Modulation of KV4.3-KChIP2 Channels by IQM-266: Role of DPP6 and KCNE2

Citation: de Benito-Bueno, A.; Socuellamos, P.G.; Merinero, Y.G.; Cercos, P.; Izquierdo, C.; Daniel-Mozo, M.; Marín-Olivero, I.; Perez-Lara, A.; Gonzalez-Vera, J.A.; Orte, A.; et al. Modulation of KV4.3-KChIP2 Channels by IQM-266: Role of DPP6 and KCNE2. Int. J. Mol. Sci. 2022,23, 9170. https://doi.org/ 10.3390/ijms23169170 Academic Editors: Antonio Ferrer-Montiel and Antonio Felipe Received: 26 July 2022 Accepted: 13 August 2022 Published: 15 August 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). International Journal of Molecular Sciences Article Modulation of KV4.3-KChIP2 Channels by IQM-266: Role of DPP6 and KCNE2 Angela de Benito-Bueno 1,† , Paula G. Socuellamos 1,† , Yaiza G. Merinero 1, Pilar Cercos 2, Carolina Izquierdo 2, Miguel Daniel-Mozo 3, Irene Marín-Olivero 4, Angel Perez-Lara 4,5 , Juan A. Gonzalez-Vera 4, Angel Orte 4, Armando Albert 3, Mercedes Martin-Martinez 2, Marta Gutierrez-Rodriguez 2,* and Carmen Valenzuela 1,6,* 1Instituto de Investigaciones Biomédicas “Alberto Sols” (CSIC-UAM), 28029 Madrid, Spain 2Instituto de Química Médica (IQM-CSIC), 28029 Madrid, Spain 3Instituto de Química Física Rocasolano, Consejo Superior de Investigaciones Científicas (IQFR-CSIC), 28006 Madrid, Spain 4 Nanoscopy-UGR Laboratory, Departamento de Fisicoquímica, Unidad de Excelencia de Química Aplicada a Biomedicina y Medioambiente, Facultad de Farmacia, Campus Cartuja, Universidad de Granada, 18071 Granada, Spain 5 Department of Neurobiology, Max Planck Institute for Multidisciplinary Sciences, 37077 Göttingen, Germany 6Spanish Network for Biomedical Research in Cardiovascular Research (CIBERCV), Instituto de Salud Carlos III, 28029 Madrid, Spain *Correspondence: [email protected] (M.G.-R.); [email protected] (C.V.); Tel.: +34-91-258-7493 (M.G.-R.); +34-91-585-4493 (C.V.) † These authors contributed equally to this work. Abstract: The transient outward potassium current (I tof ) is generated by the activation of K V 4 channels assembled with KChIP2 and other accessory subunits (DPP6 and KCNE2). To test the hypothesis that these subunits modify the channel pharmacology, we analyzed the electrophysiological effects of (3-(2-(3-phenoxyphenyl)acetamido)-2-naphthoic acid) (IQM-266), a new KChIP2 ligand, on the currents generated by K V 4.3/KChIP2, K V 4.3/KChIP2/DPP6 and K V 4.3/KChIP2/KCNE2 channels. CHO cells were transiently transfected with cDNAs codifying for different proteins (K V 4.3/KChIP2, K V 4.3/KChIP2/DPP6 or K V 4.3/KChIP2/KCNE2), and the potassium currents were recorded using the whole-cell patch-clamp technique. IQM-266 decreased the maximum peak of K V 4.3/KChIP2, K V 4.3/KChIP2/DPP6 and K V 4.3/KChIP2/KCNE2 currents, slowing their time course of inactivation in a concentration-, voltage-, time- and use-dependent manner. IQM-266 produced an increase in the charge in K V 4.3/KChIP2 channels that was intensified when DPP6 was present and abolished in the presence of KCNE2. IQM-266 induced an activation unblocking effect during the application of trains of pulses to cells expressing K V 4.3/KChIP2 and K V 4.3/KChIP2/KCNE2, but not in K V 4.3/KChIP2/DPP6 channels. Overall, all these results are consistent with a preferential IQM-266 binding to an active closed state of Kv4.3/KChIP2 and Kv4.3/KChIP2/KCNE2 channels, whereas in the presence of DPP6, IQM-266 binds preferentially to an inactivated state. In conclusion, DPP6 and KCNE2 modify the pharmacological response of KV4.3/KChIP2 channels to IQM-266. Keywords: KV4 channels; KChIP2; DPP6; KCNE; KChIP2 ligand 1. Introduction Voltage-dependent potassium channels (K V 4 subfamily) are the main contributors to the cardiac transient outward K + currents (I tof ), having a central role in controlling cardiac excitation and shaping the cardiac action potentials (AP) [ 1 – 4 ]. There are two I to components with distinct recovery kinetics: the fast (I tof ), conducted by K V 4.2 and K V 4.3 channels, and the slow (I tos ), conducted by K V 1.4 channels [ 5 ]. I tof plays a key role in the early phase of repolarization in many species, including humans [ 1 ]. In many pathological conditions such as cardiac hypertrophy, atrial fibrillation or heart failure, a reduction of Int. J. Mol. Sci. 2022,23, 9170. https://doi.org/10.3390/ijms23169170 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2022,23, 9170 2 of 18 the Itof density and the subsequent prolongation of the action potential duration has been found [ 6 – 8 ]. Therefore, the pharmacological I tof activation may have therapeutic value in those diseases. The most comprehensively studied members of the K V 4 family are K V 4.2 and the two splice variants of K V 4.3, K V 4.3S and K V 4.3L [ 9 , 10 ]. They present a complementary expression, with K V 4.2 more abundant in the ventricle and K V 4.3 in the atria [ 11 , 12 ]. In human heart failure, KV4.3L expression rises ∼33% while that of KV4.3S falls ∼75% [13]. The α subunits of K V 4.x channels are enough for the formation of functional K + channels. However, to fully reproduce the I tof current, K V 4 channels need to assemble with other accessory subunits forming channelosomes. These protein complexes can be formed by different regulatory (or β ) subunits, including KChIPs (potassium channel interacting proteins), DPPs (dipeptidyl-peptidase-like proteins), KCNEs (also termed MinK-related peptides, or MiRPs), KChAPs and KVβx subunits [14–19]. KChIPs belong to the neuronal calcium sensor superfamily [ 20 ]. Within the different KChIP members, KChIP2 is predominantly expressed in the heart [ 21 ]. Through its interaction with the amino terminus of the K V 4.3 α -subunit, KChIP2 induces an increase in the traffic of K V 4.3 channels to the plasma membrane, a delay in the macroscopic inactivation kinetics, and an acceleration of both the activation and the recovery kinetics from inactivation [ 14 , 20 ]. Interestingly, this general trend is modified by the KChIP-binding ligands. Hence, the knowledge gained from the modulation of the K V 4.3/KChIP complex by small molecules could open novel therapeutic opportunities for the treatment of cardiovascular diseases in which the Itof is reduced [19,22]. DPP6 belongs to the prolyl-oligopeptidase family of serine proteases. It is a single-pass transmembrane protein that can modify channel gating through different mechanisms, which involve direct interactions with α -subunit transmembrane core domains, including the pore and the voltage-sensing domain [ 17 , 23 , 24 ]. Recent studies have shown that DPP6 exerts its modulation through interactions with the S1 and S2 helices of the K V 4.2 voltage-sensing domain [ 25 ]. DPP6 coexpression with K V 4.3 accelerates the activation and inactivation kinetics and shifts both activation and inactivation voltage dependencies of KV4 channels to more negative potentials [17]. KCNE2, also termed MinK-related peptide 1, or MiRP1, is a single-transmembrane- domain subunit that co-assembles with the K Vα subunits, modifying, among other fundamental properties, the channel α subunit composition, trafficking, endocytosis, gating and the effects of regulation by other proteins [ 16 ]. MiRP1 slows the rates of K V 4 activation and inactivation [ 26 ] and, during channel recovery from inactivation, it induces an ‘overshoot’ of the current amplitude [ 16 , 26 ], a fact previously detected on the I tof from human subepicardial myocytes [ 27 ]. Moreover, MiRP1 is thought to interact with the pore domains of the channel and delay both channel activation and inactivation processes [26]. For all the reasons mentioned, the K V 4-KChIP2-DPP6-KCNE2 channelosome could be a promising target in the treatment of cardiac diseases. Moreover, given the fact that IQM-266 has been recently identified as a novel KChIP3 ligand that modulates K V 4 currents in rat dorsal root ganglion neurons [ 28 ], we investigated its binding to KChIP2 and the pharmacological consequences of IQM-266 on the K + currents generated by Kv4.3/KChIP2, Kv4.3/KChIP2/DPP6 and Kv4.3/KChIP2/KCNE2. 2. Results 2.1. IQM-266 Binding to KChIP2 We first tested the interaction between IQM-266 (3-(2-(3-phenoxyphenyl)acetamido)- 2-naphthoic acid) and KChIP2. IQM-266 binding was monitored by quenching of the intrinsic tryptophan fluorescence emission of KChIP2 (Figure 1a) caused by fluorescence resonance energy transfer (FRET) towards the IQM-266 chromophore. The fact that there are tryptophan residues in close proximity to the binding site and that the spectral overlap between the two chromophores is suitable allows an efficient FRET process. Using the spectral features of the emission of KChIP2 and the absorption of IQM-266, the estimated Int. J. Mol. Sci. 2022,23, 9170 3 of 18 quantum yield of the tryptophan for the parent KChIP3, and the molar absorptivity of IQM-266, we estimated a Förster distance, R 0 , of 20.4 Å (assuming free rotation of the dyes, which is likely not a totally valid assumption but provides a rough estimation of the distance) [ 29 ]. FRET is evident by the quenching in the tryptophan fluorescence and the concomitant increase in the emission of IQM-266 (Figure 1a), which indicates close proximity between the chromophore of IQM-266 and the KChIP2 tryptophan. We estimated the amount of quenching to the tryptophan emission being at saturating concentrations of IQM-266 around 0.92. This quenching efficiency corresponded to an apparent distance tryptophan–IQM-266 of circa. 13.5 Å. Next, to determine IQM-266 affinity, the quenching of tryptophan emission at 330 nm was plotted versus the concentration of IQM-266 (Figure 1a) and the dissociation constant was calculated using a Hill equation (see Methodology). Our experiment showed that IQM-266 binds to KChIP2 with a dissociation constant of 1.9 ±0.1 µM and a Hill coefficient of around 1 (Figure 1a). Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 3 of 19 resonance energy transfer (FRET) towards the IQM-266 chromophore. The fact that there are tryptophan residues in close proximity to the binding site and that the spectral overlap between the two chromophores is suitable allows an efficient FRET process. Using the spectral features of the emission of KChIP2 and the absorption of IQM-266, the estimated quantum yield of the tryptophan for the parent KChIP3, and the molar absorptivity of IQM-266, we estimated a Förster distance, R 0 , of 20.4 Å (assuming free rotation of the dyes, which is likely not a totally valid assumption but provides a rough estimation of the distance) [29]. FRET is evident by the quenching in the tryptophan fluorescence and the concomitant increase in the emission of IQM-266 (Figure 1a), which indicates close proximity between the chromophore of IQM-266 and the KChIP2 tryptophan. We estimated the amount of quenching to the tryptophan emission being at saturating concentrations of IQM-266 around 0.92. This quenching efficiency corresponded to an apparent distance tryptophan–IQM-266 of circa. 13.5 Å. Next, to determine IQM-266 affinity, the quenching of tryptophan emission at 330 nm was plotted versus the concentration of IQM-266 (Figure 1a) and the dissociation constant was calculated using a Hill equation (see Methodology). Our experiment showed that IQM-266 binds to KChIP2 with a dissociation constant of 1.9 ± 0.1 µM and a Hill coefficient of around 1 (Figure 1a). Figure 1. Concentration-dependent interaction between IQM-266 and KChIP2. (a) Interaction between IQM-266 and KChIP2. Left panel shows the plot of the quenching of tryptophan by FRET towards IQM-266. Data from the five repetitions (open symbols) are shown as well as the average value (red-filled symbols) and error bars as s.d. The red line represents the fit to a Hill equation. Right panel shows the fluorescence emission spectra (λ ex = 260 nm) from a representative titration of KChIP2 (5 µM) with increasing concentrations of IQM-266. (b) Representation of IQM266 binding pose with the homology model of KChIP2. Residues within the binding pocket, together with Trp, are indicated. For clarity, only polar hydrogens are included and the hydrogen bond is depicted as a discontinuous yellow line (PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC. New York, NY, USA). Figure 1. Concentration-dependent interaction between IQM-266 and KChIP2. ( a ) Interaction between IQM-266 and KChIP2. Left panel shows the plot of the quenching of tryptophan by FRET towards IQM-266. Data from the five repetitions (open symbols) are shown as well as the average value (red-filled symbols) and error bars as s.d. The red line represents the fit to a Hill equation. Right panel shows the fluorescence emission spectra ( λex = 260 nm) from a representative titration of KChIP2 (5 µ M) with increasing concentrations of IQM-266. ( b ) Representation of IQM266 binding pose with the homology model of KChIP2. Residues within the binding pocket, together with Trp, are indicated. For clarity, only polar hydrogens are included and the hydrogen bond is depicted as a discontinuous yellow line (PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC. New York, NY, USA). To gain further insights into the binding pocket of IQM-266, molecular docking studies were carried out. As there is no known 3D structure of KChIP2, homology models were built on the base of the NMR structure of KChIP3 (PDB ID 2JUL, 15 structures) [ 30 ], sequence identity of 77% with hKChIP2 C-terminal region. Five homology models were generated Int. J. Mol. Sci. 2022,23, 9170 4 of 18 using the Schrödinger’s Prime module. Additionally, a model was built on the base of the X-ray structure of KChIP1 (72% identity) in complex with the Kv4.3 assembly domain T1 (PDB ID 2I2R) [ 31 ]. In this structure the N-terminal helix of the channel is accommodated in a KChIP1 cleft and, thus, there is a reordering of the protein, as particularly KChIP1 H10 moves from its position in 2JUL. These models provided several protein conformations for the docking studies. Then, IQM-266 was docked in a site centered on Tyr188, Ile208 and Phe232. IFD studies identified several locations and poses of the ligand within KChIP2; as expected, a wider variety was observed for the model generated based on 2I2R due to the large pocket size. Based on the score and visual inspection, a pose was selected (Figure 1b). The IFD studies indicated that IQM-266 is located in a cleft surrounded by KChIP2 EF-hands 3 and 4 and helix H10. In this pose, the IQM-266 carboxylic acid is participating in a hydrogen bond with Ser262 from KChIP2 H10. The naphthyl ring is surrounded by hydrophobic residues, namely Met201, Ile204, Met263, Tyr188, Phe266, and a T-interaction is observed with Phe232. Similarly, the Ph-O-Ph moiety is accommodated in a hydrophobic cavity flanked by Phe136, Ile208, Met211, Met212 and Val269. IQM-266 is at 12 Å from Trp183, in agreement with the apparent distance tryptophan–IQM-266 obtained from the experimental data. 2.2. Effects of IQM-266 on KV4.3/KChIP2, KV4.3/KChIP2/DPP6 and KV4.3/KChIP2/KCNE2 Channels Human cardiac I tof is generated by the activation of K V 4.3 channels assembled with regulatory subunits, from which the most important ones are KChIP2, DPP6 and KCNE2 [16,17,24,32] . Considering that IQM-266 is a KChIP2 ligand, we analyzed the effects of IQM-266 in CHO cells transfected with K V 4.3 channels and KChIP2 together with DPP6 or KCNE2. First, we analyzed the effects of different concentrations of IQM-266 (between 0.001 and 500 µ M) on K V 4.3/KChIP2, K V 4.3/KChIP2/DPP6 and K V 4.3/KChIP2/KCNE2 channels expressed in CHO cells (Figure 2). The effects were measured at the maximum peak current and on the charge (measured as the area underneath the current). In K V 4.3/KChIP2 channels, a concentrationindependent block of the peak current between 0.001 and 1 µ M was observed, showing slighter effects on the charge (Figure 2a). Previously, we have demonstrated that IQM-266 (3 µ M) produced an activation of K V 4.3/KChIP3 [ 28 ] in the charge during the application of 250 ms depolarizing pulses from − 80 to +60 mV. This was also observed in K V 4.3/KChIP2, but to a greater extent. This effect is the result of the equilibrium between two effects: (i) block produced on the maximum peak current and (ii) a slowing effect on the inactivation kinetics. At higher concentrations, this compound produced a concentration-dependent decrease both at the maximum peak current (Supplementary Figure S1 with an EC 50 of 13 µM) and at the charge. In the presence of DPP6 (K V 4.3/KChIP2/DPP6) (Figure 2b), the IQM-266-induced block of the maximum peak current was almost negligible at concentrations lower than 100 µ M. However, under these conditions, the activating effect produced by IQM-266 in K V 4.3/KChIP2/DPP6 channels was significantly greater and appeared in a wider range of concentrations (1–10 µ M) than in K V 4.3/KChIP2 channels. These effects may be the consequence of a slight block of the maximum peak current together with a slower inactivation kinetics. Indeed, IQM-266 blocked the maximum peak current generated by K V 4.3/KChIP2/DPP6 channels exhibiting an EC 50 of 80 µ M (Supplementary Figure S1). However, lower concentrations were needed to increase the time constant of inactivation. Finally, in the presence of KCNE2 (K V 4.3/KChIP2/KCNE2), the activator effect produced by IQM-266 on K V 4.3/KChIP2 channels was abolished (Figure 2c). However, and similarly to that observed in K V 4.3/KChIP2 channels, IQM-266 slowed down the inactivation of the current. However, in contrast to that observed in K V 4.3/KChIP2 channels, the block produced by this compound, measured at the maximum peak current, was greater Int. J. Mol. Sci. 2022,23, 9170 5 of 18 (Supplementary Figure S1; with an EC 50 of 5 µ M) and the slowing of the inactivation of the current was lesser. Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 5 of 19 Figure 2. Concentration-dependent interaction between IQM-266 KV4.3/KChIP2, KV4.3/KChIP2/DPP6 and KV4.3/KChIP2/KCNE2. (a–c) Inhibition or increase of the current induced by IQM-266 in KV4.3/KChIP2 (a), KV4.3/KChIP2/DPP6 (b) and KV4.3/KChIP2/KCNE2 (c) channels measured at the maximum peak current and in the charge (measured as the area of the current during the application of a 250 ms pulse to +60 mV). Right panels show current records obtained in the absence and in the presence of IQM-266 (3 µM). Each bar represents the mean ± S.E.M. of the number of cells shown in the bars. Statics was performed by a paired Student t-test comparing the block produced at the peak current and the block/increase produced at the charge for each concentration of IQM-266 tested. *: p < 0.05. Finally, in the presence of KCNE2 (KV4.3/KChIP2/KCNE2), the activator effect produced by IQM-266 on KV4.3/KChIP2 channels was abolished (Figure 2c). However, and similarly to that observed in KV4.3/KChIP2 channels, IQM-266 slowed down the inactivation of the current. However, in contrast to that observed in KV4.3/KChIP2 channels, the block produced by this compound, measured at the maximum peak current, was greater Figure 2. Concentration-dependent interaction between IQM-266 K V 4.3/KChIP2, K V 4.3/KChIP2/DPP6 and K V 4.3/KChIP2/KCNE2. ( a – c ) Inhibition or increase of the current induced by IQM-266 in K V 4.3/KChIP2 ( a ), K V 4.3/KChIP2/DPP6 ( b ) and KV4.3/KChIP2/KCNE2 (c) channels measured at the maximum peak current and in the charge (measured as the area of the current during the application of a 250 ms pulse to +60 mV). Right panels show current records obtained in the absence and in the presence of IQM-266 (3 µ M). Each bar represents the mean ±S.E.M. of the number of cells shown in the bars. Statics was performed by a paired Student t-test comparing the block produced at the peak current and the block/increase produced at the charge for each concentration of IQM-266 tested. *: p< 0.05. Int. J. Mol. Sci. 2022,23, 9170 6 of 18 In order to characterize the increase of the current induced by IQM-266, we studied the effects of this compound at a concentration of 3 µ M on K V 4.3/KChIP2, K V 4.3/KChIP2/DPP6 and KV4.3/KChIP2/KCNE2 channels. 2.3. Time-Dependent Effects of IQM-266 on KV4.3/KChIP2, KV4.3/KChIP2/DPP6 and KV4.3/KChIP2/KCNE2 Channels The effects on the activation kinetics produced by IQM-266 were assessed by fitting the current traces of K V 4.3/KChIP2, K V 4.3/KChIP2/DPP6 and K V 4.3/KChIP2/KCNE2 to a monoexponential process, with the activation time constant ( τAct ), both in the absence and in the presence of IQM-266 (3 µM) (Figure 3). Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 6 of 19 (Supplementary Figure S1; with an EC50 of 5 µM) and the slowing of the inactivation of the current was lesser. In order to characterize the increase of the current induced by IQM-266, we studied the effects of this compound at a concentration of 3 µM on KV4.3/KChIP2, KV4.3/KChIP2/DPP6 and KV4.3/KChIP2/KCNE2 channels. 2.3. Time-Dependent Effects of IQM-266 on KV4.3/KChIP2, KV4.3/KChIP2/DPP6 and KV4.3/KChIP2/KCNE2 Channels The effects on the activation kinetics produced by IQM-266 were assessed by fitting the current traces of KV4.3/KChIP2, KV4.3/KChIP2/DPP6 and KV4.3/KChIP2/KCNE2 to a monoexponential process, with the activation time constant (τAct), both in the absence and in the presence of IQM-266 (3 µM) (Figure 3). Figure 3. Activation and inactivation kinetics of KV4.3/KChIP2 (a), KV4.3/KChIP2/DPP6 (b) and KV4.3/KChIP2/KCNE2 (c) currents in the absence and in the presence of IQM-266 (3 µM). Left panels show normalized current records under the three experimental conditions. These current records were fitted to a monoexponential equation in order to obtain the τAct values. Middle panels show the histogram representing the τAct under the different experimental conditions. Right panels show Figure 3. Activation and inactivation kinetics of K V 4.3/KChIP2 ( a ), K V 4.3/KChIP2/DPP6 ( b ) and K V 4.3/KChIP2/KCNE2 ( c ) currents in the absence and in the presence of IQM-266 (3 µ M). Left panels show normalized current records under the three experimental conditions. These current records were fitted to a monoexponential equation in order to obtain the τAct values. Middle panels show the histogram representing the τAct under the different experimental conditions. Right panels show the histograms representing the τInac values under the three experimental conditions. Each bar represents the mean ± S.E.M. of n= 8 cells (from n= 5 transfections) for K V 4.3/KChIP2 channels, n= 8 (from n= 6 transfections) for K V 4.3/KChIP2/DPP6 channels and n= 10 (from n= 9 transfections) for KV4.3/KChIP2/KCNE2 channels. *: p< 0.05. Int. J. Mol. Sci. 2022,23, 9170 7 of 18 Similarly, the inactivation kinetics ( τInac ) were analyzed by fitting the inactivation process. As it can be observed in Figure 3, the time constant of both the activation and inactivation processes were significantly increased in the presence of IQM-266 under the three experimental conditions. The slowing of the activation and inactivation kinetics produced by this compound was greater on K V 4.3/KChIP2 (Figure 3a), followed by K V 4.3/KChIP2/KCNE2 (Figure 3c) and finally by K V 4.3/KChIP2/DPP6 (Figure 3b) currents . 2.4. Voltage Dependence of the Block Produced by IQM-266 on KV4.3/KChIP2, KV4.3/KChIP2/DPP6 and KV4.3/KChIP2/KCNE2 Channels Figure 4a shows superimposed traces obtained after applying the pulse protocol represented in the top of the Figure, in the absence and in the presence of IQM-266 (3 µ M). Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 7 of 19 the histograms representing the τInac values under the three experimental conditions. Each bar represents the mean ± S.E.M. of n = 8 cells (from n = 5 transfections) for KV4.3/KChIP2 channels, n = 8 (from n = 6 transfections) for KV4.3/KChIP2/DPP6 channels and n = 10 (from n = 9 transfections) for KV4.3/KChIP2/KCNE2 channels. *: p < 0.05. Similarly, the inactivation kinetics (τInac) were analyzed by fitting the inactivation process. As it can be observed in Figure 3, the time constant of both the activation and inactivation processes were significantly increased in the presence of IQM-266 under the three experimental conditions. The slowing of the activation and inactivation kinetics produced by this compound was greater on KV4.3/KChIP2 (Figure 3a), followed by KV4.3/KChIP2/KCNE2 (Figure 3c) and finally by KV4.3/KChIP2/DPP6 (Figure 3b) currents. 2.4. Voltage Dependence of the Block Produced by IQM-266 on KV4.3/KChIP2, KV4.3/KChIP2/DPP6 and KV4.3/KChIP2/KCNE2 Channels Figure 4a shows superimposed traces obtained after applying the pulse protocol represented in the top of the Figure, in the absence and in the presence of IQM-266 (3 µM). Figure 4. Voltage dependence interaction between IQM-266 and K V 4.3/KChIP2 K V 4.3/KChIP2/DPP6 and K V 4.3/KChIP2/KCNE2 channels. ( a ) Original recordings obtained elicited by the activation of K V 4.3/KChIP2, K V 4.3/KChIP2/DPP6 and K V 4.3/KChIP2/KCNE2 channels in the absence and in the presence of IQM-266 (3 µ M) after applying the pulse protocols shown in the upper part of the figure. ( b ) I-V relationship of the currents generated by K V 4.3/KChIP2 (n= 7 cells from n= 7 transfections), K V 4.3/KChIP2/DPP6 (n= 8 cells from n= 6 transfections) and K V 4.3/KChIP2/KCNE2 (n= 10 cells from n= 8 transfections) channels in the absence and in the presence of IQM-266 at 3 µ M. ( c ) Q-V relationship of the charge through K V 4.3/KChIP2 (n= 6 cells from n= 5 transfections), KV4.3/KChIP2/DPP6 (n= 8 cells from n= 6 transfections) and K V 4.3/KChIP2/KCNE2 (n= 10 from n= 8 transfections) channels in the absence and in the presence of IQM-266 at 3 µ M, measured at the area under the current during the application of depolarizing pulse protocol shown in the upper part of the figure. Each point represents the mean ±S.E.M . *: p< 0.05. Int. J. Mol. Sci. 2022,23, 9170 8 of 18 As previously stated, IQM-266 decreased the maximum peak current and slowed down the inactivation kinetics under the three experimental conditions. By plotting the maximum peak current at each membrane potential tested for both control and after perfusion of the cells with IQM-266, the I-V relationships were obtained (Figure 4b). As it is shown, the maximum peak current block, produced by IQM-266 under the three experimental conditions, was greater in K V 4.3/KChIP2/KCNE2 channels, followed by K V 4.3/KChIP2 and K V 4.3/KChIP2/DPP6. Under the three experimental conditions, maximum peak block sharply increased in the range of voltage membrane activation. Figure 4c shows the charge–voltage (Q-V) relationship obtained after measuring the charge (measured as the area per unit time) in the absence and in the presence of IQM-266 (3 µ M). We observed that IQM-266 produced an increase in the charge in K V 4.3/KChIP2 channels that was intensified when DPP6 was present and abolished in the presence of KCNE2. From the I-V relationships, we obtained the activation curves. Their V h and the s parameters were not modified in K V 4.3/KChIP2 and K V 4.3/KChIP2/KCNE2 currents, whereas we observed a negative shift of the V h induced by IQM-266 (3 µ M) in the activation curve generated by KV4.3/KChIP2/DPP6 channels (Supplementary Figure S2). 2.5. Use-Dependent Effects of IQM-266 on KV4.3/KChIP2, KV4.3/KChIP2/DPP6 and KV4.3/KChIP2/KCNE2 Channels Although IQM-266 (3 µ M) produced an increase in the current of K V 4.3/KChIP2 and KV4.3/KChIP2/DPP6 channels, this effect may not be attained during a single action potential. Therefore, we tested whether IQM-266-induced effects displayed use dependence by applying two different pulse protocols, which may lead us to discriminate between the channel state for which IQM-266 has a greater affinity. In both protocols, 15 pulses from − 80 mV to +50 mV of 25 ms (short-pulse train) or 500 ms (long-pulse train) at frequencies of 1.4 and 1.8 Hz, respectively, were applied (Figure 5). During the short-pulse train, channels shift from the closed to the open state without inactivate, whereas during the long-pulse train, K V 4 channels passage from the closed to the open and, finally, to the inactivated state. After applying a short-pulse train under control conditions, the magnitude of the current K V 4.3/KChIP2 was not modified. However, in the presence of IQM-266 (3 µ M), the magnitude of the current elicited during the first pulse of the train exhibited the smallest amplitude and the magnitude of the current after applying the successive pulses exponentially increased until a saturated value was obtained. Similar results were obtained in K V 4.3/KChIP2/KCNE2 channels. This increase in the current during the short-pulse train application may be explained by an activation unblocking process, as it has been reported in sodium channels with other drugs [ 33 , 34 ]. Interestingly, this increase in the magnitude of the current during the application of a short-pulse train was not observed in KV4.3/KChIP2/DPP6 channels. After the application of the long-pulse train protocol to K V 4.3/KChIP2 channels in the absence of IQM-266, a decrease of a 26.0 ± 6.8% (n= 5) without changes in the inactivation kinetics of the current was observed, which is the consequence of the accumulation of inactivation. After perfusion with IQM-266, this decrease of the current during the long-pulse train was enhanced to 54.6 ± 16.4% (n= 5, p< 0.05). These results can be explained if IQM-266 also binds to the inactivated state of the channel. Moreover, during the application of the long-pulse train, the kinetics of the first pulse of the train exhibited similar monoexponential kinetics than under steady state conditions (similar to that observed in the I-V relationship), whereas the inactivation kinetics of the current became slightly faster after applying successive pulses. This small acceleration of the inactivation kinetics of the current during the application of the long-pulse train can represent the binding time constant of IQM-266 to the inactivated state of the KV4.3/KChIP2 channel. Similar results were observed when KCNE2 was present, in which the decrease of the current during the train was also greater in the presence than in the absence of IQM-266 (27.9 ± 4.9% vs. 67.8 ± 6.3% in the absence and in the presence of IQM-266, respectively, n= 6 ,p< 0.05). Furthermore, the kinetics of the current elicited after applying the first pulse Int. J. Mol. Sci. 2022,23, 9170 9 of 18 of the train were slightly slower than the currents generated after the successive currents of the train pulses. Likewise, in the presence of DPP6, a greater decrease in the magnitude of the current was observed when the long-pulse train protocol was applied ( 4.6 ±2.9% vs. 48.1 ± 10.7% in the absence and in the presence of IQM-266 respectively, n= 5 ;p< 0.05). However, under these conditions, the kinetics of the current were not changed during the application of the long-pulse train. The use-dependent effects observed during the application of the long-pulse train protocols could be explained if the recovery process in the presence of IQM-266 is slower. Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 9 of 19 inactivation. After perfusion with IQM-266, this decrease of the current during the longpulse train was enhanced to 54.6 ± 16.4% (n = 5, p < 0.05). These results can be explained if IQM-266 also binds to the inactivated state of the channel. Moreover, during the application of the long-pulse train, the kinetics of the first pulse of the train exhibited similar monoexponential kinetics than under steady state conditions (similar to that observed in the I-V relationship), whereas the inactivation kinetics of the current became slightly faster after applying successive pulses. This small acceleration of the inactivation kinetics of the current during the application of the long-pulse train can represent the binding time constant of IQM-266 to the inactivated state of the KV4.3/KChIP2 channel. Figure 5. Use-dependent effects produced by IQM-266 on K V 4.3/KChIP2, K V 4.3/KChIP2/DPP6 and K V 4.3/KChIP2/KCNE2 channels after applying the two pulse protocols shown in the top of the figure. In the three panels, the top one shows current records obtained after applying the pulse protocols shown in the upper part of the figure. The red current represents the first current record of each train. Below the current records, the normalized current versus the first pulse is shown. Each point represents the mean ± S.E.M. of n= 5 cells (from n= 5 transfections) for K V 4.3/KChIP2 channels, n= 6 cells (from n= 5 transfections) for and K V 4.3/KChIP2/KCNE2 channels and n= 6 cells (from n= 5 transfections) of KV4.3/KChIP2/DPP6 channels. *: p< 0.05. Int. J. Mol. Sci. 2022,23, 9170 16 of 18 Author Contributions: A.d.B.-B., P.G.S. and Y.G.M. conducted the electrophysiological experiments and analyzed them supervised by C.V.; P.C. and C.I. synthetized IQM-266 supervised by M.G.-R.; M.D.-M. prepared KChIP2 protein supervised by A.A.; I.M.-O. and A.P.-L. performed tryptophan FRET experiments. A.P.-L., J.A.G.-V. and A.O. designed and supervised the binding experiments and analyzed data. M.M.-M. conducted the computational studies. A.d.B.-B. and P.G.S. performed statistical analyses, generated the final figures and contributed to the manuscript writing. M.M.-M., A.A., A.P.-L., J.A.G.-V. and A.O. contributed to the manuscript writing. M.G.-R. and C.V. conceived the project, analyzed data, supervised the whole project and wrote the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This publication is the results of the: Grants SAF2016-75021-R (to C.V.), RTI2018-097189-B- C22 (to M.M.-M.) and BIO2017-89523-R (to A.A.) funded by MCIN/AEI/10.13039/501100011033 and by “ERDF A way of making Europe”; Grants PID2019-104366RB-C21 (to C.V.), PID2019-104366RB- C22 (to M.G.-R.), PID2020-114256RB-I00 (to A.O. and J.A.G.-V.), PID2020-119805RB-I00 (to A.A.) funded by MCIN/AEI/10.13039/501100011033; Grant A-FQM-386-UGR20 funded by FEDER/Junta de Andalucía-Consejería de Transformación Económica, Industria, Conocimiento (to J.A.G.-V.); Grant CB/11/00222 funded by Instituto de Salud Carlos III CIBERCV (to C.V.); Grants PIE202180E073 (to M.M.-M. and M.G.-R.), PIE201820E104 and 2019AEP148 (to C.V.) funded by Consejo Superior de Investigaciones Científicas. Grants BES-2017-080184 (to A.d.B.-B.), BES-2010-036573 (to P.C.), PRE2018- 083280 (to M.D.-M.) and RYC2018-023837-I (to A.P.-L.) funded by MCIN/AEI/10.13039/501100011033 and by “ESF Investing in your future”; Grant FPU17/02731 (to P.G.S.) funded by Ministerio de Ciencia e Innovación. Institutional Review Board Statement: All experiments shown in the present study were performed following the European Parliament 2010/63/EU and the rules of the Helsinki Declaration. All experimental procedures followed the guidelines for ethical care of the European Union (2012/63/EU). The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of the University Hospital La Paz (PI-2550). Informed Consent Statement: Not applicable for studies not involving humans. Data Availability Statement: The data that support the findings of this study are available from the corresponding author upon reasonable request. Acknowledgments: We would like to thank Susanne Kaemmerer (Technische Universität Dresden, Germany) and Dirk J. Snyders (University of Antwerpen, Belgium) for their kind help in providing us with some of the necessary constructions for this work. The authors want to thank Carmen Delgado (Instituto de Investigaciones Biomédicas Alberto Sols CSIC-UAM) for her helpful suggestions. Conflicts of Interest: The authors declare no conflict of interest. References 1. Kong, W.; Po, S.; Yamagishi, T.; Ashen, M.D.; Stetten, G.; Tomaselli, G.F. Isolation and characterization of the human gene encoding Ito: Further diversity by alternative mRNA splicing. Am. J. Physiol. 1998,275, H1963–H1970. [PubMed] 2. Nerbonne, J.M.; Kass, R.S. Molecular physiology of cardiac repolarization. Physiol. Rev. 2005 ,85, 1205–1253. [CrossRef] [PubMed] 3. Dixon, J.E.; Shi, W.; Wang, H.S.; McDonald, C.; Yu, H.; Wymore, R.S.; Cohen, I.S.; McKinnon, D. Role of the Kv4.3 K+ channel in ventricular muscle. 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