Human Kir2 Research Articles

Biochemical, biophysical, and structural investigations of two mutants (C154Y and R312H) of the human Kir2.1 channel involved in the Andersen-Tawil syndrome.

Inwardly rectifying potassium (Kir) channels play a pivotal role in physiology by establishing, maintaining, and regulating the resting membrane potential of the cells, particularly contributing to the cellular repolarization of many excitable cells. Dysfunction in Kir2.1 channels is implicated in several chronic and debilitating human diseases for which there are currently no effective treatments. Specifically, Kir2.1-R312H and Kir2.1-C154Y mutations are associated with Andersen-Tawil syndrome (ATS) in humans. We have investigated the impact of these two mutants in the trafficking of the channel to the cell membrane and function in Xenopus laevis oocytes. Despite both mutations being trafficked to the cell membrane at different extents and capable of binding PIP2 (phosphatidylinositol-4,5-bisphosphate), the main modulator for channel activity, they resulted in defective channels that do not display K+ current, albeit through different molecular mechanisms. Coexpression studies showed that R312H and C154Y are expressed and associated with the WT subunits. While WT subunits could rescue R312H dysfunction, the presence of a unique C154Y subunit disrupts the function of the entire complex, which is a typical feature of mutations with a dominant-negative effect. Molecular dynamics simulations showed that Kir2.1-C154Y mutation induces a loss in the structural plasticity of the selectivity filter, impairing the K+ flow. In addition, the cryo-EM structure of the Kir2.1-R312H mutant has been reconstructed. This study identified the molecular mechanisms by which two ATS-causing mutations impact Kir2.1 channel function and provide valuable insights that can guide potential strategies for the development of future therapeutic interventions for ATS.

Cryo-electron microscopy structure of human Kir2.1 potassium channel bound to the activator PIP2 reveals its gating mechanism

Cryo-electron microscopy structure of human Kir2.1 potassium channel bound to the activator PIP2 reveals its gating mechanism

Cryo–electron microscopy unveils the gating mechanism of the human Kir2.1 channel

Cryo–electron microscopy unveils the gating mechanism of the human Kir2.1 channel

Human Kir2.1 Potassium Channel: Protocols for Cryo-EM Data Processing and Molecular Dynamics Simulations.

Kir channels are potassium (K+) channels responsible for the mechanism of inward rectification, which plays a fundamental role in maintaining the resting membrane potential. There are seven Kir subfamilies, and their opening and closing mechanism is regulated by different regulatory factors. Genetically inherited defects in Kir channels are responsible for several rare human diseases, and for most of them, there are currently no effective therapeutic treatments. High-resolution structural information is not available for several members within the Kir subfamilies. Recently, our group achieved a significant breakthrough by utilizing cryo-EM single-particle analysis to elucidate the first structure of the human Kir2.1 channel. We present here the data processing protocol of the cryo-EM data of the human Kir2.1 channel, which is applicable to the structural determination of other ion channels by cryo-EM single-particle analysis. We also introduce a protocol designed to assess the structural heterogeneity within the cryo-EM data, allowing for the identification of other possible protein structure conformations present in the collected data. Moreover, we present a protocol for conducting all-atom molecular dynamics (MD) simulations for K+ channels, which can be incorporated into various membrane models to simulate different environments. We also propose some methods for analyzing the MD simulations, with a particular emphasis on assessing the local mobility of protein residues.

Sildenafil affects the human Kir2.1 and Kir2.2 channels at clinically relevant concentrations: Inhibition potentiated by low Ba2.

Sildenafil (Viagra), the first approved and widely used oral drug for the treatment of erectile dysfunction, was occasionally associated with life-threatening ventricular arrhythmias in patients. Since inward rectifier potassium current (I K1) may considerably contribute to this arrhythmogenesis, we investigated the effect of sildenafil on the human Kir2.1 and Kir2.2, the prevailing subunits forming the ventricular I K1 channels. Experiments were performed by the whole-cell patch clamp technique at 37°C using Chinese hamster ovary cells transiently expressing the human Kir2.1 and Kir2.2 channels. Changes of both the inward and outward current components (at -110 and -50mV, respectively) were tested to be able to consider the physiological relevance of the sildenafil effect (changes at -110 and -50mV did not significantly differ, results at -50mV are listed below). A significant Kir2.1 inhibition was observed at all applied sildenafil concentrations (16.1% ± 3.7%, 20.0% ± 2.6%, and 15.0% ± 3.0% at 0.1, 1, and 10μM, respectively). The inhibitory effect of 0.1μM sildenafil was potentiated by the presence of a low concentration of Ba2+ (0.1μM) which induced only a slight Kir2.1 inhibition by 5.95% ± 0.75% alone (the combined effect was 35.5% ± 3.4%). The subtherapeutic and therapeutic sildenafil concentrations (0.1 and 1μM) caused a dual effect on Kir2.2 channels whereas a significant Kir2.2 activation was observed at the supratherapeutic sildenafil concentration (10μM: 34.1% ± 5.6%). All effects were fully reversible. This is the first study demonstrating that sildenafil at clinically relevant concentrations inhibits both the inward and outward current components of the main human ventricular I K1 subunit Kir2.1. This inhibitory effect was significantly potentiated by a low concentration of environmental contaminant Ba2+ in agreement with recently reported data on rat ventricular I K1 which additionally showed a significant repolarization delay. Considering the similar subunit composition of the human and rat ventricular I K1 channels, the observed effects might contribute to sildenafil-associated arrhythmogenesis in clinical practice.

Empagliflozin and Dapagliflozin Increase Na+ and Inward Rectifier K+ Current Densities in Human Cardiomyocytes Derived from Induced Pluripotent Stem Cells (hiPSC-CMs).

Dapagliflozin (dapa) and empagliflozin (empa) are sodium-glucose cotransporter-2 inhibitors (SGLT2is) that reduce morbidity and mortality in heart failure (HF) patients. Sodium and inward rectifier K+ currents (INa and IK1), carried by Nav1.5 and Kir2.1 channels, respectively, are responsible for cardiac excitability, conduction velocity, and refractoriness. In HF patients, Nav1.5 and Kir2.1 expression are reduced, enhancing risk of arrhythmia. Incubation with dapa or empa (24-h,1 µM) significantly increased INa and IK1 densities recorded in human-induced pluripotent stem cell-cardiomyocytes (hiPSC-CMs) using patch-clamp techniques. Dapa and empa, respectively, shifted to more hyperpolarized potentials the INa activation and inactivation curves. Identical effects were observed in Chinese hamster ovary (CHO) cells that were incubated with dapa or empa and transiently expressed human Nav1.5 channels. Conversely, empa but not dapa significantly increased human Kir2.1 currents in CHO cells. Dapa and empa effects on INa and IK1 were also apparent in Ca-calmodulin kinase II-silenced CHO cells. Cariporide, a Na+/H+ exchanger type 1 (NHE1) inhibitor, did not increase INa or IK1 in hiPSC-CMs. Dapa and empa at therapeutic concentrations increased INa and IK1 in healthy human cardiomyocytes. These SGLT2is could represent a new class of drugs with a novel and long-pursued antiarrhythmic mechanism of action.

Cryo-electron microscopy unveils unique structural features of the human Kir2.1 channel.

We present the first structure of the human Kir2.1 channel containing both transmembrane domain (TMD) and cytoplasmic domain (CTD). Kir2.1 channels are strongly inward-rectifying potassium channels that play a key role in maintaining resting membrane potential. Their gating is modulated by phosphatidylinositol 4,5-bisphosphate (PIP2). Genetically inherited defects in Kir2.1 channels are responsible for several rare human diseases, including Andersen’s syndrome. The structural analysis (cryo–electron microscopy), surface plasmon resonance, and electrophysiological experiments revealed a well-connected network of interactions between the PIP2-binding site and the G-loop through residues R312 and H221. In addition, molecular dynamics simulations and normal mode analysis showed the intrinsic tendency of the CTD to tether to the TMD and a movement of the secondary anionic binding site to the membrane even without PIP2. Our results revealed structural features unique to human Kir2.1 and provided insights into the connection between G-loop and gating and the pathological mechanisms associated with this channel.

Structural and functional characterization of a human potassium channel, Kir2.1

Inwardly rectifying potassium (Kir) channels selectively control the permeation of K+ ions in and out of the cell. The inward rectification allows them to be associated with the control of many vital physiological functions, including heartbeat. These channels open and close in response to modulators like phosphatidylinositol 4,5-bisphosphate (PIP2)—a lipid required for function in eukaryotic channels. Dysfunctions in Kir channels often lead to diseases called channelopathies. In our case, we are interested in Andersen's syndrome, in which mutations of the human Kir2.1 protein are directly involved.

In vitro ion channel profile and ex vivo cardiac electrophysiology properties of the R(-) and S(+) enantiomers of hydroxychloroquine

Hydroxychloroquine (HCQ) is a derivative of the antimalaria drug chloroquine primarily prescribed for autoimmune diseases. Recent attempts to repurpose HCQ in the treatment of corona virus disease 2019 has raised concerns because of its propensity to prolong the QT-segment on the electrocardiogram, an effect associated with increased pro-arrhythmic risk. Since chirality can affect drug pharmacological properties, we have evaluated the functional effects of the R(-) and S(+) enantiomers of HCQ on six ion channels contributing to the cardiac action potential and on electrophysiological parameters of isolated Purkinje fibers. We found that R(-)HCQ and S(+)HCQ block human Kir2.1 and hERG potassium channels in the 1 μM–100 μM range with a 2–4 fold enantiomeric separation. NaV1.5 sodium currents and CaV1.2 calcium currents, as well as KV4.3 and KV7.1 potassium currents remained unaffected at up to 90 μM. In rabbit Purkinje fibers, R(-)HCQ prominently depolarized the membrane resting potential, inducing autogenic activity at 10 μM and 30 μM, while S(+)HCQ primarily increased the action potential duration, inducing occasional early afterdepolarization at these concentrations. These data suggest that both enantiomers of HCQ can alter cardiac tissue electrophysiology at concentrations above their plasmatic levels at therapeutic doses, and that chirality does not substantially influence their arrhythmogenic potential in vitro.

Structural and Functional Characterization of a Human Potassium Channel, Kir2.1

Inwardly rectifying potassium (Kir) channels are ubiquitously present in prokaryotic and eukaryotic cells. Kir channels selectively control the permeation of K+ ions in and out of the cell along the electrochemical gradient. Their inward rectification allows them to control the resting membrane potential and regulate many vital physiological functions such as heart rate, vascular tone, cell volume, and salt/fluid balance. Dysfunctions in Kir channels often lead to a multitude of diseases called “channelopathies”. Understanding the mechanisms involved in Kir channels and the effects of their mutations constitute a major challenge in the pharmaceutical and therapeutic research of the different channelopathies. In our case, we are interested in Andersen's syndrome, in which mutations of the human Kir2.1 protein are directly involved. By a biochemical, structural, and functional study, we aim to identify the differences between the wild type (WT) and two mutant channels in order to find links between the structure and the function of Kir2.1. This project aims at understanding the impact of clinically relevant disease-causing mutations on the structure and function of Kir2.1 potassium channels and to resolve the structure at high resolution using cryo-electron microscopy. The gating of Kir channels is modulated by various intracellular ligands that bind directly to the channel and enable its electrical activity. The lipid phosphatidylinositol-4,5-bisphosphate (PIP2) is essential to activate the eukaryotic Kir channels. We study a protein containing an arginine to histidine mutation in the putative PIP2 binding site and hypothesized that the loss of function observed in the mutated protein was due to a loss of interaction with PIP2. To test this hypothesis, we characterized the lipid-protein interaction using Surface Plasmon Resonance (SPR) and compared it with the WT protein. The SPR data shows that the mutant does not bind as well to the lipids as the WT.

Abstract 17074: Methadone Potently Blocks Cardiac IK1 Leading to Membrane Instability

Introduction: Methadone is the second most frequently reported cause of drug-induced cardiac arrest in pharmacovigilance databases, yet the mechanism of its pro-arrhythmia is unclear. Methadone-induced QTU wave prolongation has been repeatedly observed and attributed to inhibition of the delayed rectifier hERG current, but QTU fusion suggests the inwardly rectifying K + current (IK1) might also be affected by methadone. Hypothesis: Methadone pro-arrhythmia is associated with potent block of the IK1 current responsible for rapid terminal repolarization of the cardiac action potential (AP). Methods: Human Kir2.2, encoding the IK1 current, was transiently expressed in COS cells. hERG1a was stably expressed in CHO cells. Cardiac myocytes from swine were obtained by ventricular enzymatic dissociation. Ionic current and APs were measured using patch-clamp methods. Methadone HCl (R+S racemates) was dissolved in Tyrode solution. Results: Methadone suppressed IK1 current with an IC 50 of 1.47 uM (Fig 1A). Methadone also suppressed outward IK1 (-60 mV) measured in swine myocytes (Ba 2+ -sensitive current) with an IC 50 of 1.52 μM. Methadone suppressed hERG currents with an IC 50 of 2.1 μM. APs measured in swine myocytes exhibited significant prolongation (13 ± 4 % increase of APD 90 , p<0.029, n=7) as well as slowing of the rate of terminal repolarization (a specific marker of IK1 blockade) in the presence of 1 μM methadone (Fig. 1B). Fluctuations of diastolic voltage increased by 30 ± 12 and 151 ± 27 % (n=3; p<0.04) in 0.1 and 1 μM methadone, respectively, consistent with a reduction in membrane stability. Conclusions: Methadone is an equipotent blocker of IK1 and hERG. The effect of IK1 block coupled with modest hERG block has a synergistic effect on terminal repolarization that may partially explain the pro-arrhythmic impact of methadone. Moreover, this observation may be generalized to other drugs where unsuspected IK1 blockade may contribute to pro-arrhythmia and torsade de pointes.

Identification of a PEST Sequence in Vertebrate KIR2.1 That Modifies Rectification.

KIR2.1 potassium channels, producing inward rectifier potassium current (IK1), are important for final action potential repolarization and a stable resting membrane potential in excitable cells like cardiomyocytes. Abnormal KIR2.1 function, either decreased or increased, associates with diseases such as Andersen-Tawil syndrome, long and short QT syndromes. KIR2.1 ion channel protein trafficking and subcellular anchoring depends on intrinsic specific short amino acid sequences. We hypothesized that combining an evolutionary based sequence comparison and bioinformatics will identify new functional domains within the C-terminus of the KIR2.1 protein, which function could be determined by mutation analysis. We determined PEST domain signatures, rich in proline (P), glutamic acid (E), serine (S), and threonine (T), within KIR2.1 sequences using the “epestfind” webtool. WT and ΔPEST KIR2.1 channels were expressed in HEK293T and COS-7 cells. Patch-clamp electrophysiology measurements were performed in the inside-out mode on excised membrane patches and the whole cell mode using AxonPatch 200B amplifiers. KIR2.1 protein expression levels were determined by western blot analysis. Immunofluorescence microscopy was used to determine KIR2.1 subcellular localization. An evolutionary conserved PEST domain was identified in the C-terminus of the KIR2.1 channel protein displaying positive PEST scores in vertebrates ranging from fish to human. No similar PEST domain was detected in KIR2.2, KIR2.3, and KIR2.6 proteins. Deletion of the PEST domain in California kingsnake and human KIR2.1 proteins (ΔPEST), did not affect plasma membrane localization. Co-expression of WT and ΔPEST KIR2.1 proteins resulted in heterotetrameric channel formation. Deletion of the PEST domain did not increase protein stability in cycloheximide assays [T½ from 2.64 h (WT) to 1.67 h (ΔPEST), n.s.]. WT and ΔPEST channels, either from human or snake, produced typical IK1, however, human ΔPEST channels displayed stronger intrinsic rectification. The current observations suggest that the PEST sequence of KIR2.1 is not associated with rapid protein degradation, and has a role in the rectification behavior of IK1 channels.

Electrophysiological and Pharmacological Characterization of Human Inwardly Rectifying Kir2.1 Channels on an Automated Patch-Clamp Platform.

Inwardly rectifying IK1 potassium currents of the heart control the resting membrane potential of ventricular cardiomyocytes during diastole and contribute to their repolarization after each action potential. Mutations in the gene encoding Kir2.1 channels, which primarily conduct ventricular IK1, are associated with inheritable forms of arrhythmias and sudden cardiac death. Therefore, potential iatrogenic inhibition of Kir2.1-mediated IK1 currents is a cardiosafety concern during new drug discovery and development. Kir2.1 channels are part of the panel of cardiac ion channels currently considered for refined early compound risk assessment within the Comprehensive in vitro Proarrhythmia Assay initiative. In this study, we have validated a cell-based assay allowing functional quantification of Kir2.1 inhibitors using whole-cell recordings of Chinese hamster ovary cells stably expressing human Kir2.1 channels. We reproduced key electrophysiological and pharmacological features known for native IK1, including current enhancement by external potassium and voltage- and concentration-dependent blockade by external barium. Furthermore, the Kir inhibitors ML133, PA-6, and chloroquine, as well as the multichannel inhibitors chloroethylclonidine, chlorpromazine, SKF-96365, and the class III antiarrhythmic agent terikalant demonstrated slowly developing inhibitory activity in the low micromolar range. The robustness of this assay authorizes medium throughput screening for cardiosafety purposes and could help to enrich the currently limited Kir2.1 pharmacology.

Gating Mechanism of a Potassium Channel, Experimental and Theoretical Studies

Inwardly-rectifying potassium (Kir) channels are transmembrane proteins that regulate membrane electrical excitability and K+ transport in many cell types generating and controlling the membrane resting potential. The function of this channel although simple is primordial for physiological processes such as the creation and the propagation of the neuronal action's potential, the regulation of cellular volume, the muscular contraction and the cardiac pulse. Their physiological importance is highlighted by the fact that genetically inherited defects in Kir channels are responsible for a wide-range of channelopathies including Andersen's syndrome a pathology that can cause periodic paralysis or serious heart problems. To date unfortunately, this disease does not have any effective treatment. The goal of these studies is to understand how the channel gates and what are the conformational changes required. Our work focused on the KirBac3.1 channel, homologous to Kir2.1 human. We started with known crystallographic data from the KirBac3.1WT and two mutants S129R and W46R. We explored conformational changes using Molecular Dynamics using Excited Normal Modes (MDeNM), a new method which gives access to vibrational motions (Molecular Dynamics) and global motions (Normal Modes) at the same time. MDeNM successfully opened the closed state of the protein giving the opportunity to identify the key motions involved in the gating such as the role of the cytoplasmic domain, the slide-helix as well as the transmembrane helices during the opening of the channel. In parallel, HDX-MS Spectrometry studies had been performed and gave important information on the flexibility of the protein. Experimental and theoretical studies were compared for validation. In addition, human Kir2.1 was purified and imaged with a Titan Krios microcope. The structure will help to understand the gating mechanism of this human channel.

Single Molecule FRET Reveals Lipid Induced Conformational Changes in Cytoplasmic Domain of Kir2.1

Anionic phospholipids are common regulators of ion channel activity, including Kir2.1, an inward rectifying potassium channel crucial for setting and maintaining resting membrane potential in many tissues, which is activated specifically by PIP2 and phosphatidyl glycerol (PG). Crystal structures indicate that a simple upward translocation of the cytoplasmic Kir domain is induced by PIP2 and PG binding. However, recent computational and single-molecule FRET experiments show more complex intra-domain movements and rotation in the Kir domain of prokaryotic KirBac1.1 upon PIP2 binding. Single molecule FRET measurements on eukaryotic human Kir2.1 indicate similar complex motions that are not detected in static crystal structures. Our measurements indicate dynamic conformational ‘breathing’ throughout the Kir domain, suggesting greater flexibility in the intracellular domain of ion channels than may be apparent in structural studies. Specifically, our data indicate a PIP2 dependent shifts in average single molecule FRET (smFRET) distributions, with outward motions seen prior to the slide helix (residue 59) and an inward motion seen at the base (residues 286 and 288) consistent with a twisting motion of the Kir domain that was previously shown to be associated with opening in KirBac1.1. In the absence of PIP2, smFRET signals are very dynamic with relatively large (>20A) motions at specific residues. PIP2 stabilizes the conformational dynamics of the Kir domain, with less transitions between structural states, and reduction in the occurrence of large conformational transitions.

PA-6 inhibits inward rectifier currents carried by V93I and D172N gain-of-function KIR2.1 channels, but increases channel protein expression

BackgroundThe inward rectifier potassium current IK1 contributes to a stable resting membrane potential and phase 3 repolarization of the cardiac action potential. KCNJ2 gain-of-function mutations V93I and D172N associate with increased IK1, short QT syndrome type 3 and congenital atrial fibrillation. Pentamidine-Analogue 6 (PA-6) is an efficient (IC50 = 14 nM with inside-out patch clamp methodology) and specific IK1 inhibitor that interacts with the cytoplasmic pore region of the KIR2.1 ion channel, encoded by KCNJ2. At 10 μM, PA-6 increases wild-type (WT) KIR2.1 expression in HEK293T cells upon chronic treatment. We hypothesized that PA-6 will interact with and inhibit V93I and D172N KIR2.1 channels, whereas impact on channel expression at the plasma membrane requires higher concentrations.MethodsMolecular modelling was performed with the human KIR2.1 closed state homology model using FlexX. WT and mutant KIR2.1 channels were expressed in HEK293 cells. Patch-clamp single cell electrophysiology measurements were performed in the whole cell and inside-out mode of the patch clamp method. KIR2.1 expression level and localization were determined by western blot analysis and immunofluorescence microscopy, respectively.ResultsPA-6 docking in the V93I/D172N double mutant homology model of KIR2.1 demonstrated that mutations and drug-binding site are >30 Å apart. PA-6 inhibited WT and V93I outward currents with similar potency (IC50 = 35.5 and 43.6 nM at +50 mV for WT and V93I), whereas D172N currents were less sensitive (IC50 = 128.9 nM at +50 mV) using inside-out patch-clamp electrophysiology. In whole cell mode, 1 μM of PA-6 inhibited outward IK1 at −50 mV by 28 ± 36%, 18 ± 20% and 10 ± 6%, for WT, V93I and D172N channels respectively. Western blot analysis demonstrated that PA-6 (5 μM, 24 h) increased KIR2.1 expression levels of WT (6.3 ± 1.5 fold), and V93I (3.9 ± 0.9) and D172N (4.8 ± 2.0) mutants. Immunofluorescent microscopy demonstrated dose-dependent intracellular KIR2.1 accumulation following chronic PA-6 application (24 h, 1 and 5 μM).Conclusions1) KCNJ2 gain-of-function mutations V93I and D172N in the KIR2.1 ion channel do not impair PA-6 mediated inhibition of IK1, 2) PA-6 elevates KIR2.1 protein expression and induces intracellular KIR2.1 accumulation, 3) PA-6 is a strong candidate for further preclinical evaluation in treatment of congenital SQT3 and AF.

Characterization of Structural Changes via Lipid Regulation of KIR2.1 in a Lipid Environment

Regulation of ion channel activity occurs through various mechanisms, via small molecule regulators, membrane potential, and specific interaction with lipid molecules. Inward rectifying potassium (Kir) channels conduction is controlled both by small molecules, such as polyamines blocking ion conduction, and lipid molecules that stabilize a conductive state. Advancing molecular understanding of this lipid regulation has been difficult as many structural techniques rely on detergent solubilized channels, which removes specific channel:lipid interaction. FRET, both macroscopically and in single molecules, can help surmount this issue, providing measurements of structural and dynamic changes induced by lipid regulation in a membrane environment. We have used macroscopic FRET to measure lipid specific structural changes in Kir2.1 in liposomes of defined composition. We have optimized purification and fluorophore labeling of the human Kir2.1 channel, which are reconstituted into liposomes of various lipid composition. We have measured PIP2-dependent FRET changes at numerous sites, which indicate structural movements of the intracellular domain of Kir2.1 upon PIP2 binding. This work specifically investigates how movements of the slide helix contribute to activation of the channel. Further efforts will help to define the molecular details of bulk anionic lipid and PIP2-dependent structural dynamics of Kir2.1 and help to understand more generally how lipid-protein interactions regulate membrane protein function.

IS DILATED CARDIOMYOPATHY A PRIMARY FEATURE OF ANDERSEN-TAWIL SYNDROME? CHARACTERIZATION OF A NOVEL KCNJ2 MUTATION

Andersen-Tawil syndrome (ATS) is a rare autosomal dominant multisystem disorder characterized by cardiac arrhythmias, periodic paralysis, and dysmorphic features. Mutations in the KCNJ2, the gene encoding the human inward rectifier potassium channel Kir2.1 (IK1) have been linked to ATS. Currently, it remains unclear whether dilated cardiomyopathy is a primary feature of ATS.

Nav1.5 N-terminal domain binding to α1-syntrophin increases membrane density of human Kir2.1, Kir2.2 and Nav1.5 channels.

Cardiac excitability and refractoriness are largely determined by the function and number of inward rectifier K⁺ channels (Kir2.1-2.3), which are differentially expressed in the atria and ventricles, and Nav1.5 channels. We have focused on how Nav1.5 and Kir2.x function within a macromolecular complex by elucidating the molecular determinants that govern Nav1.5/Kir2.x reciprocal modulation. The results demonstrate that there is an unexpected 'internal' PDZ-like binding domain located at the N-terminus of the Nav1.5 channel that mediates its binding to α1-syntrophin. Nav1.5 N-terminal domain, by itself (the 132 aa peptide) (Nter), exerts a 'chaperone-like' effect that increases sodium (I(Na)) and inward rectifier potassium (I(K1)) currents by enhancing the expression of Nav1.5, Kir2.1, and Kir2.2 channels as demonstrated in Chinese hamster ovary (CHO) cells and in rat cardiomyocytes. Site-directed mutagenesis analysis demonstrates that the Nter chaperone-like effect is determined by Serine 20. Nav1.5-Kir2.x reciprocal positive interactions depend on a specific C-terminal PDZ-binding domain sequence (SEI), which is present in Kir2.1 and Kir2.2 channels but not in Kir2.3. Therefore, in human atrial myocytes, the presence of Kir2.3 isoforms precludes reciprocal I(K1)-INa density modulation. Moreover, results in rat and human atrial myocytes demonstrate that binding to α1-syntrophin is necessary for the Nav1.5-Kir2.x-positive reciprocal modulation. The results demonstrate the critical role of the N-terminal domain of Nav1.5 channels in Nav1.5-Kir2.x-reciprocal interactions and suggest that the molecular mechanisms controlling atrial and ventricular cellular excitability may be different.

Structural Basis for Differences in Dynamics Induced by Leu Versus Ile Residues in the CD Loop of Kir Channels.

The effect of the conserved Leu/Ile site in the CD loop on the gating dynamics of Kir channels and corresponding micro-structural mechanism remains unclear. Molecular dynamics simulations were performed to investigate the structural mechanism of chicken Kir2.2. Compared to WT, the I223L mutant channel bound to PIP2 more strongly, was activated more rapidly, and maintained the activation state more stably after PIP2 dissociation. Cellular electrophysiology assays of mouse Kir2.1 and human Kir2.2 indicated that, consistent with simulations, the Leu residue increased the channel responses to PIP2 through increased binding affinity and faster activation kinetics, and the deactivation kinetics decreased upon PIP2 inhibition. The Ile residue induced the opposite responses. This difference was attributed to the distinct hydrophobic side chain symmetries of Leu and Ile; switching between these residues caused the interaction network to redistribute and offered effective conformation transduction in the Leu systems, which had more rigid and independent subunits.Electronic supplementary materialThe online version of this article (doi:10.1007/s12035-015-9466-x) contains supplementary material, which is available to authorized users.