
==== Front
Circ Res
Circ Res
RES
Circulation Research
0009-7330
1524-4571
Lippincott Williams & Wilkins Hagerstown, MD

39166328
CIRCRES2024325009D
00004
10.1161/CIRCRESAHA.124.325009
3
10001
10003
10005
10032
Original Research
Targeting the IKs Channel PKA Phosphorylation Axis to Restore Its Function in High-Risk LQT1 Variants
https://orcid.org/0009-0005-2191-2521
Zhong Ling *
https://orcid.org/0000-0002-4416-9743
Yan Zhenzhen *
https://orcid.org/0009-0005-2146-7798
Jiang Dexiang
https://orcid.org/0000-0002-0549-755X
Weng Kuo-Chan
https://orcid.org/0000-0001-7735-5334
Ouyang Yue
https://orcid.org/0009-0002-7615-6522
Zhang Hangyu
https://orcid.org/0009-0008-0261-9794
Lin Xiaoqing
https://orcid.org/0009-0007-4702-9539
Xiao Chenxin
https://orcid.org/0000-0003-2358-1840
Yang Huaiyu
https://orcid.org/0000-0003-1844-3988
Yao Jing
https://orcid.org/0000-0003-0877-2438
Kang Xinjiang
https://orcid.org/0000-0002-5902-2277
Wang Changhe
https://orcid.org/0000-0002-7092-7587
Huang Chen
https://orcid.org/0000-0001-7263-4748
Shen Bing
https://orcid.org/0000-0002-0666-9827
Chung Sookja Kim
https://orcid.org/0000-0002-7956-2481
Jiang Zhi-Hong
https://orcid.org/0000-0003-0927-5687
Zhu Wandi
https://orcid.org/0000-0002-9758-7922
Neher Erwin
https://orcid.org/0000-0002-3696-3955
Silva Jonathan R.
https://orcid.org/0000-0001-7694-2262
Hou Panpan
Dr. Neher’s Biophysics Laboratory for Innovative Drug Discovery, State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology, Taipa, Macao SAR, China (L.Z., Z.Y., D.J., Y.O., H.Z., X.L., C.X., C.H., B.S., S.K.C., Z.-H.J., E.N., P.H.).
Macau University of Science and Technology Zhuhai MUST Science and Technology Research Institute. Zhuhai, Guangdong, China (L.Z., Z.Y., D.J., Y.O., H.Z., X.L., C.X., C.H., B.S., S.K.C., Z.-H.J., E.N., P.H.).
Department of Biomedical Engineering, Center for the Investigation of Membrane Excitability Disorders, Cardiac Bioelectricity and Arrhythmia Center, Washington University, St. Louis, MO (K.-C.W., J.R.S.).
Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University (H.Y.).
State Key Laboratory of Virology, Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, TaiKang Center for Life and Medical Sciences, Frontier Science Center for Immunology and Metabolism, Wuhan University, China (J.Y.).
Key Laboratory of Medical Electrophysiology, Ministry of Education of China, Collaborative Innovation Center for Prevention and Treatment of Cardiovascular Disease and the Institute of Cardiovascular Research, Southwest Medical University, Luzhou, China (X.K.).
Department of Neurosurgery, the Affiliated Hospital of Southwest Medical University, Luzhou, China (X.K.).
College of Life Sciences, Liaocheng University, China (X.K.).
Department of Neurology, First Affiliated Hospital, Neuroscience Research Center, Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi’an Jiaotong University, China (C.W.).
Cardiovascular Medicine Division and Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA (W.Z.).
Correspondence to: Jonathan R. Silva, PhD, Department of Biomedical Engineering, Washington University in St Louis One Brookings Dr, Whitaker Hall, Room 290G, St Louis, MO 63130, Email jonsilva@wustl.edu
Panpan Hou, PhD, Dr. Neher's Biophysics Laboratory for Innovative Drug Discovery, State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology, H710, Macau University of Science and Technology, Taipa, Macao SAR, China 999078, Email pphou@must.edu.mo
21 8 2024
13 9 2024
135 7 722738
13 6 2024
5 8 2024
9 8 2024
© 2024 The Authors.
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Circulation Research is published on behalf of the American Heart Association, Inc., by Wolters Kluwer Health, Inc. This is an open access article under the terms of the Creative Commons Attribution Non-Commercial-NoDerivs License, which permits use, distribution, and reproduction in any medium, provided that the original work is properly cited, the use is noncommercial, and no modifications or adaptations are made.

BACKGROUND:

The KCNQ1+KCNE1 (IKs) potassium channel plays a crucial role in cardiac adaptation to stress, in which β-adrenergic stimulation phosphorylates the IKs channel through the cyclic adenosine monophosphate (cAMP)/PKA (protein kinase A) pathway. Phosphorylation increases the channel current and accelerates repolarization to adapt to an increased heart rate. Variants in KCNQ1 can cause long-QT syndrome type 1 (LQT1), and those with defective cAMP effects predispose patients to the highest risk of cardiac arrest and sudden death. However, the molecular connection between IKs channel phosphorylation and channel function, as well as why high-risk LQT1 mutations lose cAMP sensitivity, remain unclear.

METHODS:

Regular patch clamp and voltage clamp fluorometry techniques were utilized to record pore opening and voltage sensor movement of wild-type and mutant KCNQ1/IKs channels. The clinical phenotypic penetrance of each LQT1 mutation was analyzed as a metric for assessing their clinical risk. The patient-specific–induced pluripotent stem-cell model was used to test mechanistic findings in physiological conditions.

RESULTS:

By systematically elucidating mechanisms of a series of LQT1 variants that lack cAMP sensitivity, we identified molecular determinants of IKs channel regulation by phosphorylation. These key residues are distributed across the N-terminus of KCNQ1 extending to the central pore region of IKs. We refer to this pattern as the IKs channel PKA phosphorylation axis. Next, by examining LQT1 variants from clinical databases containing 10 579 LQT1 carriers, we found that the distribution of the most high-penetrance LQT1 variants extends across the IKs channel PKA phosphorylation axis, demonstrating its clinical relevance. Furthermore, we found that a small molecule, ML277, which binds at the center of the phosphorylation axis, rescues the defective cAMP effects of multiple high-risk LQT1 variants. This finding was then tested in high-risk patient-specific induced pluripotent stem cell–derived cardiomyocytes, where ML277 remarkably alleviates the beating abnormalities.

CONCLUSIONS:

Our findings not only elucidate the molecular mechanism of PKA-dependent IKs channel phosphorylation but also provide an effective antiarrhythmic strategy for patients with high-risk LQT1 variants.

arrhythmias, cardiac
death, sudden
long QT syndrome
phosphorylation
potassium channel, voltage-gated
stem cells
National Natural Science Foundation of China (CN)50110000180932171221 Not ApplicableNational Key Research and Development Program of China (CN)5011000121662023YFF1204000 Not ApplicableFundo para o Desenvolvimento das CiÃªncias e da Tecnologia (MO)5011000064690074/2022/A2 Not ApplicableFundo para o Desenvolvimento das CiÃªncias e da Tecnologia (MO)5011000064690098/2023/RIA2 Not ApplicableFundo para o Desenvolvimento das CiÃªncias e da Tecnologia (MO)501100006469002/2023/ALC Not ApplicableFundo para o Desenvolvimento das CiÃªncias e da Tecnologia (MO)501100006469006/2023/SKL Not ApplicableMacau University of Science and Technology (MO)501100015260FRG-23-030-SKL Not ApplicableNational Heart, Lung, and Blood Institute (US)100000050R01HL148803 Not ApplicableNational Heart, Lung, and Blood Institute (US)100000050R01NS092570 Not ApplicableOPEN-ACCESSTRUE
SDCT
==== Body
pmcNovelty and Significance

What Is Known?

The cyclic adenosine monophosphate (cAMP)/PKA (protein kinase A)-dependent phosphorylation of IKs channel under stress condition plays a key role in fight-or-flight response.

Long-QT syndrome type (LQT1) variants that lose the cAMP sensitivity predispose patients to the highest risk of cardiac arrest and sudden death under stress conditions.

What New Information Does This Article Contribute?

IKs channel PKA phosphorylation axis’, bridging the N-terminus phosphorylation of KCNQ1 and the central pore region of IKs, was proposed to explain the molecular mechanism of IKs channels.

The distribution of the most high-penetrance LQT1 variants was found to extend across the IKs channel PKA phosphorylation axis, demonstrating its clinical relevance.

Small molecule compounds ML277 and R-L3 were found to modulate the IKs channel PKA phosphorylation axis, and to rescue the defective cAMP sensitivity of high-risk LQT1 mutations.

The IKs channel plays crucial roles in controlling heart rhythm under fight-or-flight response. Under stress conditions, the β-adrenergic stimulation phosphorylates IKs and increases its current through the cAMP/PKA pathway. LQT1 variants with defective cAMP effects predispose patients to the highest risk of cardiac arrest and sudden death. However, the molecular connection between IKs channel phosphorylation and channel function, as well as why high-risk LQT1 mutations lose cAMP sensitivity, remain unclear. Here, by systematically elucidating mechanisms of a series of LQT1 variants that lack cAMP sensitivity, we identified molecular determinants of IKs channel regulation by phosphorylation and proposed the IKs channel PKA phosphorylation axis. Next, by examining LQT1 variants from clinical databases containing 10,579 LQT1 carriers, we found that the distribution of the most high-penetrance LQT1 variants extends across the IKs channel PKA phosphorylation axis, demonstrating its clinical relevance. Furthermore, we found that small molecules, ML277 and R-L3, can effectively modulate the phosphorylation axis and rescue the defective cAMP effects of multiple high-risk LQT1 variants. This finding was then tested in high-risk patient-specific iPSC-derived cardiomyocytes, where ML277 remarkably alleviated the beating abnormalities. Together, our findings provide an effective antiarrhythmic strategy for patients with high-risk LQT1 variants.

In This Issue, see p 705

Meet the First Author, see p 706

Long-QT syndrome (LQTS) is a congenital or acquired cardiac disorder characterized by a prolongation of the QT interval of the ECG and a propensity to ventricular tachyarrhythmia that may lead to syncope, cardiac arrest, or sudden death.1–4 Long-QT syndrome type 1 (LQT1), caused by loss-of-function variants in the voltage-gated KCNQ1 potassium (KVLQT1, KV7.1) channel, is the most common form of LQTS, accounting for 30% to 35% of all cases.5,6

In the heart, KCNQ1 associates with the KCNE1 auxiliary subunit to form the slowly activating delayed rectifier KCNQ1+KCNE1 (IKs) channel.1–3 IKs currents contribute to the repolarization of cardiac action potentials and thereby play a key role in regulating the heart rhythm.7–9 Under stress conditions, the IKs channel is crucial for preventing prolonged action potentials and cardiac events in response to β-adrenergic stimulation. This process is part of cardiac adaptation (or fight-or-flight response), during which the neurotransmitter norepinephrine binds to cardiac β-adrenergic receptors and increases the intracellular cyclic adenosine monophosphate (cAMP) concentration. Subsequently, the elevated cAMP activates the PKA (protein kinase A) cascade and phosphorylates the IKs channel. The phosphorylation of IKs drastically increases channel current to facilitate repolarization, preventing increased action potential duration (APD) under adrenergic stimulation.10–12

Adrenergic tone rises during wakefulness and spikes further in stressful situations. Consequently, patients with LQT1 have an increased risk of developing arrhythmias and cardiac sudden death when the β-adrenergic pathway is activated, for example, during strenuous morning exercise or emotional stress.13 Hundreds of KCNQ1 variants are associated with LQT1,14–16 among which variants that impair the β-adrenergic (cAMP) regulation of IKs channels, such as R190Q and A341V, predispose patients to a much higher risk of arrhythmia and sudden death.13,17–21 However, the detailed molecular mechanism underlying IKs channel phosphorylation and why these high-risk LQT1 mutations lose cAMP sensitivity remain unclear, which significantly hinders the development of new antiarrhythmic therapeutics.

KCNQ1 belongs to the homotetrameric voltage-gated potassium (KV) channel subfamily.1–3 Each KCNQ1 subunit contains 6 transmembrane segments (S1–S6), with the S1-S4 forming the voltage sensing domain (VSD) and the S5 to S6 constituting the pore domain.22–25 The N- and C-termini of KCNQ1 are cytoplasmic domains that can interact with multiple intracellular co-factors including CaM (calmodulin)26–29 and phosphatidylinositol 4,5-bisphosphate (PIP2).30–32 These interactions mediate the KCNQ1 response to different physiological conditions.33–38 KCNQ1 has 2 different open states, intermediate-open (IO) and activated-open (AO) states, following a dynamic Hand-and-Elbow gating process.14,23,39–42 The IO and AO states exhibit different gating and modulation properties, including distinct coupling mechanisms between the VSD and the channel pore. For example, (1) KCNQ1 channel predominantly opens in the IO state. The KCNE1 subunit suppresses the IO state VSD-pore coupling but enhances the AO state VSD-pore coupling, so that IKs channels open only in the AO state.14,39,40,42 (2) A small molecule compound ML27743,44 can increase the AO state current by specifically enhancing the VSD-pore coupling, which partially mimics KCNE1 function.14,23,41

In this study, we used an integrative approach combining the voltage clamp fluorometry (VCF) technique, patient-specific–induced pluripotent stem cell (iPSC)–derived cardiomyocytes, and clinical data analyses. We elucidated crucial molecular determinants responsible for the channel phosphorylation and proposed an IKs channel PKA phosphorylation axis that forms a continuous pathway from the N-terminal of KCNQ1 to the central pore region of IKs. Leveraging these molecular insights, we were able to predict the high clinical penetrance of LQT1 variants with defective cAMP sensitivity. We also found that the distribution of high-risk LQT1 variants mirrors the IKs channel phosphorylation axis, demonstrating the clinical importance of this phosphorylation axis. Furthermore, we demonstrated that the ML277 compound, binding at the center of the phosphorylation axis, could rescue the impaired cAMP response of different groups of high-risk LQT1 variants.

METHODS

Data Availability

The data that supports the findings of the study are available from the corresponding author upon reasonable request.

Constructs and Mutagenesis

Overlap extension and high-fidelity PCR kits (P521, Vazyme International LLC) were used for making each KCNQ1 channel point mutation, which was confirmed by DNA sequencing (Genewiz, Inc). The cRNAs of WT KCNQ1 and all mutants were synthesized using the mMessage T7 Transcription kit (AM1344, Applied Biosystems-Thermo Fisher Scientific) for oocyte injections. RNAs were kept in −80 °C.

Oocyte Preparation and Ion Channel Expression

Mature oocytes (at stage V or VI) were obtained from more than one-and-a-half-year-old female Xenopus laevis (Nasco) by laparotomy. Please see the Major Resources Table in the Supplemental Material. Frogs were kept at room temperature in a fresh-water tank with a 12-hour light/12-hour dark cycle, feeding with pork liver twice a week. All animal-related experimental protocols follow the guidelines of the Institutional Animal Care and Use Committee of Macau University of Science and Technology. Collagenase (C9891, Sigma-Aldrich) at 0.5 mg/mL concentration was used to digest oocytes. For cRNA microinjection, WT or mutant KCNQ1 cRNAs (9.2 ng) with or without KCNE1 cRNA were injected into each oocyte with a 4:1 KCNQ1:KCNE1 weight ratio. This allows a saturate KCNE1 association to KCNQ1.41,45 The KCNQ1:Ciona intestinalis voltage-sensitive phosphatase (CiVSP) weight ratio was kept at 4:1 to obtain a suitable PIP2 depletion rate. Injected cells were incubated in ND96 solution (in millimoles): 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, 5 HEPES, 2.5 CH3COCO2Na, 1:100 Pen-Strep, pH 7.6 at 18 °C for 2 to 6 days for electrophysiology recordings. Unless otherwise stated, all chemicals were purchased from Sigma-Aldrich.

Two-Electrode Voltage Clamp and VCF

Microelectrodes (BF150-86-10, Sutter Instrument) were made with a puller (P-1000, Sutter Instrument) with 1 to 3 MΩ resistances when filled with 3 mol/L KCl. The extracellular solution was ND96 solution without CH3COCO2Na (S8750, Sigma-Aldrich). Currents were recorded with an OC-725D two-electrode voltage clamp amplifier (Warner). Currents were sampled at 1 kHz and low-pass-filtered at 2 kHz. Membrane-permeable cAMP (B7880, Sigma-Aldrich) was applied to mimic the β-adrenergic stimulation. For KCNQ1+CiVSP and KCNQ1+KCNE1+CiVSP experiments, the pulse interval was 5 minutes at −80 mV holding potential to replenish PIP2. For VCF experiments, pseudo-WT KCNQ1 (KCNQ1-C214A/G219C/C331A, KCNQ*)40,46,47 or mutations on top of this VCF background were injected. Oocytes were incubated on ice for 30 minutes in labeling solution: 10 μM Alexa 488 C5-maleimide (A10254, Thermo Scientific) in 100 mmol/L K+ solution. After labeling, oocytes were washed 3× with the ND96 solution before VCF recordings. All recordings were performed at room temperature of 20 °C to 22 °C.

Whole-Cell Patch Clamp Recordings in Chinese Hamster Ovary Cells

Chinese hamster ovary cells up to passage 30 were cultured in Ham F-12 culture medium (C11765500BT, Gibco) supplemented with 10% fetal bovine serum (10270106, Gibco) and 1% penicillin-streptomycin-glutamine solution (10378016, Gibco) and were maintained at 37 °C in the presence of 5% CO2. Chinese hamster ovary cells were transiently transfected with human KCNQ1 (WT or mutant), human KCNE1, human Yotiao, and GFP. GFP-fluorescent cells were used for whole-cell patch clamp experiments after overnight expression. Cells were plated in culture dishes placed on the stage of an inverted microscope (Olympus IX73), and currents were recorded by using a whole-cell patch clamp technique with a HEKA EPC 10 amplifier. Currents were sampled at 20 kHz and low-pass-filtered at 2.9 kHz. Microelectrodes (BF150-86-10, Sutter Instrument) were prepared with the P-1000 puller (Sutter Instrument) with 3 to 7 MΩ resistance when filled with pipette solution containing (millimoles): 130 KCl, 20 KOH, 5 ATP-K2, 1 MgCl2, 5 EGTA, 10 HEPES, and 0.2 µmol/L okadaic acid (OA), pH=7.3. The extracellular solution was the F-12 solution containing (millimoles) 130 NaCl, 3.8 KCl, 30 NaHCO3, 1.6 MgSO4, 0.9 CaCl2, 7.0 d-glucose, 2 sodium pyruvate, with necessary nutrients, pH=7.5. Cells were incubated at 37 °C for 30 minutes in extracellular solution with 0.1 µmol/L ML277 and 0.2 mmol/L cAMP to activate and phosphorylate the channels. ML277 and cAMP were also added to the extracellular solution when doing experiments. Currents were recorded at 30 °C temperature with a temperature controller (TC-344C, Warner Instruments).

Action Potential Recording From Guinea Pig Ventricular Myocytes

Adult (male and female) guinea pigs (body weight, 350–500 g, around 2 months old) were kept at room temperature in a specific pathogen-free (SPF) level animal facility with a 12-hour light/12-hour dark cycle, feeding with regular food and water. Guinea pig hearts were quickly excised after anesthetization with a peritoneal injection of sodium pentobarbitone (150 mg/kg). A Langendorff perfusion system was utilized to isolate heart cells. Isolated ventricular myocytes were kept in KB solution containing (in millimoles): 83 KCl, 30 K2HPO4, 5 MgSO4, 5 Na-pyruvate, 5 beta-hydroxy-butyric acid, 20 taurine, 5 creatine, 10 glucose, 0.5 EGTA, 5 HEPES, and 5 Na2ATP, and pH 7.4. For action potential recordings, freshly isolated guinea pig ventricular myocytes were kept in a bath solution containing (in millimoles): 140 NaCl, 3 KCl, 1 MgCl2, 1.8 CaCl2, 10 HEPES, 10 Glucose, pH 7.4. The pipette solution contains (in millimoles): 115 K-aspartic acid, 35 KOH, 3 MgCl2, 10 HEPES, 11 EGTA, 5 glucose, 3 MgATP, pH 7.4. Cells were paced at 1 Hz with a 180 pA pulse current for 10 ms duration to generate action potentials. The APD90 was determined at 90% repolarization from the peak amplitude. All recordings were performed at room temperature (21–23 °C).

Electrophysiology Data Analysis

Data were analyzed with Clampfit (Axon Instruments), Sigmaplot (SPSS), and IGOR (Wavemetrics). Because of photo-bleaching, fluorescence signals were baseline subtracted. G-V and F-V curves were fitted with single or double Boltzmann equations in the form of 1/(1+exp(−z*F*(V−V1/2)/RT)), where V is the voltage, z is the equivalent valence, V1/2 is the half-maximal voltage, F is the Faraday constant, R is the gas constant, and T is the absolute temperature.

High-Risk LQT1 Variants Prediction

Based on our proposed PKA phosphorylation axis in the IKs channel, we predicted that the following LQT1 variants would pose patients to elevated clinical risk: (1) S182K (by breaking the VSD-CaM interaction); (2) R190Q, R195Q, H258N, R259Q, K354N, R360Q, H363Q, and R366Q (by affecting PIP2 binding); (3) V254M, H258W, A341V, P343A, G345A, W248R, L251A, V255W, Y267A, I268W, L271E, F335A, S338F, F339S, and L342A (by affecting the Hand-and-Elbow modulation). Penetrance data of each variant was obtained from the KCNQ1 Variant Browser15 and compared with that of other LQT1 variants. Blinding procedures were not considered in the study design.

iPSC Cardiomyocyte Differentiation and Multi-/Microelectrode Array Recording

Human iPS cell lines from a LQT1 R190Q patient and a health control were maintained in Essential 8 medium (A1517001, Thermo Fisher) on matrigel-coated (354230, Corning) plates at 37 °C with 5% CO2. The iPS cell lines were differentiated using a previously described protocol,48 with minor modifications. Briefly, on the day 0 of differentiation, culture medium was switched to RPMI/B-27 medium without insulin (-ins; A1895601, Thermo Fisher) with 6 μM CHIR99021 (C-6556, LC Laboratories); on the day 2 (48 hours later), the medium was replaced with RPMI/B-27(-ins); on the day 3, the medium was replaced with RPMI/B-27(-ins) with 5 µmol/L IWP2 (3553, Tocris); on the day 5 (48 hours later), the medium was replaced with RPMI/B-27(-ins); on the day 7, day 10, and day 13, the medium was replaced with RPMI/B-27 with insulin (+ins; 17504044, Thermo Fisher). The induced cardiomyocytes were then treated with an 8-day lactate purification protocol (4 mmol/L lactate and 25 mmol/L HEPES in glucose-free RPMI). After day 21, pure iPS-CM was kept in RPMI/B-27(+ins) for microelectrode array experiments. Briefly, iPS-CMs were dissociated with 200 U/mL of collagenase II (17101015, Thermo Fisher) for 2 hours followed by TrypLE Express for 5 minutes, then 90 000 cells in 3 µL of droplet were plated to the matrigel-coated microelectrode array chamber (60MEA200/10iR-Ti-gr). ML277 and cAMP were prepared in the RPMI/B27(+ins) solution with 10× of the working concentration. At the time to add compound (ML277 and cAMP), replace 50 µL recording solution from the total 500 µL volume with the 10× concentrated compound solution. Field potentials were recorded at 37 °C using MEA2100 from Multichannel Systems, and data were analyzed with MC_Rack software.

Statistical Analysis

Averaged data were presented as mean±SEM with n specifying the number of independent experiments. Differences between 2 groups with normal distribution (examined with Shapiro-Wilk normality test) were compared by using t test or paired t test. Otherwise, differences between groups were compared by using 2-sample Wilcoxon rank-sum test, Wilcoxon matched-pairs signed-ranks test, Kruskal-Wallis rank-sum test, and post hoc mean comparison Tukey test or Dunnett test.49 Statistical analyses were performed with Sigmaplot (SPSS) and R software (4.1.2 version, multcomp package). Statistical significance was set as *P<0.05, **P<0.01, and ***P<0.001.

RESULTS

To solve the long-standing puzzle of molecular mechanisms underlying IKs channel phosphorylation, we directly applied membrane-permeable cAMP (8-Br-cAMP) to mimic β-adrenergic stimulation of KCNQ1 and IKs channels. We used the Xenopus oocyte heterologous expression system, in which KCNQ1 and IKs channels showed cAMP responses comparable to those at mammalian cells11,50,51 (Figure S1): cAMP (0.5 mmol/L) had only minimal effect on the amplitude of KCNQ1 current (18.0±5.2% increase) but significantly increased the KCNQ1+KCNE1 current amplitude (242.4±24.8% increase; P value=4.3×10−3; Figure 1A and 1C). These results imply that the KCNE1 subunit is required for the cAMP-induced current increase.13,17,19,20,52

Figure 1. The high-risk long-QT syndrome type 1 (LQT1) mutation R190Q loses both cyclic adenosine monophosphate (cAMP) and phosphatidylinositol 4,5-bisphosphate (PIP2) sensitivities. A, Representative currents of KCNQ1 and KCNQ1+KCNE1 channels before (black) and after (red) adding 0.5 mmol/L cAMP. Currents were recorded at +40 mV for 4 seconds. B, Representative currents of R190Q and R190Q+KCNE1 channels before (black) and after (red) adding 0.5 mmol/L cAMP, recorded at the same condition. C, Summary of the cAMP-induced current increases ([I+cAMP-IControl]/IControl %), or ∆I+cAMP/IControl %): 18.0±5.2% for KCNQ1 (n=6), 242.4±24.8% for KCNQ1+KCNE1 (n=6), 18.3±6.6% for R190Q (n=5), 70.0±12.4% for R190Q+KCNE1 (n=5). Significant differences were assessed via Wilcoxon rank-sum test. D, Voltage clamp fluorometry (VCF) results of R190Q. The F-V relationship (blue) was fitted with a double Boltzmann equation, and the G-V relationship (black) was fitted with a single Boltzmann equation. VCF results of WT KCNQ1 were shown in gray. E, Averaged currents of KCNQ1+CiVSP (ciona intestinalis voltage-sensitive phosphatase; black) and R190Q+CiVSP (red). The cell membrane was depolarized to +60 mV (for 4 seconds) to deplete the membrane PIP2, and the currents of each group showed decay due to the dynamic PIP2 depletion process. τd indicates the current decay rate. F, Normalized peak currents (1±0.14 for KCNQ1+CiVSP, n=5; and 0.38±0.06 for R190Q+CiVSP, n=5) and τd (τd>4 seconds for KCNQ1+CiVSP, n=5 and τd=0.4±0.08 seconds for R190Q+CiVSP, n=5). Significant differences were assessed via Wilcoxon rank-sum test.

The High-Risk LQT1 Mutation R190Q Impairs Both cAMP and PIP2 Sensitivities

Prior clinical studies found that the lethality of LQT1 is dependent on the variant’s cAMP sensitivity and its location within the channel protein.13,18 For instance, a high-risk LQT1 mutation R190Q, located on the S2–S3 linker, largely reduces the cAMP effect. We found that in Xenopus oocytes, 0.5 mmol/L cAMP-induced R190Q-IKs channel current increases by only 70.0±12.4%, significantly lower than that of the WT IKs channel (P=4.3×10−3; Figure 1B and 1C). When expressed alone, R190Q displays a minimal response to cAMP (18.3±6.6% increase; Figure 1B and 1C), similar to WT KCNQ1. As β-adrenergic stimulation under stressful conditions is the major trigger of cardiac events in LQT1 patients,13,53 this defective cAMP response may lead to a higher risk of arrhythmia and sudden death.

To uncover the mechanism underlying the reduced cAMP response of R190Q, we first characterized the voltage-dependent activation of R190Q using the VCF technique.30,40,47,54 Compared with WT KCNQ1, the mutant showed almost identical voltage dependence of fluorescence and conductance (F-V and G-V; Figure 1D), suggesting that R190Q does not alter the voltage dependence of VSD activation or pore conductance. These results indicate that the defective cAMP effect of R190Q arises from alterations in channel function that are not linked to voltage-dependent activation.

PIP2 is a membrane lipid that is critical for KCNQ1 activation,23,24,30,31,55,56 especially for the VSD-pore coupling.23,30 In high-resolution structures,23,24 the R190 residue was found close to the PIP2 molecule, and previous studies suggested that it is involved in PIP2 binding.30 To test its PIP2 sensitivity, we coexpressed R190Q with the voltage-sensitive phosphatase CiVSP, which dynamically depletes PIP2 upon depolarization,57 and assessed the current in response to a 60-mV pulse depolarization (4 seconds). A channel with lower PIP2 affinity was shown to be more sensitive to PIP2 depletion, leading to a faster rate of current decay.30,39 This phenotype provides a reliable functional readout for assessing PIP2 affinity of KCNQ1 channels. For the WT KCNQ1+CiVSP channel, the current initially increased and then decayed over time upon depolarization due to the dynamic process of PIP2 depletion (τdecay (τd)>4 seconds; Figure 1E and 1F. In contrast, R190Q+CiVSP showed a lower current amplitude (31±11%) and a much faster current decay than that of the WT KCNQ1+CiVSP (τd=0.3±0.04 seconds; P=7.5×10−3; Figure 1E and 1F). These results suggest that the R190Q variant has an impaired PIP2 response, which may lead to its reduced cAMP sensitivity.

PIP2 Modulates the Channel cAMP Sensitivity

PIP2 is required for the VSD-pore coupling23,30 (Figure 2A and 2C). When we coexpressed KCNQ1* (KCNQ1-C214A/G219C/C331A, pseudo-WT KCNQ1 of the VCF background40,46,47) with CiVSP, we observed normal 2-step VSD activation that is similar to that of KCNQ1* channels but a closed pore upon depolarization (Figure 2B), which is consistent with previous studies.30,46 To dissect the relationship between PIP2 modulation and cAMP-mediated channel phosphorylation, we next examined cAMP effects on mutant KCNQ1 channels with varying PIP2 sensitivities. We utilized 2 pairs of mutations, surrounding the PIP2 binding site, that exhibit either down- or upregulated PIP2 sensitivity: R190Q and K362N showed decreased PIP2 sensitivity, while K196Q and R249Q showed increased PIP2 affinity30 (Figure 2A). Compared with WT IKs channels, we found that K362N+KCNE1 exhibited clearly decreased cAMP sensitivity (from 242.4±24.8% to 79.7±10.7%; P=3.2×10−4; Figure 2D and 2E), similar to that of R190Q+KCNE1 channels (Figure 1); whereas K196Q+KCNE1 and R249Q+KCNE1 both showed significantly increased cAMP sensitivity (from 242.4±24.8% to 387.1±43.6% and 406.8±48.6%; P=3.9×10−3 and 1.9×10−3; Figure 2D and 2E). These results demonstrate that the PIP2 sensitivity of IKs channels is correlated with its cAMP response. As PIP2 was found to mediate the VSD-pore coupling process30 (Figure 2B and 2C), these results further suggest that PIP2-mediated VSD-pore coupling modulates the cAMP sensitivity of KCNQ1 and IKs channels.

Figure 2. Phosphatidylinositol 4,5-bisphosphate (PIP2) modulates cyclic adenosine monophosphate (cAMP) sensitivity. A, Structures of KCNQ1 and the PIP2 (green) binding (PDB:7XNL23). R190 and K362 were shown in red (R190Q and K362N were found to decrease the PIP2 sensitivity30), and K196 and R249 were shown in blue (K196Q and R249Q were found to increase the PIP2 sensitivity30). B, Voltage clamp fluorometry (VCF) results of KCNQ1+CiVSP (ciona intestinalis voltage-sensitive phosphatase). Before the recording, cells were depolarized to a train of +60 mV (for 4 seconds) to deplete the membrane PIP2, so that currents were eliminated. The F-V relationship (blue) was fitted with a double Boltzmann equation, and the I-V relationship (black, no current, n.c.) was normalized to the WT KCNQ1 currents. VCF results of WT KCNQ1 were shown in gray. C, Cartoon model to show that PIP2 mediates the voltage sensor domain (VSD)-pore coupling of both IO (intermediate open) and AO (activated open) states of KCNQ1. D, Representative currents of K362N+KCNE1, KCNQ1+KCNE1, and R249Q+KCNE1 channels before (black) and after (red) adding cAMP. The test pulse was +40 mV for 4 seconds. E, Summary of the cAMP-induced current increases: 71.6±4.6% for R190Q+KCNE1 (n=5), 79.7±10.7% for K362N+KCNE1 (n=6), 242.4±24.8% for KCNQ1+KCNE1 (n=6), 387.1±43.6% for K196Q+KCNE1 (n=6), and 406.8±48.6% for R249Q+KCNE1 (n=5). Significant differences were assessed via 1-way ANOVA, and P values were obtained via Tukey post hoc test.

The Hand-and-Elbow Sites Are Necessary for cAMP Modulation

We previously proposed a Hand-and-Elbow gating mechanism, in which 2 distinct groups of interactions, from the hand and the elbow sites respectively, are responsible for the dynamic VSD-pore coupling of KCNQ114,23,41 (Figure 3A). Here, we found that these 2 groups of VSD-pore coupling interactions are necessary for cAMP modulation.

Figure 3. The Hand-and-Elbow sites are important for cyclic adenosine monophosphate (cAMP) modulation. A, Structures of KCNQ1 (PDB:7XNL23) to show the Hand-and-Elbow voltage sensor domain (VSD)-pore coupling mechanism. The residue A341, at the hand site, was shown in purple, and the residue W248, at the elbow pocket, was shown in orange. B, Voltage clamp fluorometry (VCF) results of A341V. The F-V relations were fitted with a double Boltzmann function. The F-V relation of WT KCNQ1 was shown in gray. Normalized currents of A341V (small current [sc]) were shown in black. C, Cartoon model to show that A341V disrupts the VSD-pore coupling of both IO (intermediate open) and AO (activated open) states of KCNQ1. D, Representative currents of KCNQ1+KCNE1 and A341V+KCNE1 before (black) and after (red) adding cAMP. E, Cartoon model to show that W248R suppresses the AO state VSD-pore coupling of KCNQ1. F, The cAMP-induced current increase of KCNQ1+KCNE1 (black, 242.4±24.8%, n=6), A341V+KCNE1 (red, 64.7±16.1%, n=5), V254M+KCNE1 (red, 77.0±16.4%, n=5), W248R+KCNE1 (red, 86.6±17.0%, n=5), R243C+KCNE1 (red, 105.3±7.9%, n=5), and F256A+KCNE1 (red, 97.4±2.9%, n=5). Significant differences were assessed via 1-way ANOVA, and P values were obtained via Tukey post hoc test.

Within the hand site (the canonical VSD-pore coupling site), we found that interactions between the S4-S5 linker and S6 are required for cAMP modulation. As shown in Figure 3B and 3C, the LQT1 mutation A341V, located in the hand site, showed normal stepwise VSD activation, but the pore remained closed upon VSD activation (Figure S2A and S2B), suggesting a disrupted VSD-pore coupling. When coexpressed with KCNE1, the A341V+KCNE1 channel also showed suppressed IKs currents with severely reduced cAMP response (Figure 3D and 3F; Figure S2C and S2D). Further scanning identified 4 additional mutations in the hand site (V254M, H258W, P343A, and G345A) that showed a phenotype similar to that of A341V with suppressed IKs currents and reduced cAMP response (Figure 3F; Figure S2D).

While exploring the elbow site, we found that interactions between the S4/S4–S5 linker and the neighboring pore (S5′ and S6′) are also essential for cAMP modulation. As illustrated in Figure 3A and 3E, the LQT1 mutation W248R, in the elbow site, showed normal activation current but significantly suppressed IKs current when coexpressed with KCNE1 (Figure S2E), indicating a disrupted VSD-pore coupling for the AO state.14 We found that the W248R+KCNE1 channel exhibited a defective cAMP response (86.6±17.0%, P=7.4×10−6; Figure 3F; Figure S2F). Further scanning of the elbow site revealed that mutations R243W, L250W, L251A, V255W, F256A, Y267A, I268W, L271E, G272A, F335A, S338F, F339S, and L342A showed a phenotype similar to that of W248R with suppressed IKs currents and reduced cAMP response (Figure 3F, Figure S2G). Additionally, even for the F256A mutation, which showed mildly suppressed IKs currents (Figure S2G), the cAMP response of F256A+KCNE1 was largely reduced (from 242.4±24.8% to 97.4±2.9%; P=6.9×10−6; Figure 3F). These results suggest that interactions within the Hand-and-Elbow VSD-pore coupling sites are essential for the cAMP response.

LQT1 Mutations That Disturb the KCNQ1-KCNE1 Interaction Lose cAMP Effects

Previous studies have shown that the SFF motif (S338, F339, and F340) in the S6 segment interacts with KCNE146,58 (Figure 4A). The fact that KCNE1 can boost the cAMP response of KCNQ1 suggests that these KCNQ1-KCNE1 interactions may also play a key role in cAMP responses. To validate this, we tested cAMP response of mutations that disturb these interactions and found that S338F+KCNE1 and F339S+KCNE1 channels also showed largely suppressed mutant-IKs currents and reduced cAMP responses (from 242.4±24.8% to 101.1±9.2%; P=4.6×10−5; and to 74.0±12.1%, P=4.4×10−5; Figure 4B and 4C). Notably, both S338F and F339S are LQT1 mutations with increased arrhythmic risk.18,59 Their loss-of-function phenotype of mutant IKs currents and the defective cAMP effects may predispose patients at high risk of cardiac events under stress.

Figure 4. The KCNE1 subunit is required for the cyclic adenosine monophosphate (cAMP)-induced current increase. A, Schematic structure of KCNQ1+KCNE1 (KCNQ1 structure from PDB:7XNL23 with the KCNE3 subunit from PDB:6V01,24 KCNE3 was used to indicate the possible location of KCNE1) to show interactions between KCNQ1 SFF motif (S338, F339, and F340) and the KCNE1 subunit. B, Representative currents of KCNQ1+KCNE1 and F339S+KCNE1 before (black) and after (red) adding cAMP. C, Summary of cAMP-induced current increases: 242.4±24.8% for KCNQ1+KCNE1 (n=6), 101.1±9.2% for S338F+KCNE1 (n=6), and 74.0±12.1% for F339S+KCNE1 (n=5). Significant differences were assessed via 1-way ANOVA and P values were obtained via Tukey post hoc test. D, Current rundown of KCNQ1+KCNE1+CiVSP before (black) and after adding cAMP (red). The cell membrane was depolarized to a double pulse protocol at +60 mV (for 4 seconds) to induce a dynamic PIP2 depletion process. The current amplitudes of the first and second pulses were defined as I1 and I2. E, The I2/I1 ratio of KCNQ1+KCNE1+CiVSP before (black, 0.29±0.02) and after (red, 0.42±0.03) adding cAMP (n=5). Significant differences were assessed via Wilcoxon matched-pairs signed-ranks test.

To further explore why KCNE1 boosts the cAMP response of KCNQ1, we coexpressed the KCNQ1+KCNE1 channel with CiVSP and examined the PIP2 sensitivity before and after the cAMP application. A 60-mV depolarization was applied to induce PIP2 depletion, and we utilized the IKs current reduction rate (the I2/I1 ratio) as a metric to evaluate PIP2 sensitivity. Strikingly, we observed a substantial increase in the I2/I1 ratio following cAMP application (from 0.29±0.02 to 0.42±0.03; P=3.2×10−4; Figure 4D and 4E), indicating an enhanced PIP2 sensitivity due to cAMP-induced phosphorylation. Of note, co-expression and activation of CiVSP reduced the overall membrane PIP2 concentration, so that cAMP induced less current increase than in WT (Figure S3). This result further underscores the importance of PIP2 in cAMP modulation. Taken together, these results suggest that the KCNE1 subunit is another key determinant for cAMP effects.

CaM-VSD Interactions Contribute to the cAMP-Induced Current Increase

CaM is another cofactor necessary for the KCNQ1 channel assembly and function.23–27,29,60,61 This dumbbell-shaped protein clamps helix A (HA) and helix B (HB) of the KCNQ1 C-terminal and interacts with the bottom part of the VSD (the S2-S3 linker) during channel gating23–25,29 (Figure 5A).

Figure 5. A potential IKs channel phosphorylation axis is responsible for the cyclic adenosine monophosphate (cAMP)-induced current increase. A, Structure of KCNQ1 (protein data bank [PDB]:7XNL23) to show direct interactions between CaM (calmodulin) and voltage sensor domain (VSD). The S182 residue was shown in purple, and the phosphatidylinositol 4,5-bisphosphate (PIP2) was shown in green. B, Representative currents of KCNQ1+KCNE1 and S182K+KCNE1 before (black) and after (red) adding cAMP. C, The cAMP-induced current increases were 43.9±10.6% for S182K+KCNE1 (n=6), and 28.3±4.4% for S27D/S92D+KCNE1 (n=9). Significant differences were assessed via 1-way ANOVA, and P values were obtained via least significant difference test. D, A potential molecular pathway of cAMP-induced IKs channel current increase: PKA (protein kinase A)-dependent phosphorylation occurs at N-terminal (S27/S92) of KCNQ1 → CaM and VSD interaction → PIP2-binding site → ML277-binding site → KCNE1 subunit, and finally increases the IKs current. This is a schematic structural complex with KCNQ1 structure from PDB:7XNL,23 and the KCNE3 subunit from PDB:6V0124 to mimic the KCNE1 subunit.

To probe whether the CaM-VSD interactions are involved in cAMP modulation, we generated a mutation at the S2–S3 linker, S182K, which was found to disrupt interactions between CaM and the VSD.29 When testing its cAMP effect, we observed a significant reduction in the cAMP response of S182K+KCNE1 (from 242.4±24.8% to 43.9±10.6%; P=8.6×10−6. Figure 5B and 5C), indicating that the CaM-VSD interactions are also essential for maintaining cAMP sensitivity of the IKs channel.

A Potential IKs Channel PKA Phosphorylation Axis

Two Serine residues (S27/S92) at the N-terminal of KCNQ1 are sites for PKA-dependent phosphorylation.10,11,31,50,62,63 Consistent with previous reports,31,50,62,63 we observed that the cAMP-induced current increase in the phosphomimetically mutated S27D/S92D(SSDD)+KCNE1 channel was almost abolished (28.3±4.4%; P=3.3×10−9; Figure 5C). Taken together, by systematically elucidating mechanisms underlying a series of LQT1 mutants lacking cAMP sensitivity, our findings pinpoint key determinants of the IKs channel phosphorylation axis within the channel protein: the cAMP/PKA induced phosphorylation occurs at the N-terminal (S27/S92) of KCNQ1, then regulates the following parts/functions of the IKs channel: interactions between CaM and VSD → PIP2 modification → Hand-and-Elbow modulation (the small molecule ML277 binding site) → the KCNE1 subunit, which finally increases the IKs channel current (Figure 5D). This phosphorylation axis establishes a continuous pathway extending from the peripheral phosphorylation at the N-terminal to the central pore region of the IKs channel (Figure 5D).

LQT1 Variants, Which Disturb the IKs Channel Phosphorylation Axis, Exhibit Increased Clinical Risk

Given the critical role of IKs channels in the cardiac fight-or-flight response, LQT1 variants with disrupted PKA phosphorylation could predispose individuals to an elevated risk of arrhythmia.13,17–21 Therefore, we hypothesized that LQT1 variants located near the IKs channel phosphorylation axis, as defined in this study, are associated with increased arrhythmia risk. To test this, we utilized a curated clinical database consisting of 10,579 LQT1 carriers, sourced from multiple international arrhythmia genetic centers.15 We used the clinical phenotypic penetrance of each LQT1 locus as a metric for assessing clinical risk.64

Based on our detailed understanding of the IKs channel PKA phosphorylation axis, we predicted that the following LQT1 variants would pose patients to elevated clinical risk: (1) S182K (by breaking VSD-CaM interactions; Figure 5); (2) R190Q, R195Q, H258N, R259Q, K354N, R360Q, H363Q, and R366Q (by affecting PIP2 binding, Figures 1 and 2); (3) V254M, H258W, A341V, P343A, G345A, W248R, L251A, V255W, Y267A, I268W, L271E, F335A, S338F, F339S, and L342A (by affecting the Hand-and-Elbow modulation, and KCNE1 modulation; Figures 3 and 4). Examination of penetrance data from related LQT1 variants (with at least 5 carriers) revealed that these PKA phosphorylation-related variants demonstrated higher clinical penetrance compared with other LQT1 variants (Figure 6A). These findings suggest that LQT1 variants proximal to the IKs phosphorylation axis are linked to an elevated clinical risk and our mechanistic insights could potentially be utilized to predict the clinical severity of LQT1 variants.

Figure 6. Long-QT syndrome type 1 (LQT1) variants located in the IKs channel phosphorylation axis exhibited increased clinical phenotypic penetrance. A, Prediction of LQT1 mutations (at residues S182, R190, R195, H258, R259, K354, R360, H363, R366, V254, H258, A341, P343, G345, W248, L251, V255, Y267, I268, L271, F335, S338, F339, and L342) that disturb the IKs channel phosphorylation axis may show increased clinical penetrance. The penetrance density of these related LQT1 variants were shown in red, and penetrance density of other LQT1 variants is in gray. B, Penetrance analysis of LQT1 variants from 10 579 carriers,15 with >5 carriers for each LQT1 mutation. Two parts (later portion of the voltage sensor domain [VSD], the pore domain, Helix A [HA], and Helix B-Helix C [HB-HC]) with a high frequency of high-penetrance LQT1 variants were labeled in red, and the rest parts of the channel protein, showing low-penetrance, were labeled in green. LQT1 variants with penetrance >75%, reflecting a high clinical risk, were highlighted in red dots. C, The distribution of high-risk LQT1 mutations with penetrance >75% (red spheres) mirrors the IKs channel phosphorylation axis. LQT1 mutations from the neighboring pore domain (cyan spheres) were also shown to better exhibit potential interactions at the transmembrane segments.

Consistent with this prediction, we further analyzed the penetrance data of all LQT1 variants, with at least 5 carriers, against their amino acid positions on the KCNQ1 protein, and found that high-penetrance (>75%) LQT1 variants clearly concentrated in the latter portion of VSD, pore domain, HA, HB, and HC (Figure 6B). Intriguingly, the distribution of these high-penetrance LQT1 variants showed a clear correlation with the IKs channel phosphorylation axis, surrounding the CaM, the PIP2-binding site, the ML277-binding site, and the KCNE1 subunit (Figure 6C). These findings collectively shed light on the clinical importance of the IKs channel phosphorylation axis and provide insights into understanding why mutations in proximity to the phosphorylation axis manifest higher clinical risk under stressful conditions.

ML277 Enhances PIP2 Affinity and cAMP Sensitivity of KCNQ1

Multiple modulators targeting KCNQ1 and IKs channels have been developed.32,65–74 Based on our findings above, we sought to develop a new strategy, by the application of exogenous small molecules, to modulate the cAMP sensitivity of the IKs channel.

At the center of the IKs channel phosphorylation axis, the small molecule ML277 was found to bind at the Elbow pocket14,23,41 (Figure 7A and 7B). From structural data, its binding site is just above the PIP2 molecule (Figure 7A). We therefore tested whether ML277 is capable of enhancing the PIP2 sensitivity of KCNQ1. We coexpressed KCNQ1 with CiVSP and measured current response to PIP2 depletion at 60-mV depolarization in the absence and presence of ML277. Between each 60-mV test pulse, cells were kept at −80 mV holding potential for 5 minutes to replenish membrane PIP2. ML277 was added and tested every 5 minutes until a stable current was observed. We found that, following the dynamic PIP2 depletion, KCNQ1+CiVSP showed a clear current rundown during the 60 mV/4 second test pulse (black; Figure 7C). However, after ML277 addition, the current amplitude of KCNQ1+CiVSP+ML277 almost doubled and gradually increased over time (Figure 7C and 7D), indicating that the current is less sensitive to PIP2 depletion. In other words, ML277 increases the PIP2 sensitivity of KCNQ1. Together with our previous structural and functional studies,14,23,41 these results further support that ML277 can mimic the KCNE1 function on KCNQ1.

Figure 7. ML277 enhances the phosphatidylinositol 4,5-bisphosphate (PIP2) affinity and cyclic adenosine monophosphate (cAMP) sensitivity. A, KCNQ1 structure to show the binding sites of PIP2 (green) and ML277 (blue; protien data bank [PDB]:7XNL23). B, Cartoon scheme to show that ML277 selectively enhances the VSD-pore coupling of the AO (activated open) state of KCNQ1.14,23,41 C, Averaged currents of KCNQ1+CiVSP before (black) and after (blue) adding ML277. The cell membrane was depolarized to +60 mV (for 4 seconds) to gradually deplete the membrane PIP2 and kept at −80 mV holding potential for 5 minutes to replenish the PIP2. ML277 was added at the end of the 5 minutes, and stable results (2−3 rounds) after adding ML277 were analyzed. D, Averaged current sizes of KCNQ1+CiVSP before (black) and after (red) adding ML277 (n=6). Significant differences were assessed via Wilcoxon matched-pairs signed-ranks test. E, Representative currents of KCNQ1 before (black) and after adding ML277 (blue) and then after adding cAMP (red). F, Current increases of KCNQ1 after adding cAMP (18.0±5.2%, n=6), and KCNQ1 after adding ML277 (blue, 77.9±4.2%, n=6) and then after adding ML277+cAMP (red, 206.3±7.0%, n=6). G, The cAMP-induced current increases of KCNQ1 (18.0±5.2%, n=6) and KCNQ1+ML277 (99.2±8.2%, n=10). Significant differences were assessed via t test.

Next, we tested whether ML277 increases the cAMP sensitivity of the KCNQ1 channel. Although KCNQ1 alone showed little cAMP sensitivity (18.0±5.2%; Figure 7E), 1 µmol/L ML277 largely sensitized the channel to cAMP, so that cAMP induced a much larger current increase (99.2±8.2%; P=2.5×10−4; Figure 7E and 7G). These results not only confirm the key roles of the elbow pocket and PIP2 along the phosphorylation axis but also suggest new strategies for modulating the phosphorylation axis by exogenous ligands to rescue the defective cAMP effects of high-risk LQT1 mutations.

ML277 Rescues the Defective cAMP Effects in High-Risk LQT1 Variants

Next, we investigated if we could apply this strategy to rescue the impaired cAMP response in the high-risk LQT1 variant R190Q (Figure 1). We observed that 1 µmol/L ML277 not only increased the current of R190Q but also effectively sensitized the channel to cAMP. Specifically, under treatment with 1 µmol/L ML277, we observed a cAMP-induced current increase of 77.4±4.1%, compared with 18.2±3.1% in untreated conditions (P=7.9×10−3; Figure 8A and 8C).

Figure 8. ML277 rescues the defective cyclic adenosine monophosphate (cAMP) sensitivity of high-risk long-QT syndrome type 1 (LQT1) mutations. A, Representative currents of R190Q before (black) and after adding ML277 (blue) and then adding cAMP (red). B, Averaged results of cAMP-induced current increases of R190Q (18.3±6.6%, n=5), R190Q+KCNE1 (70.0±12.4%, n=5), R190Q (63.3±11.6% for +ML277, n=5; and 192.1±19.7% for +ML277+cAMP, n=5). C, The cAMP-induced current increases of R190Q alone (18.3±6.6%, n=5) and R190Q+ML277 (77.4±4.1%, n=5). Significant differences were assessed via t test. D, Representative field potentials of the healthy control iCMs before (black) and after (red) adding cAMP, and the R190Q iCMs before (black) and after (red) adding cAMP, and then after (blue) adding ML277 with cAMP. E, The cAMP shortened the field potential duration (FPD) of control iCMs (from 1.00±0.09 to 0.83±0.08, n=4; P=1.7×10−3), while R190Q iCMs showed little cAMP sensitivity (from 1.39±0.06 to 1.35±0.06, n=5, P=0.14), but ML277 shortened the FPD (from 1.35±0.06 to 0.99±0.04, n=5, P=0.010). Significant differences were assessed via Wilcoxon matched-pairs signed-ranks test. F, Representative field potentials of the healthy control before and after adding cAMP and ML277 with cAMP. G, Representative field potentials of the R190Q group before and after adding cAMP and ML277 with cAMP. The R190Q group developed an abnormal missing beating (shown as *). This missing beating phenotype was aggravated after adding cAMP, and ML277 accelerated the beating rate and corrected the arrhythmic-like phenotype. H, Representative currents of A341VHet before (black) and after adding ML277 (blue) and then adding cAMP with ML277 (red). (I) Averaged results of ML277 (blue) and ML277+cAMP (red) induced current increases of WT KCNQ1 and LQT1 mutations. J, The cAMP-induced current increases of KCNQ1 alone (18.0±5.2%, n=6), A341VHet+ML277 (103.8±13.7%, n=5), V254MHet+ML277 (95.5±5.5%, n=5), G189R+ML277 (100.0±5.2%, n=5), R243C+ML277 (68.1±8.9%, n=5), W392R+ML277 (80.7±12.9%, n=5), Y522S+ML277 (121.8±11.3%, n=5), R539W+ML277 (64.8±3.7%, n=5), R555H+ML277 (72.7±6.9%, n=5), R562M+ML277 (68.4±5.8%, n=5). Significant differences were assessed via 1-way ANOVA, and P values were obtained via Tukey post hoc test.

To test whether ML277 can rescue R190Q’s dysfunction under physiological conditions, we utilized patient-specific iPSC-derived cardiomyocyte models. We differentiated iPSCs from a healthy control and an R190Q patient75 into cardiomyocytes (control iPSC-derived cardiomyocyte [iCM] and R190Q iCM, respectively) and measured their action potentials at the single cell level by patch clamp as well as at the tissue level by recording field potentials with multielectrode arrays. Compared with the control iCM group, the R190Q iCM group exhibited distinct electrophysiological features with a noticeably prolonged APD and field potential durations with defective cAMP sensitivity75 (Figure 8D; Figure S4), suggesting that this R190Q iCM model successfully recapitulates the clinical phenotype. Furthermore, application of ML277 effectively shortened APDs and field potential durations of both the Control and the R190Q groups (Figure 8D and 8E; Figure S4). We also observed that, unlike the control group, which showed normal cAMP sensitivity and uniform beating pattern (Figure 8F), the R190Q group developed abnormally missing beats, as indicated by an asterisk in Figure 8G. This arrhythmic-like beating phenotype was aggravated under adrenergic stress following cAMP administration, while the beating rate failed to increase (Figure 8G). However, the addition of ML277 was able to accelerate the beating and correct the arrhythmic-like phenotype to a level similar to that of the healthy control group (Figure 8G).

To determine whether this strategy is applicable to a broader set of LQT1 variants, we further tested 2 other groups of high-risk LQT1 variants expressed in Xenopus oocytes (Figure 8H and 8J). The first group was comprised of high-risk LQT1 variants from different determinants along the phosphorylation axis that still show measurable current sizes, such as G189R, R243C, W392R, Y522S, R539W, R555H, and R562M. The application of ML277 successfully enhanced the cAMP sensitivity of these mutant channels (Figure 8I and 8J). We then confirmed that this strategy also worked in Chinese hamster ovary cells. ML277 effectively enhanced the cAMP sensitivity of WT KCNQ1, and high-risk LQT1 mutations R243C and Y522S (Figure S5). The second group contained the top 2 lethal LQT1 mutations, A341V (301 LQT1/375 carriers) and V254M (148 LQT1/174 carriers),15 which completely lose channel function (Figure 3; Figure S6A). The application of ML277 did not rescue the impaired cAMP effect of homozygous mutants (A341VHom and V254MHom; Figure S6B through SBD). R-L3 is the first potent and selective KCNQ1 activator discovered by the Sanguinetti laboratory,66,76,77 with similar effects to ML277 on KCNQ1.41,77,78 We found that R-L3 also successfully enhanced cAMP effects of WT KCNQ1 and the high-risk LQT1 R190Q (Figure S7A through S7C, S7F), but, likewise, could not rescue currents or the cAMP sensitivity of A341VHom and V254MHom (Figure S7D and S7E). On the other hand, we found that ML277 was able to enhance the cAMP effects of A341VHet (A341V+KCNQ1) and V254MHet (V254M+KCNQ1) under heterozygous conditions (103.8±13.7% and 95.5±5.5% current increase; P=7.0×10−4 and P=6.3×10−4, respectively, Figure 8H and 8J). Heterologous expression of these LQT1 variants is more commonly observed in patients.79

DISCUSSION

The human heart displays remarkable adaptability during physical exertion or stress through a fight-or-flight response, which facilitates stable increases in chronotropy, inotropy, and lusitropy. Central to the stability of this response is the cAMP-induced IKs current increase, which accelerates myocyte repolarization, countering increased inward Ca2+ currents.11,50 Consequently, LQT1 mutations that cause reduced or lost cAMP sensitivity of IKs currents pose the highest risk to patients.13,17–20 However, the detailed molecular mechanisms underlying the cAMP-induced IKs current increase and its relation to clinical high-risk of LQT1 mutations have puzzled scientists and clinicians for decades. Recently, a targeted protein phosphorylation approach was developed for restoring the N-terminal (S27/S92) phosphorylation of IKs.80 And the AO state of KCNQ1 was found to have higher cAMP sensitivity than IO.81 Nevertheless, currently resolved Xenopus or human KCNQ1 structures did not include the N-terminal of KCNQ1 or the KCNE1 subunit,22–25 leaving the structural basis and subsequent activation pathway following the N-terminal phosphorylation still largely unclear. This structural and functional gap impedes the development of new therapeutics for LQT1 patients, especially for those high-risk variants with reduced cAMP sensitivity.

In this study, we identify an activation pathway of IKs channel phosphorylation, involving different determinants of the channel. This phosphorylation axis propagates the phosphorylation-induced conformational changes from the N-terminal of KCNQ1 to the channel pore. Multiple lines of evidence suggest that this phosphorylation axis is central to the regulation of VSD-pore coupling: (1) key determinants PIP2, ML277, and the KCNE1 subunit were previously found to significantly enhance the VSD-pore coupling of KCNQ114,23,30,39,41,42; (2) from clinical penetrance analysis, no LQT1 variants (>75% penetrance with at least 5 carriers) were found affecting the middle or upper part of VSD (Figure 5C) and suggesting that phosphorylation may not affect VSD activation. In line with this observation, the G-V relationship of IKs after phosphorylation only shows a ≈10 mV left-shift, despite a >200% increase in current size; (3) Thompson et al50,51 have shown that cAMP does not increase the number of IKs channels on the cell membrane or the single channel conductance but reduces first latencies to channel opening and increases the occupancy of higher-subconductance levels. These results consistently support the notion that modulation of the VSD-pore coupling can be an effective way to alter the phosphorylation of IKs. Due to structural similarity82,83 (Figure S8), this mechanism may also provide insight when studying the phosphorylation of neuronal KCNQ2-5 channels.

Previous studies have demonstrated a cross talk between cAMP modulation and PIP2 sensitivity.63,84,85 Further studies found that LQT1 mutations from the S2–S3 linker, the S4–S5 linker, and the lower S6 lose cAMP sensitivity and constitute increased risk to patients.13,18,19 However, the relationship between these scattered mutations and their defective cAMP response remained unclear. In this study, our experimental data identify a detailed chain reaction following the N-terminal phosphorylation. We also scoured the LQT1 clinical databases and found that (1) it can be anticipated that LQT1 variants disrupting the IKs channel phosphorylation axis are associated with an elevated clinical risk; (2) the distribution pattern of high-risk LQT1 variants closely aligns with the IKs channel phosphorylation axis. These findings have significant implications for predicting the physiological function and clinical phenotype of numerous unexplored variants.

Of note, ML277 can regulate both the KCNQ1 channel and IKs channels associated with unsaturated numbers (1, 2, and 3) of KCNE1 subunits but not on IKs channels with saturated (4) KCNE1 subunits.41,44,86,87 Accumulating evidence demonstrates that in native IKs channels, the KCNE1 association to KCNQ1 is not saturated.45,88–91 Consistent with this observation, our microelectrode array data show that ML277 can shorten the field potential duration in iCMs. We also confirmed this in adult guinea pig ventricular myocytes under both control and an acquired LQTS condition (by adding 100 µmol/L Moxifloxacin, a hERG channel blocker). ML277 effectively shortened their APD90s (Figure S9), suggesting that the KCNQ1 component of native IKs channels provides a promising new antiarrhythmia target. However, ML277 itself (or R-L3) is not able to rescue currents of LQT1 mutations that totally disrupt VSD-pore coupling in homozygous conditions (such as A341V and V254M). Hence, further biophysical and pharmacological studies on these high-risk mutations will be of great importance. In addition, ML277 also increases currents of LQT1 mutations that are distant from the phosphorylation axis,14 which expands application scenarios of this new strategy. In summary, our findings open a new therapeutic avenue, demonstrating that enhancing VSD-pore coupling of native IKs channels by exogenous small molecules can be a promising strategy to rescue cAMP sensitivity, especially for high-risk LQT1 variants where KCNE1 loses its ability to boost cAMP sensitivity.

Limitations of This Study

In this study, we combine biophysical approaches with clinical databases and h-iPSC techniques to improve our understanding of IKs channel phosphorylation. However, it is important to note that there are limitations. For the R190Qpatient iPSC model, a more appropriate control would be the genetic correction of the R190Q mutation. Recent advances in cardiac ion channel gene therapy suggest that this is a promising treatment for arrhythmias.92,93 Experimentally, the averaged IKs current size is smaller than 2 pA/pF in iPSC models.75,94–96 This tiny current size makes it challenging to conduct drug-adding experiments. For the LQT1 clinical database, we were using the clinical phenotypic penetrance of each LQT1 locus as a metric for assessing clinical risk. More direct and comprehensive analysis of cardiac events (cardiac arrest, hospitalization, defibrillation surgery, etc) and event-free survival curves of each LQT1 carrier may provide new insight into the mechanistic understanding of the IKs channel phosphorylation axis and its relation to clinical high-risk LQT1 variants. Detailed structural analysis of conformational changes of IKs channel induced by phosphorylation is needed to fill the gap between channel gating and its clinical relevance.

ARTICLE INFORMATION

Acknowledgments

The authors thank Prof. Karl-Ludwig Laugwitz from Technical University of Munich for generously sharing the R190Qpatient and control iPSC lines.

Author Contributions

P. Hou and J.R. Silva conceived the project, designed the research, and supervised the study. L. Zhong, Z. Yan, D. Jiang, K.-C. Weng, W. Zhu, Y. Ouyang, H. Zhang, X. Lin, C. Xiao, and P. Hou performed experiments. L. Zhong, Z. Yan, W. Zhu, D. Jiang, K.-C. Weng, C. Huang, B. Shen, S.K. Chung, X. Kang, C. Wang, J.R. Silva, and P. Hou analyzed data. W. Zhu, H. Yang, J. Yao, X. Kang, C. Wang, C. Huang, B. Shen, S.K. Chung, Z.-H. Jiang, E. Neher, and J.R. Silva provided key intellectual expertise and methodologies. P. Hou, W. Zhu, J.R. Silva, E. Neher, S.K. Chung, L. Zhong, and Z. Yan wrote and revised the article with input from all authors.

Sources of Funding

This work was supported by the Joint Funding of the Macau Science and Technology Development Fund and the Ministry of Science and Technology of the People’s Republic of China (0006/2021/AMJ to P. Hou), by the National Natural Science Foundation of China (32171221 to P. Hou), by the National Key Research and Development Program of China (2023YFF1204000 to J. Yao), by the Macau Science and Technology Development Fund (0074/2022/A2 and 0098/2023/RIA2 to P. Hou, 002/2023/ALC, and 006/2023/SKL), and by Macau University of Science and Technology (FRG-23-030-SKL to P. Hou). J.R. Silva (National Institutes of Health, R01HL148803 and R01NS092570) and W. Zhu was funded by the Drs Morton and Toby Mower Science Innovation Fund Fellowship, United States.

Disclosures

None.

Supplemental Material

Extended Materials

Major Resources Table

Supplementary Material

Nonstandard Abbreviations and Acronyms

APD action potential duration

CaM calmodulin

cAMP cyclic adenosine monophosphate

CiVSP Ciona intestinalis voltage-sensitive phosphatase

iCM iPSC-derived cardiomyocyte

IKs KCNQ1+KCNE1

iPSC induced pluripotent stem cell

LQT1 long-QT syndrome type 1

PIP2 phosphatidylinositol 4,5-bisphosphate

PKA protein kinase A

VCF voltage clamp fluorometry

VSD voltage sensor domain

* L. Zhang and Z. Yan contributed equally.

For Sources of Funding and Disclosures, see page 735–736.

Supplemental Material is available at https://www.ahajournals.org/doi/suppl/10.1161/CIRCRESAHA.124.325009.
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