudes measured at â40 mV in control and 1 Î¼M ML277 and 1 Î¼M 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 Î¼M ML277 and 1 Î¼M R-L3. Data in each cell are connected by straight lines. (E). Summary of measurement of slow deactivation time constant (Ï50) in the absence and presence of both drugs. ML277: n = 10; R-L3: n = 9.
	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 Î¼M 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 Î¼M R-L3. The inset shows representative currents recorded at +60 mV. (C). Deactivation of tail current amplitude measured at â40 mV in control and 1 Î¼M ML277 and 1 Î¼M R-L3 solutions. (D). Summary data for peak KCNQ1/KCNE1-G52R channel activity measured 2 s at +60 mV in control and 1 Î¼M ML277 and 1 Î¼M R-L3 solutions. The currents measured in each cell are connected by straight lines. (E). Summary of slow deactivation time constant (Ï50) measured at â40 mV in the absence and presence of 1 Î¼M solutions of both drugs: ML-277: n = 6; R-L3: n = 10, R-L3.
	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 Î¼M ML277 (A) and 1 Î¼M 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 Î¼M 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 (Ï50) in the absence and presence of 1 Î¼M of both drugs. ML277: n = 14; R-L3: n = 10.
	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 Î¼M ML277 (A) and 1 Î¼M 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 Î¼M ML 277 (red). (E). Normalized KCNQ1/KCNE1/KCNE1-G52R conductance/voltage curves measured in the absence (black) and presence of 1 Î¼M RL-3 (red). (F). Summary of slow deactivation time tail constant (Ï50) measured at â40 mV in the absence and presence of 1 Î¼M each drug. ML277: n = 14; R-L3: n = 20.
	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 ÂµM ML277. (C). KCNQ1-S546L-KCNE1 channel activity measured at +60 mV in the absence (boack) and presence (red) of 1 Î¼M R-L3. (D). Normalized conductance/voltage curves measured in the absence (black curves) and presence (red curves) of 1 Î¼M 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 ÂµM ML277 or 1 Î¼M R-L3 with straight lines joining recordings for individual cells.
	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 ÂµM ML277. (C). KCNQ1-T265I-KCNE1 channel activity at +60 mV in the absence (black trace) and presence (red trace) of 1 Î¼M R-L3. (D). Normalized KCNQ1-T265I-KCNE1 channel conductance/voltage curves measured in the absence (black) and presence of 1 Î¼M 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 Î¼M of each drug with straight lines joining recordings for individual cells.
	FIGURE 10. Cd2+ sensitivity of KCNQ1 and KCNE1 mutants. (A) Currents in response to voltage step to +40 mV from â80 mV with a tail voltage of â40 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 Dunnettâs test for multiple comparisons with Control (Q1+E1-G55C). Ns = non-significant, *p < 0.05.
	Supplementary Figure S1. Examples of currents elicited by step depolarization to +60 mV in cells expressing WT KCNQ1 (A); WT KCNQ1 expressed with WT KCNE1 (B); WT KCNQ1 expressed with KCNE1-L51H (C); and WT KCNQ1 expressed with KCNE1-G52R.

References [+] :

Barhanin, K(V)LQT1 and lsK (minK) proteins associate to form the I(Ks) cardiac potassium current. 1996, Pubmed, Xenbase