KCNE1 (IKs)

YJMCC-08204; No. of pages: 14; 4C: Journal of Molecular and Cellular Cardiology xxx (2015) xxx–xxx

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BACE1 modulates gating of KCNQ1 (Kv7.1) and cardiac delayed rectifier KCNQ1/KCNE1 (IKs) Marianne Agsten a,1, Sabine Hessler a,1, Sandra Lehnert a,1, Tilmann Volk b, Andrea Rittger c, Stephanie Hartmann a, Christian Raab c, Doo Yeon Kim d, Teja W. Groemer e, Michael Schwake c,f, Christian Alzheimer a,2, Tobias Huth a,⁎,2 a

Institut für Physiologie und Pathophysiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91054 Erlangen, Germany Institut für Zelluläre und Molekulare Physiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91054 Erlangen, Germany c Institute of Biochemistry, Christian-Albrechts-Universität, 24098 Kiel, Germany d Genetics and Aging Research Unit, MassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA e Department of Psychiatry and Psychotherapy, University Hospital, 91054 Erlangen, Germany f Biochemie III, Fakultät für Chemie, Universität Bielefeld, 33501 Bielefeld, Germany b

a r t i c l e

i n f o

Article history: Received 2 April 2015 Received in revised form 17 September 2015 Accepted 6 October 2015 Available online xxxx Keywords: KCNQ1 KCNE1 Kv7.1 BACE1 IKs Cardiomyocytes

a b s t r a c t KCNQ1 (Kv7.1) proteins form a homotetrameric channel, which produces a voltage-dependent K+ current. Coassembly of KCNQ1 with the auxiliary β-subunit KCNE1 strongly up-regulates this current. In cardiac myocytes, KCNQ1/E1 complexes are thought to give rise to the delayed rectifier current IKs, which contributes to cardiac action potential repolarization. We report here that the type I membrane protein BACE1 (β-site APP-cleaving enzyme 1), which is best known for its detrimental role in Alzheimer's disease, but is also, as reported here, present in cardiac myocytes, serves as a novel interaction partner of KCNQ1. Using HEK293T cells as heterologous expression system to study the electrophysiological effects of BACE1 and KCNE1 on KCNQ1 in different combinations, our main findings were the following: (1) BACE1 slowed the inactivation of KCNQ1 current producing an increased initial response to depolarizing voltage steps. (2) Activation kinetics of KCNQ1/E1 currents were significantly slowed in the presence of co-expressed BACE1. (3) BACE1 impaired reconstituted cardiac IKs when cardiac action potentials were used as voltage commands, but interestingly augmented the IKs of ATP-deprived cells, suggesting that the effect of BACE1 depends on the metabolic state of the cell. (4) The electrophysiological effects of BACE1 on KCNQ1 reported here were independent of its enzymatic activity, as they were preserved when the proteolytically inactive variant BACE1 D289N was co-transfected in lieu of BACE1 or when BACE1-expressing cells were treated with the BACE1-inhibiting compound C3. (5) Co-immunoprecipitation and fluorescence recovery after photobleaching (FRAP) supported our hypothesis that BACE1 modifies the biophysical properties of IKs by physically interacting with KCNQ1 in a β-subunit-like fashion. Strongly underscoring the functional significance of this interaction, we detected BACE1 in human iPSC-derived cardiomyocytes and murine cardiac tissue and observed decreased IKs in atrial cardiomyocytes of BACE1-deficient mice. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Abbreviations: AP, action potential; APP, amyloid precursor protein; BACE1, β-site APP-cleaving enzyme 1; DMEM, Dulbecco's modified Eagle's medium; DTT, dithiothreitol; EGFP, enhanced green fluorescent protein; FRAP, fluorescence recovery after photobleaching; HEK293T, human embryonic kidney 293 cells with large T antigen; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; IKs, slow delayed rectifier potassium current; IP-buffer, immunoprecipitation buffer; KCNE1, potassium voltagegated channel subfamily E member 1; KCNQ1, potassium channel, voltage gated KQTlike subfamily Q, member 1; NP-40, nonyl phenoxypolyethoxylethanol; PBS, phosphate buffered saline; SDS, sodium dodecyl sulfate; TRIS, tris(hydroxymethyl)aminomethane. ⁎ Corresponding author at: Institut für Physiologie und Pathophysiologie, FriedrichAlexander-Universität Erlangen-Nürnberg, Universitätsstr 17, 91054 Erlangen, Germany. E-mail address: [email protected] (T. Huth). 1 These authors share first authorship. 2 These authors share senior authorship.

KCNQ1 (Kv7.1) proteins constitute the pore-forming α-subunits of a homotetrameric, voltage-gated K+ channel that is widely expressed in cardiac myocytes and many non-excitable tissues, in particular epithelial cells [1,2]. In the heart, KCNQ1 channels co-assemble with KCNE1 βsubunits [3] to generate the delayed rectifier current IKs, which is partially responsible for terminating cardiac action potentials (reviewed in [4]). Loss-of-function mutations in KCNQ1 give rise to the long QT syndrome, which may lead to serious heart arrhythmias and sudden death [4,5]. KCNE1 subunits alter the biophysical features of KCNQ1 in several important aspects [3,6–8]: (i) They delay channel opening after depolarization, (ii) they shift the voltage dependence of current activation

http://dx.doi.org/10.1016/j.yjmcc.2015.10.006 0022-2828/© 2015 Elsevier Ltd. All rights reserved.

Please cite this article as: M. Agsten, et al., BACE1 modulates gating of KCNQ1 (Kv7.1) and cardiac delayed rectifier KCNQ1/KCNE1 (IKs), J Mol Cell Cardiol (2015), http://dx.doi.org/10.1016/j.yjmcc.2015.10.006

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M. Agsten et al. / Journal of Molecular and Cellular Cardiology xxx (2015) xxx–xxx

to more positive potentials, (iii) they enhance single-channel conductance, and (iv) they abrogate current inactivation. The exact stoichiometry of KCNQ1/E1 channel complexes is a long-standing matter of dispute. While some argued that IKs might arise from KCNQ1 channels with a variable number of KCNE1 subunits attached (ranging from one to four), others favored a fixed stoichiometry where a functional IKs channel is composed of four KCNQ1 and two KCNE1 subunits ([reviewed in [9,10]). Recently, we reported that KCNE1 is also a substrate of proteolytic cleavage by β-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1) [11]. BACE1 is best known for its essential role in the amyloidogenic pathway that leads to the formation of toxic amyloid-β peptides in Alzheimer's disease, but APP is by no means the only substrate. In fact, a number of protein targets of BACE1 have been identified over the last years [12], indicating that the enzyme is involved in a broad spectrum of developmental and physiological processes in excitable and presumably in non-excitable tissues [13–19]. With respect to ion channels, β-subunits of voltage-dependent Na+ channels were the first to be identified as targets of BACE1 [20]. Subsequent studies highlighted the functional significance of this interaction for channel expression and gating [21–25]. In the course of those studies, we made the unexpected finding that many of the functional effects of BACE1 on Na+ channels, as well as on neuronal KCNQ channels, were independent of its proteolytic activity. Rather, BACE1 acted like an accessory subunit and modified channel gating through direct protein– protein interaction [22,26]. In our previous study on the interactions between BACE1 and KCNE1, we obtained first evidence that BACE1 alters electrophysiological properties of heterologously expressed KCNQ1/E1 channels [11]. Here, we report that BACE1 strongly augments IKs in murine atrial cardiomyocytes. We provide a detailed analysis of the effects of BACE1 on KCNQ1 channels and reconstituted IKs. Unexpectedly, we also found that the regulation of IKs by BACE1 depends on the level of intracellular ATP. 2. Methods 2.1. Animals BACE1−/− mice (BACE1tm1Psa) were generated by insertion of a neo expression cassette from pMC1neopA into exon 1 of BACE1, resulting in a premature stop codon [21]. Mice were housed and fed according to federal guidelines and had ad libitum access to food and water. Mice were genotyped by either detecting wild type allele or the neo cassette at P8 to P16 using PCR amplification. 2.2. Antibodies and Plasmids The following antibodies were used in this work: rabbit anti-BACE1 (ab108394, Abcam, Cambridge, UK); mouse anti-Flag (M2, SigmaAldrich, Schnelldorf, Germany); rat anti-HA-POD (3F10, Roche, Basel, Switzerland); rabbit anti-actin (A2066, Sigma-Aldrich); mouse anti-βactin-HRP (A3854, Sigma-Aldrich); goat anti-rabbit-IgG-HRP (ab6721, Abcam). The following cDNA constructs were used in this work: hKCNQ1 (NM_000218.2), hKCNQ1-FLAG, and hKCNQ1-HA in pFROG; hKCNE1 (NM_001127670.1) in pFROG; hBACE1 (NM_012104.4) and hBACE1 D289N in pcDNA3.1 were kindly provided by Michael Willem (Adolf-Butenandt-Institute, Ludwig-Maximilians-Universität München, Germany); hBACE1-FLAG in pcDNA3.1; hBACE2 (NM_012105.3) in pCMV6-XL5; hKCNE1, hBACE1, and hKCNQ1, all fused to EGFP (U55761.1) in pcDNA3.1 + hygro; mCherry (Clonetech, Mountain View, CA, USA), pEGFP-C1 (Clonetech) and hENaC1α (NM_001038.5, kindly provided by Christoph Korbmacher, Institut für Zelluläre und Molekulare Physiologie, Friedrich-Alexander-Universität ErlangenNürnberg, Germany, with permission from Harry Cuppens, Centre For Human Genetics, University of Leuven, Belgium) fused to mCherry in

pcDNA3.1 + hygro; Flag-hKCNQ2 (NM_004518.4 with rs1801475) in pcDNA3.1. 2.3. Cell culture and transfection HT22 cells (hippocampal neuronal cell line) and HEK293T cells (ATCC accession number CRL-11,268) were cultured and maintained at 37 °C in 5% CO2 in DMEM medium (HT22: 4,5 g/l glucose, HEK293T: 1 g/l glucose, Gibco, Karlsruhe, Germany), supplemented with 10% fetal bovine serum (Biochrom, Berlin, Germany) and 1% penicillin/ streptomycin solution (Biochrom). One day prior to transfection, HEK293T cells were plated in 3.5 cm dishes (Falcon; BD Biosciences Discovery Labware, Bedford, MA) for electrophysiology, in 6 cm dishes (Falcon) for Western blot analysis or onto 1.5 H coverslips (VWR, Darmstadt, Germany) for FRAP experiments. For transient transfection for patch-clamp experiments, Nanofectin (PAA, Pasching, Austria) was applied, using 1 μg of cDNA of each construct and 0.5 μg of enhanced green fluorescent protein (pEGFP-C1; Clontech, Mountain View, CA) or jetPEI (Peqlab, Erlangen, Germany), using 250 ng of BACE1, 150 ng of KCNQ1, KCNE1 and 75 ng of pEGFP-C1. For FRAP experiments, 150 ng EGFP-tagged protein (hKCNE1-EGFP, hBACE1-EGFP, or hKCNQ1-EGFP) and 450 ng mCherry, hENaC1α-mCherry, hKCNQ1, or Flag-hKCNQ2 were co-transfected using jetPEI (Peqlab). For Western blot analysis, HEK293T cells were transfected with 1750 ng BACE1 and 250 ng pEGFP-C1 using jetPEI (Peqlab). 2.4. iPSC-derived cells iPSC-derived cardiomyocytes (Cor.4U® colorless MPC hIPSC-derived cardiomyocytes, LOT CB155CL_SN_250k) were obtained from Axiogenesis (Cologne, Germany). 7.5 ∗ 105 cardiomyocytes were plated as a monolayer into one cavity of a 12 well plate coated with fibronectin (Sigma-Aldrich) and cultured in provided culture medium supplemented with 2 μg/ml Ciprofloxacin (Sigma-Aldrich). The medium was exchanged every second day. After 5 days, cells were harvested. We observed a syncytial contraction with a frequency of about 1 Hz at that time. iPSC-derived neurons were differentiated and maintained as described elsewhere [27]. 2.5. Co-immunoprecipitation HEK293T cells were harvested 48 h after transfection and lysed in IPbuffer (120 mM NaCl, 50 mM TRIS pH 7.4, 0.5% NP-40, protease inhibitor cocktail (Roche). Lysates (600 μg protein) were incubated with anti-Flag M2 antibody (Sigma-Aldrich) at 4 °C for four hours and proteinantibody complexes were precipitated with Protein G-coupled magnetic beads (Life Technologies, Darmstadt, Germany) for 15 min at room temperature. The beads were washed 4 times with IP-buffer and proteins were eluted by incubation at 55 °C for 20 min in 2× SDS sample loading buffer (0.125 Tris HCl (pH 6.8 at 25 °C), 4% SDS, 0.02% bromophenol blue, 20% glycerol) supplemented with 1 mM DTT. 2.6. Cell lines and iPSC-derived cell lysates Cells were lysed in a buffer containing 10 mM Tris–HCl (pH 7.6), 2 mM EDTA (pH 8.0), 150 mM NaCl, 0.2% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, 1 mM 1–10-phenanthroline and protease inhibitor cocktail (Roche) and incubated for 5 min. For homogenization, lysates were centrifuged in shredder columns (Qiagen, Hilden, Germany) at 1000 rpm, 6 min and subsequently sonicated for 5 min. Insoluble fractions were removed by centrifuging at 13,000 rpm for 15 min. Protein concentration was measured using the BCA Protein Assay Kit (Pierce, Darmstadt, Germany). Samples were prepared with 4 × loading dye (Lonza, Basel, Switzerland) and 5% DTT for SDS-PAGE and Western blot analysis.

Please cite this article as: M. Agsten, et al., BACE1 modulates gating of KCNQ1 (Kv7.1) and cardiac delayed rectifier KCNQ1/KCNE1 (IKs), J Mol Cell Cardiol (2015), http://dx.doi.org/10.1016/j.yjmcc.2015.10.006

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2.7. Western blot analysis

2.10. Patch-clamp recordings of atrial myocytes

Samples were heated at 55 °C for IP-samples or 95 °C for cell lysates for 5 min. Proteins were separated on 10% SDS gels (Bio-Rad, Munich, Germany), 12.5% or 4–12% Bis-Tris NuPage (Life Technologies) and transferred onto PVDF membranes (Bio-Rad) or nitrocellulose membranes (Carl Roth, Karlsruhe, Germany). After blocking, the indicated primary antibodies were incubated over night at 4 °C in 1% BSA and 0.1% NaN3. The membranes were incubated with secondary antibody coupled to horseradish peroxidase in 5% milk for 1 h at room temperature. The signal was visualized by enhanced chemiluminescence using Clarity Western ECL Substrate (Bio-Rad) and imaged with the ChemoStar Imager (INTAS, Göttingen, Germany). Membranes were re-probed after stripping (6 M Guanidine-HCl (Carl Roth), 20 mM Tris, 0.2% Triton X-100, (pH 7.5), and 0.8% 2-Mercaptoethanol (Carl Roth)).

For recording of atrial mouse cardiomyocytes, the pipette solution contained (in mM) 120 K-glutamate, 10 KCl, 4 MgCl2, 10 EGTA, 5 Na2ATP, 10 HEPES, titrated to pH = 7.20 with KOH and the bath solution was modified Tyrode's solution containing (in mM) 138 NaCl, 4 KCl, 1 MgCl2, 0.33 NaH2PO4, 2 CaCl2, 10 glucose, 10 HEPES, titrated to pH = 7.30 with NaOH. Chromanol 293B (Sigma-Aldrich) was prepared as stock solution at 100 mM in DMSO and diluted 1:10,000 in modified Tyrode's solution for a final concentration of 10 μM. DMSO 1:10,000 was present under control conditions as well. Pipette resistance was 2– 4 MΩ in bath solution. Currents were recorded using an EPC-10 amplifier (HEKA Electronik, Lambrecht, Germany), controlled by a PentiumIV based computer and the PULSE-Software (HEKA Elektronik). Membrane capacitance (Cm) and series resistance (Rs) were calculated using the automated capacitance compensation procedure of the EPC10 amplifier. Rs was kept below 10 MΩ and was compensated by 85% leading to an average effective Rs of b1.5 MΩ. Pipette potentials were corrected for the liquid junction potential of 13.3 mV. Whole-cell currents were low-pass filtered at 1 kHz and sampled at 5 kHz. Wholecell currents were elicited by depolarizing voltage steps from a holding potential of −90 mV to +60 mV for 5000 ms. Whole-cell data were analyzed using the PULSE-FIT software (HEKA Elektronik). All experiments were performed at room temperature (23 ± 1 °C).

2.8. Fluorescence Recovery after Photobleaching (FRAP) Two days after transfection, samples were transferred into custommade imaging chambers and covered with HEPES-buffered saline (containing, in mM: 150 NaCl, 3 KCl, 2 CaCl2, 2 MgCl2, 10 HEPES, 10 Dglucose, pH 7.4). FRAP experiments were performed within 1 h on a LSM 780 (Zeiss, Jena, Germany) with a Plan-Apochromat 63×/1.40 NA oil objective and LASOS argon laser LGN 3001 (488 nm laser line) at 20 ± 1 °C. EGFP intensity was recorded from a 104 × 49 pixel (8.4 μm × 3.9 μm) area at 16 bit, 1 airy unit, and an interval of 500 ms for 120 frames in total. After 5 frames, EGFP was bleached within a 33 × 22 pixel region. EGFP intensity within the bleach region was normalized to the mean intensity before bleaching. Intensity after bleaching (frame 6) was set to 0. EGFP recovery was fitted to the equation I(t) = I0 + A ∗ e−t/τ, where I(t) is the EGFP intensity at time point t, I0 the mobile fraction, A the amplitude factor of the fitted curve, t the time after bleaching, and τ the recovery time constant, using OriginPro 9.0G (OriginLab Corp., Northhampton, MA, USA). Statistical significance was tested on logarithmically transformed data. Recovery time constants are given as geometric mean ± back-transformed S.E.M. No significant differences were observed between the mobile fractions of all sample groups. To control for recovery of EGFP fluorophore, cells were fixed with methanol pre-cooled to −20 °C at −20 °C for 10 min and acetone pre-cooled to −20 °C for 1 min and subjected to FRAP recordings. No EGFP recovery was observed (n = 7).

2.9. Isolation of atrial myocytes Wild type and BACE1−/− mice of either sex, 6–8 weeks old, were anesthetized by sevoflurane (AbbVie, Wiesbaden, Germany) and decapitated. The heart was quickly excised and placed into ice-cold phosphate buffered saline (D8537, Sigma-Aldrich), where it stopped beating immediately. Subsequently, the heart was perfused via the ascending aorta at 37 °C for 5 min with modified Tyrode's solution containing 4.5 mM Ca2+ and 5 mM EGTA (~1 μM free Ca2+). Perfusion was continued for 17 min, recirculating 25 ml of the same solution containing collagenase (CLS type II, 160 U/ml, Biochrom KG, Berlin, Germany) and protease (type XIV, 0.6 U/ml, Sigma-Aldrich). Finally, the heart was perfused with storage solution [28] containing 100 μM Ca2 + for 5 min. After separation of the atria from the ventricles, atrial tissue pieces were minced and gently agitated to obtain single cardiomyocytes. After adaption to physiological Ca2 + levels, cells were transferred to cell culture dishes containing storage solution supplemented with 100 IU/ml penicillin and 0.1 mg/ml streptomycin, stored at 37 °C in a water-saturated atmosphere containing 5% CO2 and used for experiments for up to 8 h. Only quiescent single cells were used for experiments.

2.11. Patch-clamp recordings of transfected HEK293T cells Transfected cells were identified using an inverted fluorescence microscope (Axiovert 40, Zeiss, Jena, Germany) with a fiber optic coupled light source (UVICO, Rapp OptoElectronic, Hamburg, Germany). Current signals were recorded in whole-cell voltage-clamp mode 2–3 days post transfection at room temperature (22 ± 1 °C), if not otherwise stated. To reduce expression differences between groups, we used the same batch of cells, the same DNA preparation, and the same transfection protocol. Recordings were started 3 min after whole-cell access was obtained. Data were sampled at 20 kHz and filtered at 5 kHz, using an Axopatch 200B or 700B amplifier in combination with a Digidata 1322A interface and pClamp10 software (all from Molecular Devices/MDS Analytical Technologies, Sunnyvale, CA, USA). Electrodes were made from borosilicate glass (Harvard Apparatus, Edenbridge, UK or BioMedical Instruments, Zoellnitz, Germany), using a DMZ-Universal Puller (Zeitz, Munich, Germany). Pipette resistance in bath solution was 1.8–3.3 MΩ and access resistance was typically b5 MΩ before series resistance compensation (75%). Solutions were exchanged by means of a rapid, gravity-driven Y-tube system. External solution contained (in mM): 145 NaCl, 4 KCl, 2 CaCl2, 2 MgCl2, 10 D-Glucose, 10 HEPES, adjusted to pH 7.4 with NaOH. For recording of deactivation kinetics, high potassium bath solution was used, containing (in mM): 5 NaCl, 150 KCl, 2 MgCl2, 2 CaCl2, 10 HEPES and 10 D-glucose, adjusted to pH 7.4 with KOH. The standard internal solution was composed of (in mM) 5 NaCl, 120 KCl, 2 MgCl2, 1 CaCl2, 5 EGTA and 10 HEPES, adjusted to pH 7.2 with KOH. The ATPcontaining internal solution was composed of (in mM) 135 K-gluconate, 4 NaCl, 3 MgCl2, 5 HEPES, 5 EGTA, 2 Na2-ATP, 0.3 Na3-GTP adjusted to pH 7.25 with KOH. Chemicals were purchased from Sigma-Aldrich. For some experiments, the BACE1 inhibitor C3 (10 μM, Merck, Darmstadt, Germany) was applied to HEK293T cells after transfection and then added every 12 h until cells were used for electrophysiological experiments [29]. 2.12. Voltage-clamp experiments with heart action potentials waveforms The waveform of a heart action potential (AP) was taken from a MATLAB simulation (MathWorks, Ismaning, Germany) of undiseased human ventricle [30]. Thirty consecutive APs at 1 Hz were applied as command potential before and 180 s after treatment with 100 μM chromanol (Sigma-Aldrich). Recordings were performed at 34 ± 1 °C using poly-D-

Please cite this article as: M. Agsten, et al., BACE1 modulates gating of KCNQ1 (Kv7.1) and cardiac delayed rectifier KCNQ1/KCNE1 (IKs), J Mol Cell Cardiol (2015), http://dx.doi.org/10.1016/j.yjmcc.2015.10.006

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lysine coated FluoroDishes (WPI, Sarasota, FL, USA) for better thermal conduction in conjunction with a custom-made heatable and feedbackcontrolled dish mount. 2.13. Modeling Single-channel noise-free time series were simulated using a given Markov model and subsequently averaged (2000 simulations, 1000 ms each) with the program KIEL-PATCH [31].

2.14. Data analysis To determine the voltage dependence of current activation, a leak correction was performed. The potassium conductance at each command potential was calculated with the use of the equation G = I/ (V − Erev), where I is the mean amplitude of the potassium current at the end of the depolarizing step at voltage V and where Erev is the equilibrium potential for potassium ions under our experimental conditions. G was fitted by a standard sigmoidal (Boltzmann) relation of the form G/

Please cite this article as: M. Agsten, et al., BACE1 modulates gating of KCNQ1 (Kv7.1) and cardiac delayed rectifier KCNQ1/KCNE1 (IKs), J Mol Cell Cardiol (2015), http://dx.doi.org/10.1016/j.yjmcc.2015.10.006

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Gmax = 1/[1 + exp.((Vmid − V) / k)], where Vmid is the midpoint of activation and k is the slope factor. KCNQ1 activation and inactivation time constants were determined with the use of a bi-exponential fit on the whole activation timescale, where τi with negative amplitude factor was associated with activation and τi with positive amplitude factor with inactivation. Inactivation time constants were determined if applicable. Activation time constants of KCNQ1/E1 recordings were fitted with a bi-exponential function. The first fitting cursor was set after the initial plateau phase. The second cursor was set at the end of the activation pulse at 5 s or if the current was declining (with ATP in the pipette) at the time of peak. The slow component was taken as activation time constant. The faster component with a small amplitude accounted for the initial transition from the plateau to the rising phase in some recordings. Data analysis was performed using pClamp 10 (Molecular Devices) and OriginPro 8.1G software. All data are expressed as mean ± standard error of the mean (S.E.M.), with differences to be considered significant at p b 0.05. Statistical analysis was performed with OriginPro 9.0 software (OriginLab). Tests to determine statistical significance are stated in text or figure legends. 3. Results 3.1. BACE1 slows the inactivation kinetics of KCNQ1 channels in a proteolysis-independent fashion To explore the possible impact of BACE1 on KCNQ1 channels, we used HEK293T cells as a heterologous expression system and performed whole-cell voltage-clamp recordings from cells expressing KCNQ1 alone or in combination with its KCNE1 β-subunit and/or BACE1. In good agreement with previous reports [3], expression of homomeric KCNQ1 channels yielded voltage-dependent K+ currents that were activated around −50 mV, increased with membrane depolarization and displayed no or little apparent inactivation during prolonged depolarization (Fig. 1A1). Co-expression of BACE1 produced a substantial change in the shape of KCNQ1 currents. Compared to control recordings, the depolarizing voltage steps evoked much stronger outward currents after the onset of the depolarizing step that were followed by prominent inactivation before reaching steady-state levels (Fig. 1A2). Importantly, this effect of BACE1 on channel gating occurred independently of its enzymatic activity, since it was preserved when the catalytically inactive BACE1 D289N variant was co-expressed with KCNQ1 (Fig. 1A3), or when the enzymatic activity of BACE1 was pharmacologically suppressed by the specific BACE1 inhibitor C3 (Fig. 1A4). These results, together with the finding that co-expression of BACE2 failed to alter KCNQ1 currents (Fig. 1B), support a specific, non-catalytic action of BACE1. The selective KCNQ1 blocker chromanol [32] suppressed both the inactivating and the steady-state component of KCNQ1/BACE1 currents (Fig. 1C), indicating that the prominent inactivating component introduced by co-

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expression of BACE1 is specific to its interaction with KCNQ1 and does not result from effects of the secretase on intrinsic currents of HEK293T cells. From activation experiments shown in Fig. 1A, we determined the I– V relationships for KCNQ1 currents in the absence and presence of BACE1. To avoid contamination by intrinsic currents of HEK293T cells, I–V curves were constructed after subtracting chromanol-insensitive currents from control recordings (cf. Fig. 1C). Whereas BACE1 strongly enhanced peak KCNQ1 currents (Fig. 1D, red triangles), the I-V -relationship of steady-state KCNQ1 currents (Fig. 1D, red and black boxes) as well as their voltage dependence of activation remained unaffected (Fig. 1E). In a different, non-chromanol-subtracted dataset, which also included the enzymatically inactive BACE1 variant D289N, both BACE1 constructs caused a small, but significant increase of the activation time constants of KCNQ1 currents (Fig. 1F). In comparison, the effect of the two BACE1 variants on the almost voltage-independent time constant of inactivation was much more prominent (Fig. 1G). Thus, the apparently “overshooting” current response to depolarizing voltage steps in KCNQ1 and BACE1 co-expressing cells can be attributed to the pronounced BACE1-mediated slowing of inactivation, leading to an increased peak current. Since BACE1 seemed to act predominantly on inactivation, we explored this feature in more detail. We quantified the fraction of current inactivation by normalizing steady-state to peak currents. Whereas apparent current inactivation was negligible in HEK293T cells expressing KCNQ1 alone (Fig. 1H, black squares), co-expression of BACE1 significantly enhanced the fraction of current inactivation (Fig. 1H, red squares), initially allowing much higher peak currents. Again, this effect could be fully replicated with inactive BACE1 D289N (Fig. 1H, green squares) and the BACE1 inhibitor C3 (Fig. 1H, blue squares) [29]. Recovery from inactivation with BACE1 was investigated in two different paradigms, in which we varied either the voltage (at fixed duration) or the duration (at fixed voltage) of the repolarizing steps between the two depolarizing test pulses. Representative examples are illustrated in Fig. 2A,B and summarized in Fig. 2C,D. These experiments demonstrated that in the presence of BACE1, recovery from inactivation is strongly dependent on voltage (Fig. 2C) and time (Fig. 2D), making it, compared to inactivation, a relatively slow process that also required very negative membrane potentials. Again, the effects were virtually identical for normal BACE1 and its mutant variant D289N (Fig. 2C,D). 3.2. Assembly of KCNQ1 and BACE1 in the same multi-protein channel complex Our electrophysiological findings strongly suggested that BACE1 regulates an essential feature of KCNQ1 gating, namely its inactivation kinetics, independently of its catalytic activity. The most direct explanation would be that the BACE1 molecule can physically associate with

Fig. 1. Co-expression of BACE1 with KCNQ1 produces current phenotype with apparent inactivation. Whereas stepwise depolarization of HEK293T cells expressing KCNQ1 alone evoked currents that appeared to lack appreciable inactivation (A1), co-transfection of BACE1 or its catalytically inactive variant BACE1 D289N introduced a prominent inactivating component (A2,A3). The appearance of an inactivating component was not abrogated by compound C3, which blocks the proteolytic activity of BACE1 (A4), but was absent when cells were cotransfected with BACE2 instead of BACE1 (B). Inset in (A1) depicts voltage protocol. (C), the KCNQ1 channel blocker chromanol (100 μM) was used to demonstrate that the inactivating component introduced by BACE1 resulted indeed from altered KCNQ1 gating. Subtraction of recordings obtained in the absence (C1) and presence of chromanol (C2) served to isolate KCNQ1/BACE1 current (C3). (D), current–voltage (I–V) curves of chromanol-subtracted KCNQ1 currents were determined in the absence and presence of co-transfected BACE1 using the activation protocol of (A1). Although co-expression of BACE1 gave rise to large peak current after onset of depolarization (red triangles), inactivation brought current amplitude down to steady-state values (red boxes) not different from those recorded with KCNQ1 alone (black boxes). We did not determine peak I–V curves for KCNQ1 alone, since those currents had no appreciable peaks (A1). (E), normalized conductance was derived from steady-state I–V relationship and fitted with a Boltzmann equation before calculation of means, yielding half-activation voltages that did not differ significantly (KCNQ1: Vh = −12.6 ± 1.9 mV, n = 11; +BACE1: Vh = −9.0 ± 1.3 mV, n = 10). Time constants of activation (F) and inactivation (G) were determined from a different set of data without chromanol subtraction. Time constants were determined with the use of a bi-exponential fit, where τi with negative amplitude factor was associated with activation and τi with positive amplitude factor with inactivation. Inactivation time constants of KCNQ1 were determined if applicable. KCNQ1 n = 15 (F), n = 4 (G); +BACE1 n = 40 (F), n = 38 (G); +BACE1 D289N n = 17 (F), n = 16 (G). *p b 0.05, **p b 0.01, ***p b 0.001. Statistics were performed using a pairwise Mann–Whitney-test with Bonferroni correction. (H), apparent fractional inactivation of KCNQ1 currents was enhanced by BACE1. Inactivation was quantified from activation recordings by 1− IIPS with steadystate current IS and peak current IP. Co-transfection of BACE1 or BACE1 D289N caused a substantial fraction of the initially evoked current to become inactivated. This effect was not sensitive to the BACE1 inhibiting compound C3. KCNQ1 n = 14; +BACE1 n = 14, +BACE1 D289N n = 14; BACE1 +C3 treatment n = 14. ***p b 0.001. Statistical analysis was performed using a pairwise Mann–Whitney-test with Bonferroni correction.

Please cite this article as: M. Agsten, et al., BACE1 modulates gating of KCNQ1 (Kv7.1) and cardiac delayed rectifier KCNQ1/KCNE1 (IKs), J Mol Cell Cardiol (2015), http://dx.doi.org/10.1016/j.yjmcc.2015.10.006

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Fig. 2. Recovery from inactivation of KCNQ1 current in the presence of BACE1 is time- and voltage-dependent. Recovery from inactivation was determined with a double-pulse protocol in which either inter-pulse voltage (A) or inter-pulse duration were systematically varied (B). Relative inactivation was quantified from above recordings by dividing fraction of inactivation of second pulse by that of first pulse for different inter-pulse voltages (C) and intervals (D). KCNQ1 +BACE1 n = 15 (C), n = 13 (D); +BACE1 D289N n = 5 (C), n = 5 (D).

KCNQ1 channels in a β-subunit-like fashion as we have shown previously for KCNQ2/Q3 heteromers [26]. Ample evidence from established auxiliary subunits of numerous voltage-dependent ion channels clearly indicates that such a physical interaction is well capable of modifying the gating properties of the α-subunit. To support the notion that KCNQ1 and BACE1 physically interact, we performed co-immunoprecipitation and fluorescence recovery after photobleaching (FRAP) experiments in HEK293T cells. The coimmunoprecipitation blots demonstrate that it was indeed possible to precipitate BACE1 together with KCNQ1 (Fig. 3A) and vice versa (Fig. 3B). Interestingly, expression of KCNQ1 augmented the occurrence of a shorter, possibly immature variant of BACE1, which is also predominantly co-precipitated (Fig. 3A). The FRAP experiments aimed to analyze the diffusion behavior of BACE1 or KCNE1 in the absence or presence of KCNQ1. Therefore, either BACE1 or KCNE1 was tagged with EGFP. To assess surface mobility at the cell membrane, a small region of the membrane (Fig. 3C 1, red ROI) was bleached (Fig. 3C2) and subsequent recovery of fluorescence (Fig. 3C3), i.e. diffusion, was monitored over time. Time traces of fluorescence recovery were fitted to a mono-exponential equation, as illustrated in Fig. 3D. Previously, Roura-Ferrer et al. [33] demonstrated that KCNQ1 restricts KCNE1 diffusion, which was confirmed in our experiments (Fig. 3E, left panel). Strongly resembling its effect on KCNE1 diffusion, co-expression of KCNQ1 also slowed BACE1 diffusion, whereas co-expression of the epithelial sodium channel (ENaC), as a control, did not affect BACE1 diffusion (Fig. 3E, middle panel). Since we had recently identified the neuronal KCNQ family member KCNQ2 as a new interaction partner of BACE1 [26], we also tested its effect on BACE1 mobility. Co-expression of KCNQ2 indeed slowed BACE1 movement in a fashion very similar to that of KCNQ1 (Fig. 3E, middle panel). Notably, the mobility of KCNE1 or BACE1 upon co-expression of KCNQ1 was indistinguishable from the diffusion behavior of KCNQ1 itself (Fig. 3E, right panel).

3.3. Effects of BACE1 on IKs-like current mediated by KCNQ1/E1 channel complexes In cardiac myocytes, KCNQ1 subunits assemble with KCNE1 βsubunits to generate the delayed rectifier current IKs, which contributes to the repolarization of the cardiac action potential. To unravel the effects of BACE1 on this channel complex, we performed whole-cell recordings from HEK293T cells co-transfected with KCNQ1 and KCNE1 alone or in combination with BACE1. Co-assembly with KCNE1 modifies the gating of KCNQ1 channels in several aspects (see Introduction). At the whole-cell level, these effects of KCNE1 translate into current responses that are strikingly different from those mediated by KCNQ1 alone. The characteristic alterations can be readily detected from a comparison of the current trajectories of Fig. 1A1, which were generated by KCNQ1, with those of Fig. 4A, which were conducted by KCNQ1/E1. Note that currents kept growing even though step duration was longer (5 s) than that for recordings of KCNQ1 alone (1 s). Upon co-transfection of BACE1, we observed a moderate enhancement of KCNQ1/E1 currents (Fig. 4B). KCNQ1/E1 channels require intracellular ATP binding to open properly [34]. The dramatic effect that addition of ATP to the pipette solution had on the amplitude of KCNQ1/E1 currents is exemplified in Fig. 4C,D and summarized in the I/V-relationships determined in the absence and presence of 2 mM intracellular ATP (Fig. 4E,F). Notably, intracellular ATP reversed the effect of BACE1 on KCNQ1/E1 currents from an enhancement without the nucleotide to a reduction when ATP was present (Fig. 4E vs. F). By contrast, the slowing of current activation by BACE1 was not appreciably affected by ATP (Fig. 4G,H). As reported previously [11], BACE1 shifted the voltage dependence of KCNQ1/E1 current in the depolarizing direction. This effect was particularly pronounced in the presence of intracellular ATP (KCNQ1/E1: ΔV = 4.8 mV, p b 0.09; KCNQ1/E1ATP: ΔV = 7.2 mV, p b 0.003). Compared to our previous study [11], the BACE1-mediated voltage-shift (in the

Please cite this article as: M. Agsten, et al., BACE1 modulates gating of KCNQ1 (Kv7.1) and cardiac delayed rectifier KCNQ1/KCNE1 (IKs), J Mol Cell Cardiol (2015), http://dx.doi.org/10.1016/j.yjmcc.2015.10.006

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Fig. 3. Co-immunoprecipitation and protein surface diffusion provide evidence for physical interactions between KCNQ1 and BACE1. (A,B) HEK293T cells were mock-transfected or transfected with tagged constructs as indicated above the blots. After 48 h, cells were lysed and immunoprecipitated (IP) with anti-Flag antibody. Lysates (input) and immunoprecipitates were immunoblotted and membranes were probed with antibodies as indicated. (A), BACE1 could be precipitated with KCNQ1. (B), vice versa, KCNQ1 could be precipitated with BACE1. (C), fluorescence recovery after photobleaching (FRAP) assessed mobility of EGFP-tagged transmembrane proteins at the plasma membrane. A small region of the membrane (C1, red ROI) was bleached (C2) and subsequent recovery of fluorescence (C3) was monitored over time. (D), example traces of BACE1-EGFP fluorescence upon co-expression of mCherry or KCNQ1 are shown (green traces) with corresponding fit curves using the equation I(t) = A ∗ e−t/τ + I0. (E), recovery time constants τ were determined from the constructs stated in the graph and with constructs co-expressed indicated below the graph. Transfected cDNA levels were equalized with mCherry plasmid. Statistics were performed using one-way ANOVA with post-hoc Tukey test. **p b 0.01, ***p b 0.001.

absence of ATP) was appreciably smaller in size. The reason for this variation is not immediately obvious, but might be related to different batches of HEK293T cells or to changes in transfection procedure. In any case, we qualitatively reproduced here the right-ward shift of the activation curve by BACE1 reported in our previous study. Since peak current measurements for the construction of the I/Vrelationships of Fig. 4E,F could not be made under steady-state conditions, it seems plausible to assume that the delayed activation contributes to the apparent decrease of KCNQ1/E1ATP current by BACE1. Because the current is so enormously amplified by intracellular ATP, the effect of BACE1 on the activation time constant might well override its small augmenting effect on current amplitude, which was observed in the absence of the nucleotide. Although this kinetic scheme should offer a reasonable explanation for the apparent reversal by ATP of how BACE1 alters KCNQ1/E1 current amplitude, additional effects of BACE1 on the pore opening mechanism of ATP are conceivable. To explore the role of BACE1 in the inactivation process, we performed recordings in high extracellular K+ (see Methods) and varied systematically the duration of the depolarization to + 50 mV before stepping back to −100 mV. A representative example of this set of experiments is illustrated in Fig. 5A, which was performed in the presence of 2 mM ATP. With activation complete and no inactivation occurring,

tail currents from channel closing should decay in a virtually identical, monotonic fashion irrespective of how long the cell was depolarized. As predicted by the notion that KCNE1 abrogates inactivation of KCNQ channels (see Introduction), such tail kinetics were indeed observed for KCNQ1/E1 currents (Fig. 5A3). By contrast, monotonic decay was not observed in traces with KCNQ1 alone or with co-transfected BACE1, since tails became broader as depolarization was extended (Fig. 5A1,2,4). A biphasic time course of tail current emerged, with an initial rise in amplitude before it exponentially declined. This “hook” in the tail current trajectory can be best explained by assuming that, after a strong repolarizing step, exit from the inactivated state proceeds faster than deactivation, so that initially more inactivated channels (re-)open than open channels close. Thus, kinetic analysis of hooked tail currents allows one to infer on how channel inactivation has proceeded during the preceding depolarization. We quantified tails by determining the time of the inflection point as illustrated in Fig. 5B. This time point is depending on the fraction and kinetics of re-opening channels vs. their deactivation kinetics and serves as an overall measure of the magnitude of the “hook”. As expected, the lowest values were obtained with KCNQ1/E1 (Fig. 5C, black trace), although, with extended depolarizations, some channels accumulated in an inactivated state. Recordings with KCNQ1 alone showed

Please cite this article as: M. Agsten, et al., BACE1 modulates gating of KCNQ1 (Kv7.1) and cardiac delayed rectifier KCNQ1/KCNE1 (IKs), J Mol Cell Cardiol (2015), http://dx.doi.org/10.1016/j.yjmcc.2015.10.006

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Fig. 4. BACE1 interacts with KCNQ1/E1 channel complexes. KCNQ1/E1 currents with typical slow activation kinetics were evoked in the absence (A) and presence of BACE1 (B). (C,D), activation recordings were repeated with 2 mM ATP in the patch pipette. (E,F), I–V relations were quantified at the end of current traces (A–D) at t = 5 s. (G,H), activation time constants from above recordings (A–D) were fitted with a bi-exponential function. The slow component is depicted in the graph and represents the very slow activation process after the plateau phase. The faster component with a small amplitude accounted for the initial transition from the plateau to the rising phase in some recordings. KCNQ1 +KCNE1 n = 22; KCNQ1 +KCNE1 +ATP n = 43; +BACE1 n = 22; +BACE1 +ATP n = 47; Statistics were performed using a Mann–Whitney-test. *p b 0.05, **p b 0.01, ***p b 0.001.

appreciable hooks and the inactivation process reached steady-state after about 800 ms. Co-transfection of BACE1 delayed the inactivation process considerably, as predicted by the previously illustrated

appearance of strongly decaying peak currents during depolarizing voltage steps in physiological K+ gradient (Fig. 1A). Interestingly, the tail currents of KCNQ1/E1 with BACE1 were initially (up to 400 ms)

Please cite this article as: M. Agsten, et al., BACE1 modulates gating of KCNQ1 (Kv7.1) and cardiac delayed rectifier KCNQ1/KCNE1 (IKs), J Mol Cell Cardiol (2015), http://dx.doi.org/10.1016/j.yjmcc.2015.10.006

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Fig. 5. BACE1 delays and increases inactivation in KCNQ1 and KCNQ1/E1. (A), tail currents were recorded after a variable activation time period Δt at +50 mV in high extracellular potassium solution and 2 mM intracellular ATP. Transfections are indicated in the graph. The command protocol is depicted above current traces. (B), illustrates analysis of tail currents. Tails were obtained from recordings shown in (A) and overlaid for illustration starting at the step to −100 mV. Colored traces indicate a multi-exponential fit to obtain noise-free time series (B1). Subsequently, the time of peak of the first derivative was determined (B2), yielding the inflection point of the hooked tail current (arrows), if applicable, otherwise set to zero (red trace). (C,D), inflection time points of hooked tail currents were plotted against Δt of the activation period indicating the duration and strength of the inactivation process without (C) and with 2 mM ATP (D) in the pipette solution. Brackets indicate groups that were statistically compared. KCNQ1 n = 26 (C), n = 22 (D); KCNQ1 +BACE1 n = 30 (C), n = 20 (D); KCNQ1 +KCNE1 n = 29 (C), n = 20 (D); KCNQ1 +KCNE1 +BACE1 n = 30 (C), n = 26 (D); Statistics were performed using a pairwise Mann–Whitney-test with Bonferroni correction. *p b 0.05, **p b 0.01, ***p b 0.001.

Please cite this article as: M. Agsten, et al., BACE1 modulates gating of KCNQ1 (Kv7.1) and cardiac delayed rectifier KCNQ1/KCNE1 (IKs), J Mol Cell Cardiol (2015), http://dx.doi.org/10.1016/j.yjmcc.2015.10.006

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Fig. 6. Effects of BACE1 on KCNQ1/E1 currents evoked by cardiac action potential-like waveforms at near-physiological temperature. (A), using the waveform of a cardiac action potential as voltage command (blue trace), KCNQ1/E1 currents expressed in HEK293T cells were examined at 34 °C with 2 mM ATP in the pipette solution. 30 consecutive current responses were superimposed (black traces) and averaged (red trace). Total protocol duration was 1000 ms. (B), the experiment outlined in (A) was repeated with a pipette solution containing no ATP. (C,D), mean current responses to cardiac action potential waveform from cells transfected with KCNQ1/E1 (black traces) or KCNQ1/E1 +BACE1 (red traces) in the presence (C) or absence of intracellular ATP (D). (E,F), the above experiments were repeated with elevated extracellular potassium solution to monitor decay of KCNQ1/E1 current after repolarization was completed. Note slower time scale as compared to recordings in (C,D). KCNQ1 +KCNE1 n = 15 (C), n = 29 (D), n = 22 (E), n = 16 (F); +BACE1 n = 14 (C), n = 33 (D), n = 23 (E), n = 20 (F). The averaged current traces were filtered using a median percentile filter to remove shot noise. Significant differences at a given time point are assumed for non-overlapping error ranges.

indistinguishable from those of KCNQ1/E1 alone, before hooks became increasingly pronounced. The different time windows in which BACE1 affects inactivation of KCNQ1 channels alone vs. that of KCNQ1/E1 channels can be taken as evidence for a genuine effect of BACE1 on the latter. Intracellular ATP did not fundamentally alter the inactivation processes of the various channel combinations, but made the unblocking process more prominent except for KCNQ1/E1 assemblies (Fig. 5D). 3.4. Effects of BACE1 on reconstituted cardiac IKs We next explored how the multiple effects of BACE1 on KCNQ1/E1 channel complexes might combine to alter the behavior of cardiac delayed rectifier current IKs in a physiologically more realistic scenario. For this purpose, we used the approximated waveform of a heart action potential (AP) as voltage command, with or without 2 mM ATP in the

pipette solution, and performed the recordings in warmed extracellular medium. Under these conditions, the cardiac AP waveform evoked a delayed outward current which peaked during the repolarizing phase, thereby reproducing the characteristic feature of IKs (Fig. 6A,C black trace). Co-transfection of BACE1 yielded a current response with a substantially altered trajectory. As expected from its effects on KCNQ1/E1 currents during square depolarizing steps (Fig. 4C,D), BACE1 augmented the initial current response following the onset of the depolarization. However, owing to its pronounced decelerating effect on the slow activation process of KCNQ1/E1 channels, BACE1 strongly attenuated the characteristic late rise in current amplitude (Fig. 6C, red trace). The previous tail current recordings (Fig. 5A) demonstrated that KCNQ1/E1 channels are endowed with slow deactivation kinetics. The AP-evoked current recordings might therefore underestimate the impact of the deactivation kinetics on cellular excitability, because the

Please cite this article as: M. Agsten, et al., BACE1 modulates gating of KCNQ1 (Kv7.1) and cardiac delayed rectifier KCNQ1/KCNE1 (IKs), J Mol Cell Cardiol (2015), http://dx.doi.org/10.1016/j.yjmcc.2015.10.006

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Fig. 7. Effect of the proteolytically inactive BACE1 variant D289N on KCNQ1/E1 currents evoked by cardiac action potential-like waveforms at room temperature. Using the waveform of a cardiac action potential as voltage command (outlined in Fig. 6A), KCNQ1/E1 currents expressed in HEK293T cells were examined with 2 mM ATP in the pipette solution. KCNQ1 +KCNE1 n = 43; +BACE1 n = 45; +BACE1 D289N n = 32. Significant differences at a given time point are assumed for non-overlapping error ranges.

driving force becomes negligible when the cell is repolarized to rest within 300 ms of AP onset. Even after that time point, however, membrane conductance remains transiently elevated as channels are gradually

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closed by deactivation. As a consequence, depolarizing currents, which would interfere with regular cardiac rhythmogenesis, would be attenuated by shunting inhibition. To estimate the time course of such post-AP inhibition through deactivating KCNQ1/E1 channels, we recorded current responses in high K+ bath solution. The averaged recordings clearly indicated that the deactivation process outlasts the cardiac AP waveform by several 100 ms (Fig. 6E, black trace, note different time scale compared to Fig. 6C). As expected, co-transfection of BACE1 reduced peak current amplitude without an appreciable effect on current decline (Fig. 6E, red trace). Given the boosting effect of ATP on KCNQ1/E1 currents [34], we wondered how the current response would be altered if we simulated the drop in intracellular ATP that is experienced by metabolically compromised cardiac cells. The effect of omitting ATP from the pipette solution is depicted in Fig. 6B,D,F (black trace). The overall current amplitude was strongly reduced and, functionally even more important, the late rise in current amplitude during the repolarizing phase of the AP waveform was almost completely abrogated. In the absence of intracellular ATP, co-expression of BACE1 augmented both early and late KCNQ1/E1 current (Fig. 6D, red trace). Recordings in high external K + showed that BACE1 increased peak current and slowed its deactivation after repolarization to rest (Fig. 6F, black vs. red trace). These data suggest that

Fig. 8. Physiological relevance of endogenous BACE1 in cardiomyocytes. (A,B), BACE1 is endogenously expressed in mouse heart tissue and in human iPSC-derived cardiomyocytes. (A), hearts were prepared from wild type and BACE1 knockout mice. Total heart lysate was analyzed by immunoblotting using the indicated antibodies. For comparison, total brain lysates were subjected to the same procedure. (B), cell lysates with 60 ng protein of HEK293T cells transiently transfected with BACE1, 60 μg protein of HT22 cells and hiPSC-derived cardiomyocytes, and 30 μg protein of hiPSC-derived neurons were probed with the same antibody as in (A) after immunoblotting. All lysates show a robust BACE1 expression. (C–E) Currents were recorded from acutely isolated murine atrial cardiomyocytes of wild type or BACE1−/− mice (inset of D) using a step protocol. The cell capacitance was comparable in both groups (wt 84.0 ± 7.5 pF; BACE1−/− 89.0 ± 5.4 pF). Individual currents were quantified as mean between 950 and 990 ms after depolarization to +60 mV. After baseline recordings, 10 μM chromanol was applied and chromanol-sensitive currents (E) were calculated by subtraction. Wild type n = 24 cells from 3 mice; BACE1−/− n = 12 cells from 3 mice. Statistics were performed using a two-sample t test. *p b 0.05.

Please cite this article as: M. Agsten, et al., BACE1 modulates gating of KCNQ1 (Kv7.1) and cardiac delayed rectifier KCNQ1/KCNE1 (IKs), J Mol Cell Cardiol (2015), http://dx.doi.org/10.1016/j.yjmcc.2015.10.006

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Fig. 9. Simulation of how changes in single-channel kinetics influence apparent macroscopic current inactivation. (A), KCNQ1 currents were simulated with a Markov model of singlechannel kinetics that includes the two open states O1 and O2 and the inactivated state I. Closed states before opening were merged into Cstart. Activation remained invariant using the rate constants indicated (in Hz). (B), noise-free time series were simulated 2000 times for 1000 ms, every time starting from state Cstart. The open probability P(open) was calculated from ensemble averages and plotted against time. The rate constants attached to the inactivated state I were varied according to the parameters denoted on the graph. Rate constants are given in Hz.

the direction in which BACE1 alters IKs is coupled to the metabolic state of the cell. To determine whether the effects of BACE1 on reconstituted IKs, like those on KCNQ1 alone, are non-proteolytic in nature, we conducted experiments with the catalytically inactive BACE1 variant D289N, using an AP-waveform protocol (Fig. 7). To better resolve possible kinetic differences, we performed these recordings at room temperature. Both BACE1 and its mutant augmented the current over the whole AP duration in a highly similar fashion, suggesting that the BACE1 effects on KCNQ1/E1 are predominantly attributable to a direct, non-enzymatic interaction. This finding does not exclude an effect of KCNE1 proteolysis on a biophysical feature of IKs (as reported before [11]), but it shows that the physiologically relevant regulation of IKs function by BACE1 is heavily dominated by its direct, non-proteolytic interaction with the channel complex. 3.5. Physiological relevance To show that BACE1 is a likely interaction partner of native KCNQ1/ E1 channels, we explored its presence in cardiac tissue using two approaches. First, we analyzed the expression of BACE1 in heart tissue of normal and BACE1−/− mice at postnatal day 30 (P30). The Western blots of Fig. 8A show that BACE1 is indeed expressed in mouse cardiac tissue, as it is in brain tissue. Second, we analyzed cell lysates from BACE1-transfected HEK293T cells, from cardiac myocytes derived from human iPS cells, from the murine immortalized hippocampal cell line HT22, and from neuronal hiPS cells-derived cells using the same antibody as in Fig. 8A. BACE1 was robustly expressed in all cell types including human cardiac myocytes (Fig. 8B). To directly demonstrate the relevance of BACE1 as a component of physiological IKs, we performed patch-clamp recordings from acutely isolated murine atrial cardiomyocytes of wild type and BACE1-deficient mice. A previous study had already shown that KCNQ1/E1 give rise to a chromanol-sensitive current in these cells [35], and this finding was confirmed here. We quantified total and chromanol-sensitive K+ currents from their response to a depolarizing step to +60 mV, where both Na+ and Ca2+ currents reversed, leaving K+ channels as the main contributor to the recorded currents (Fig. 8C,D). Compared to the wild type preparation, atrial myocytes from BACE1−/− mice showed decreased total steady-state currents (wild type cells 22.7 ± 2.4 pA/pF, n = 12; mutant cells 17.4 ± 1.2 pA/pF, n = 24; p = 0.031, two-sample t test). Most importantly in the context of this study, the chromanol-sensitive current of BACE1-deficient myocytes was reduced roughly by half (Fig. 8E) when compared to their wild type counterparts, emphasizing the importance of BACE1 for regular murine atrial IKs. Although chromanol might not have fully suppressed IKs, it seems plausible from the reduction of total vs. chromanol-sensitive currents that further currents are altered

in BACE1−/− mice. Two possible explanations come to mind, which are not mutually exclusive. Firstly, this phenomenon might reflect a rebalancing of cardiac conductances in response to the reduction of IKs. Secondly, BACE1 may also interact with other cardiac ion channels to modulate their gating properties. In any case, our observation that the genetic disruption of an accessory subunit of KCNQ1 has also an impact on other currents in atrial myocytes is not without precedent, since deletion of KCNE1 was reported to similarly affect both, chromanol-sensitive current and total current [35]. 4. Discussion When co-expressed with KCNQ1 alone or together with its accessory subunit KCNE1, BACE1 profoundly modified key biophysical features of the respective K+ current, so that their response to depolarizing and repolarizing voltage commands was substantially changed. We will first discuss the effects of BACE1 on KCNQ1 alone, which appear rather straight forward, and then move on to the more complicated interactions of BACE1 with KCNQ1/E1 channel complexes. The most striking effect of BACE1 on homomeric KCNQ1 was the appearance of a prominent inactivating peak current after the onset of a larger depolarization. This effect did not require the proteolytic activity of BACE1, given that it was, firstly, reproduced by its enzymatically inactive variant BACE1 D289N and because it was, secondly, not affected by pre-incubation with C3, a compound that blocks its proteolytic activity [29]. Also, the effect was highly specific in that it was absent when KCNQ1 was co-expressed with BACE2 in lieu of BACE1. The most parsimonious explanation for the electrophysiological changes introduced by BACE1 that is also supported by our co-immunoprecipitation experiments is to assume that BACE1 interacts with KCNQ1 in a β-subunitlike fashion and that this interaction alters the gating kinetics of KCNQ1. Such interaction is not without precedent, since we have previously found a similar effect of BACE1 on the gating of voltage-dependent Na+ channels [22] and neuronal KCNQ channels [26]. Fluorescence recovery after photobleaching (FRAP) experiments, which assessed surface mobility of BACE1 or KCNE1, further strengthened the hypothesis of BACE1 acting like a β-subunit. When KCNQ1 was co-expressed, diffusion of both KCNE1 and BACE1 was decelerated. Roura-Ferrer et al. [33] had previously described this effect for KCNE1, lending support to the notion that BACE1 acts like an accessory subunit. Earlier studies on the activation and inactivation of macroscopic KCNQ1 currents proposed a sequential gating scheme, where KCNQ1 channels have two kinetically distinct open states and where inactivation is a fast and intrinsically voltage-independent process [36]. Employing such a scheme, we used here a linear Markov model to simulate how the BACE1-induced changes in macroscopic KCNQ1 current might be explained in terms of altered gating transitions (Fig. 9A). Rate constants

Please cite this article as: M. Agsten, et al., BACE1 modulates gating of KCNQ1 (Kv7.1) and cardiac delayed rectifier KCNQ1/KCNE1 (IKs), J Mol Cell Cardiol (2015), http://dx.doi.org/10.1016/j.yjmcc.2015.10.006

M. Agsten et al. / Journal of Molecular and Cellular Cardiology xxx (2015) xxx–xxx

associated with the non-conducting state I for inactivation were adjusted to simulate inactivation processes that set in with different kinetics. As required by our experimental data without intracellular ATP (Fig. 1D), all simulations with an inactivated state I should reach the same steadystate open probability of ensemble averages. We therefore kept the ratio of the rate constants λ and μ, which govern the transitions between O2 and I, at a constant value. When inactivation kinetics were fast, the simulated ensemble current mimicked the behavior of macroscopic KCNQ1 current, where inactivation is so rapid that it escapes macroscopic inspection (blue trace in Fig. 9B). As we gradually slowed inactivation, a transient peak of increasing amplitude appeared within the first few 100 ms of simulation (red and black traces in Fig. 9B). This behavior is strongly reminiscent of the effect of BACE1 on the kinetic phenotype of macroscopic KCNQ1 current. Unlike KCNE1, which abrogates inactivation (simplified as purple trace in Fig. 9B) [6], BACE1 appears to interfere with the inactivation process of KCNQ1 channels in a more moderate fashion, slowing the rate constants for inactivation without affecting the equilibrium between inactivated and open states during large sustained depolarization. In functional terms, the up-regulation of KCNQ1 currents by BACE1 might prove physiologically important in all tissues that co-express KCNQ1 and the rather ubiquitous BACE1 [37,38]. For example, BACE1 has a broad spectrum of substrates in pancreatic β cells [39] and has been specifically implicated in the regulation of plasma insulin levels [18]. KCNQ1 is also present in pancreatic β cells and, importantly, has been linked to insulin secretion [40,41] and type-2 diabetes [42,43]. Thus, it seems conceivable that the modulation of KCNQ1 activity by BACE1 that we report here, might, among other effects, also play a role in the regulation of insulin secretion. Obviously, the different actions of BACE1 are difficult to explain solely by means of the above gating scheme (Fig. 9). One way to approach this issue is to suppose that the macroscopic K+ current from HEK293T cells co-transfected with KCNQ1, KCNE1, and BACE1 is not produced by a uniform population of channel complexes. If we elaborate on the above idea that BACE1 can physically interact with KCNQ1, we have to ask what will happen if KCNQ1 has now two binding partners, namely KCNE1 and BACE1, with one of them at least partially shedding the other. Do the subunits compete for binding to KCNQ1? Does binding of one partner facilitate or impair binding of the second partner? What fraction of KCNE1 is cleaved by BACE1? With all these questions unresolved, we cannot rule out that the whole-cell current is generated by two or more subpopulations of channel complexes. In such a scenario, KCNE1, BACE1, and perhaps C- and N-terminal fragments of KCNE1 might assemble with KCNQ1 to form heterodimeric and/or -trimeric complexes of unknown stoichiometries. Given that the exact stoichiometry of KCNQ1/E1 is still a matter of dispute [9,10], it will be a rather daunting task to disentangle the multitude of interaction options when BACE1 comes into play. To determine the physiological significance of our findings from the heterologous expression system for the electrophysiology of cardiac cells, we embarked on current recordings from mouse atrial myocytes which are endowed with functionally relevant IKs, as reported previously by Temple et al. [35]. We found indeed that the genetic disruption of Bace1 reduced the normalized current amplitude of the chromanolsensitive current by about 50% when compared to that of wild type myocytes. This finding clearly establishes BACE1 as a significant player in cardiac electrophysiology. Owing to the high beating frequency of mouse hearts, the voltagedependent currents shaping their AP-waveform are differentially tuned when compared to those of mammals with slower beating frequencies. As a consequence, individual currents might exhibit properties that deviate from predictions based on the electrophysiological behavior of human cardiac channels when expressed in a heterologous system. With respect to human IKs, which we have reconstituted in HEK293T cells, its murine equivalent seems peculiar in that it appears to lack the characteristic slow activation upon depolarization, as

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reported here and in the previous study mentioned above, and in that it is substantially enhanced when KCNE1 is missing [35]. This raises the possibility that a considerable fraction of IKs in murine atrial myocytes is produced by KCNQ1 complexes lacking KCNE1. This notion receives support from our data showing that heterologously expressed KCNQ1/E1 currents are reduced by co-expressed BACE1 under physiological condition, i.e. with ATP in the pipette solution (Fig. 4F). Thus, if carried predominantly by KCNQ1/E1 complexes, IKs of BACE1-null mice should be increased in comparison to wild type cells. The fact that we observed the exact opposite prompted us to re-examine the effect of BACE1 on KCNQ1 alone, now with ATP in the pipette solution, as it was present in the myocyte recordings. Under this condition, BACE1 produced indeed a strong increase in KCNQ1 current (Suppl. Fig. 1), thereby offering a plausible explanation for the reduced IKs in BACE1deficient myocytes. While our recordings from wild type and BACE1null mice establish BACE1 as a significant constituent of IKs, the mouse equivalent of this current exhibits some particular features, which hamper direct predictions of how BACE1 will influence cardiac electrophysiology in humans. To extrapolate the effects of BACE1 on cardiac IKs in human heart, we used a typical cardiac action potential waveform as voltage command at near physiological temperature and added ATP to the pipette solution to ensure proper channel gating (Fig. 6). From our experiments with reconstructed IKs-like currents, we predict that high levels of BACE1 should impair the characteristic function of IKs in normal cardiomyocytes, namely to provide sufficient outward current to secure action potential repolarization in due time. These data suggest that BACE1 levels or the likelihood of its interaction with KCNQ1/E1 channels should be low under physiological conditions. However, when intracellular ATP levels are down in metabolically stressed cardiac cells, a dramatic decline in IKs ensues. Our recapitulation of BACE1 effects in ATP-deprived cells suggests that its interaction with KCNQ1/E1 channels should now strengthen IKs by augmenting its amplitude and prolonging its duration, thereby shortening a particularly vulnerable time window and counteracting afterdepolarizing currents. Our data show that BACE1 is present in heart tissue and, more specifically, in human iPSC-derived cardiomyocytes, but we do not know yet whether BACE1 activity and expression is up-regulated by cellular stress resulting from e.g. hypoxia and ischemia. Such an up-regulation has already been demonstrated in the brain [44–46] and made responsible for a vicious cycle, in which co-morbidity factors drive BACE1, thereby enhancing the formation of toxic amyloid β-peptides and thus accelerating the progression of cognitive impairment and dementia. It will be interesting to determine, whether BACE1 activity in heart tissue is also susceptible to pathophysiological conditions. Whereas the enhanced activity of BACE1 seems to be detrimental to the diseased brain, our data hint at the possibility that the functional consequences might be different in damaged cardiac tissue, where up-regulation of BACE1 would be expected to exert an antiarrhythmogenic effect by augmenting IKs. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.yjmcc.2015.10.006. Disclosures None. Author contributions M.A. and S.He. performed electrophysiological recordings of HEK293T cells and analyzed data. S.L. performed and analyzed FRAP experiments with contributions from T.W.G., T.V. performed electrophysiological recordings of isolated cardiomyocytes and analyzed data, A.R. performed co-immunoprecipitation. S.Ha. performed Western blot of iPSC-derived cardiomyocytes, C.R. performed immunoblots of mouse tissue. T.H. and C.A. initiated the study and designed research, to

Please cite this article as: M. Agsten, et al., BACE1 modulates gating of KCNQ1 (Kv7.1) and cardiac delayed rectifier KCNQ1/KCNE1 (IKs), J Mol Cell Cardiol (2015), http://dx.doi.org/10.1016/j.yjmcc.2015.10.006

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M. Agsten et al. / Journal of Molecular and Cellular Cardiology xxx (2015) xxx–xxx

which D.Y.K. and M.S. contributed. C.A. and T.H. wrote the paper with contributions from all co-authors. All authors approved the final version of the manuscript. The present work was performed in (partial) fulfillment of the requirements for obtaining the degree Dr. med. for M.A. and Dr. rer. nat. for S.He., S.L., and S.Ha. Funding This work was supported by the Johannes und Frieda MarohnStiftung (to T.H. and C.A.), the Deutsche Forschungsgemeinschaft (INST 90/675-1 FUGG to C.A.), the Staedtler-Stiftung (to C.A.), the ELAN-program of the Universitätsklinikum Erlangen (to T.H. and T.W.G.), National Institute of Health (1RF1AG048080-01 to D.Y.K.), and Studienstiftung des deutschen Volkes (to S.L. and S.Ha.). Acknowledgments iPSC-derived neurons were kindly provided by Iryna Prots (group of Beate Winner, IZKF, Universitätsklinikum Erlangen, Germany). We are grateful to Iwona Izydorczyk, Annette Kuhn, and Anette WirthHücking for technical assistance. We thank Paul Saftig for helpful comments on the manuscript. References [1] C. Chouabe, N. Neyroud, P. Guicheney, M. Lazdunski, G. Romey, J. Barhanin, Properties of KvLQT1 K+ channel mutations in Romano-Ward and Jervell and Lange-Nielsen inherited cardiac arrhythmias, EMBO J. 16 (1997) 5472–5479. [2] R. Rapetti-Mauss, F. O'Mahony, F.V. Sepulveda, V. Urbach, B.J. Harvey, Oestrogen promotes KCNQ1 potassium channel endocytosis and postendocytic trafficking in colonic epithelium, J. Physiol. 591 (2013) 2813–2831. [3] J. Barhanin, F. Lesage, E. Guillemare, M. Fink, M. Lazdunski, G. Romey, K(V)LQT1 and lsK (minK) proteins associate to form the I(Ks) cardiac potassium current, Nature 384 (1996) 78–80. [4] F. Charpentier, J. Merot, G. Loussouarn, I. Baro, Delayed rectifier K(+) currents and cardiac repolarization, J. Mol. Cell. Cardiol. 48 (2010) 37–44. [5] M.V. Soldovieri, F. Miceli, M. Taglialatela, Driving with no brakes: molecular pathophysiology of Kv7 potassium channels, Physiology (Bethesda) 26 (2011) 365–376. [6] M. Tristani-Firouzi, M.C. Sanguinetti, Voltage-dependent inactivation of the human K + channel KvLQT1 is eliminated by association with minimal K + channel (minK) subunits, J. Physiol. 510 (Pt 1) (1998) 37–45. [7] E. Wrobel, D. Tapken, G. Seebohm, The KCNE tango — how KCNE1 interacts with Kv7.1, Front. Pharmacol. 3 (2012) 142. [8] M. Tristani-Firouzi, M.C. Sanguinetti, Structural determinants and biophysical properties of HERG and KCNQ1 channel gating, J. Mol. Cell. Cardiol. 35 (2003) 27–35. [9] M. Wang, R.S. Kass, Stoichiometry of the slow I(ks) potassium channel in human embryonic stem cell-derived myocytes, Pediatr. Cardiol. 33 (2012) 938–942. [10] L.D. Plant, D. Xiong, H. Dai, S.A. Goldstein, Individual IKs channels at the surface of mammalian cells contain two KCNE1 accessory subunits, Proc. Natl. Acad. Sci. U. S. A. 111 (2014) E1438–E1446. [11] C.C. Sachse, Y.H. Kim, M. Agsten, T. Huth, C. Alzheimer, D.M. Kovacs, et al., BACE1 and presenilin/gamma-secretase regulate proteolytic processing of KCNE1 and 2, auxiliary subunits of voltage-gated potassium channels, FASEB J. 27 (2013) 2458–2467. [12] P.H. Kuhn, K. Koroniak, S. Hogl, A. Colombo, U. Zeitschel, M. Willem, et al., Secretome protein enrichment identifies physiological BACE1 protease substrates in neurons, EMBO J. 31 (2012) 3157–3168. [13] X. Hu, X. Zhou, W. He, J. Yang, W. Xiong, P. Wong, et al., BACE1 deficiency causes altered neuronal activity and neurodegeneration, J. Neurosci. 30 (2010) 8819–8829. [14] C. Cheret, M. Willem, F.R. Fricker, H. Wende, A. Wulf-Goldenberg, S. Tahirovic, et al., Bace1 and neuregulin-1 cooperate to control formation and maintenance of muscle spindles, EMBO J. 32 (2013) 2015–2028. [15] B.D. Hitt, T.C. Jaramillo, D.M. Chetkovich, R. Vassar, BACE1−/− mice exhibit seizure activity that does not correlate with sodium channel level or axonal localization, Mol. Neurodegener. 5 (2010) 31. [16] T.W. Rajapaksha, W.A. Eimer, T.C. Bozza, R. Vassar, The Alzheimer's beta-secretase enzyme BACE1 is required for accurate axon guidance of olfactory sensory neurons and normal glomerulus formation in the olfactory bulb, Mol. Neurodegener. 6 (2011) 88. [17] M. Willem, A.N. Garratt, B. Novak, M. Citron, S. Kaufmann, A. Rittger, et al., Control of peripheral nerve myelination by the beta-secretase BACE1, Science 314 (2006) 664–666. [18] A. Hoffmeister, J. Tuennemann, I. Sommerer, J. Mossner, A. Rittger, D. Schleinitz, et al., Genetic and biochemical evidence for a functional role of BACE1 in the regulation of insulin mRNA expression, Obesity (Silver Spring) 21 (2013) E626–E633.

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Please cite this article as: M. Agsten, et al., BACE1 modulates gating of KCNQ1 (Kv7.1) and cardiac delayed rectifier KCNQ1/KCNE1 (IKs), J Mol Cell Cardiol (2015), http://dx.doi.org/10.1016/j.yjmcc.2015.10.006