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High-throughput experiments suggest that almost 20% of human proteins may be S-palmitoylatable, a post-translational modification (PTM) whereby fatty acyl chains, most commonly palmitoyl chain, are linked to cysteine thiol groups that impact on protein trafficking, distribution and function. In human, protein S-palmitoylation is mediated by a group of 23 palmitoylating 'Asp-His-His-Cys' domain-containing (DHHC) enzymes. There is no information on the scope of protein S-palmitoylation, or the pattern of DHHC enzyme expression, in the heart. We used resin-assisted capture to pull down S-palmitoylated proteins from human, dog, and rat hearts, followed by proteomic search to identify proteins in the pulldowns. We identified 454 proteins present in at least 2 species-specific pulldowns. These proteins are operationally called 'cardiac palmitoylome'. Enrichment analysis based on Gene Ontology terms 'cellular component' indicated that cardiac palmitoylome is involved in cell-cell and cell-substrate junctions, plasma membrane microdomain organization, vesicular trafficking, and mitochondrial enzyme organization. Importantly, cardiac palmitoylome is uniquely enriched in proteins participating in the organization and function of t-tubules, costameres and intercalated discs, three microdomains critical for excitation-contraction coupling and intercellular communication of cardiomyocytes. We validated antibodies targeting DHHC enzymes, and detected eleven of them expressed in hearts across species. In conclusion, we provide resources useful for investigators interested in studying protein S-palmitoylation and its regulation by DHHC enzymes in the heart. We also discuss challenges in these efforts, and suggest methods and tools that should be developed to overcome these challenges.

Junctophilins (JPH1–JPH4) are expressed in excitable and nonexcitable cells, where they tether endoplasmic/sarcoplasmic reticulum (ER/SR) and plasma membranes (PM). These ER/SR–PM junctions bring Ca-release channels in the ER/SR and Ca as well as Ca-activated K channels in the PM to within 10–25 nm. Such proximity is critical for excitation–contraction coupling in muscles, Ca modulation of excitability in neurons, and Ca homeostasis in nonexcitable cells. JPHs are anchored in the ER/SR through the C-terminal transmembrane domain (TMD). Their N-terminal Membrane-Occupation-Recognition-Nexus (MORN) motifs can bind phospholipids. Whether MORN motifs alone are sufficient to stabilize JPH–PM binding is not clear. We investigate whether S-palmitoylation of cysteine (Cys), a critical mechanism controlling peripheral protein binding to PM, occurs in JPHs. We focus on JPH2 that has four Cys residues: three flanking the MORN motifs and one in the TMD. Using palmitate–alkyne labeling, Cu(I)-catalyzed alkyne-azide cycloaddition reaction with azide-conjugated biotin, immunoblotting, proximity–ligation–amplification, and various imaging techniques, we show that JPH2 is S-palmitoylatable, and palmitoylation is essential for its ER/SR–PM tether function. Palmitoylated JPH2 binds to lipid–raft domains in PM, whereas palmitoylation of TMD-located Cys stabilizes JPH2's anchor in the ER/SR membrane. Binding to lipid–raft domains protects JPH2 from depalmitoylation. Unpalmitoylated JPH2 is largely excluded from lipid rafts and loses the ability to form stable ER/SR–PM junctions. In adult ventricular myocytes, native JPH2 is S-palmitoylatable, and palmitoylated JPH2 forms distinct PM puncta. Sequence alignment reveals that the palmitoylatable Cys residues in JPH2 are conserved in other JPHs, suggesting that palmitoylation may also enhance ER/SR–PM tethering by these proteins.

Key points In adult ventricular myocytes, the IKs channels are distributed on the surface sarcolemma, not t-tubules. In adult ventricular myocytes, KCNQ1 and KCNE1 have distinct cell surface and cytoplasmic pools. KCNQ1 and KCNE1 traffic from the endoplasmic reticulum to plasma membrane by separate routes, and assemble into IKs channels on the cell surface. Liquid chromatography/tandem mass spectrometry applied to affinity-purified KCNQ1 and KCNE1 interacting proteins reveals novel interactors involved in protein trafficking and assembly. Microtubule plus-end binding protein 1 (EB1) binds KCNQ1 preferentially in its dimer form, and promotes KCNQ1 to reach the cell surface. An LQT1-associated mutation, Y111C, reduces KCNQ1 binding to EB1 dimer. Abstract Slow delayed rectifier (IKs ) channels consist of KCNQ1 and KCNE1. IKs functions as a 'repolarization reserve' in the heart by providing extra current for ventricular action potential shortening during β-adrenergic stimulation. There have been debates as to how KCNQ1 and KCNE1 traffic in cells, where they associate to form IKs channels, and the distribution pattern of IKs channels relative to β-adrenergic signaling complex. We used experimental strategies not previously applied to KCNQ1, KCNE1 or IKs , to provide new insights into these issues. 'Retention-using-selected-hook' experiments showed that newly translated KCNE1 constitutively trafficked through the conventional secretory path to the cell surface. KCNQ1 largely stayed in the endoplasmic reticulum, although dynamic KCNQ1 vesicles were observed in the submembrane region. Disulfide-bonded KCNQ1/KCNE1 constructs reported preferential association after they had reached cell surface. In-situ proximity ligation assay (PLA) detected IKs channels in surface sarcolemma but not t-tubules of ventricular myocytes, similar to the reported location of adenylate cyclase 9/yotiao. Fluorescent protein (FP)-tagged KCNQ1 and KCNE1, in conjunction with antibodies targeting their extracellular epitopes, detected distinct cell surface and cytoplasmic pools of both proteins in myocytes. We conclude that in cardiomyocytes KCNQ1 and KCNE1 traffic by different routes to surface sarcolemma where they assemble into IKs channels. This mode of delayed channel assembly helps IKs fulfill its function of repolarization reserve. Proteomic experiments revealed a novel KCNQ1 interactor, microtubule plus-end binding protein 1 (EB1). EB1 dimer (active form) bound KCNQ1 and increased its surface level. An LQT1 mutation, Y111C, reduced KCNQ1 binding to EB1 dimer. This article is protected by copyright. All rights reserved.

Introduction: More that 10% of human proteins can be S-palmitoylated, a post-translational modification (PTM) whereby palmitoyl chains are covalently linked to cysteine thiol groups. S-palmitoylation influences protein trafficking, distribution and function. There is no information on the scope of protein S-palmitoylation in the heart, or how this enzyme-mediated reversible PTM is regulated. Hypothesis: S-palmitoylation occurs to a wide spectrum of proteins in cardiomyocytes, and is coordinated by membrane-embedded palmitoylating (DHHC) enzymes. DHHC enzymes are subject to remodeling during chronic hypertension. Methods: We used resin-assisted capture to purify S-palmitoylated proteins from ventricular myocardium of 3 species: human, dog, and rat. We used global unbiased proteomic search to identify S-palmitoylated proteins. We validated DHHC antibodies and used them to monitor protein level and subcellular distribution of native DHHC enzymes in ventricular myocytes. Results: We built a 'composite' cardiac palmitome composed of 462 S-palmitoylatable proteins identified in ≥ 2 species-specific cardiac palmitomes. Enrichment analysis based on GO term 'cellular component' indicated that they are mainly involved in cell-cell and cell-substrate associations, sarcolemma and sarcomere organization, vesicular trafficking, G-protein function, ATP-dependent transmembrane transport, and mitochondria inner and outer membrane organization. Among the 23 DHHC enzymes, we detected ten in hearts across species. In ventricular myocytes with well-defined subcellular compartments, DHHC enzymes exhibited distinct distribution patterns: peripheral sarcolemma (DHHC1), M-lines (DHHC2), Z-lines (DHHC5), vesicles (DHHC7) and intercalated disc (DHHC9). In aging spontaneously hypertensive rats (a model of chronic hypertension, some in heart failure), seven DHHC enzymes were upregulated in the heart, accompanied by a higher degree of S-palmitoylation of CaMK II, caveolin3, Na/Ca exchanger, and Na/K pump α-subunit. Conclusion: S-palmitoylation is involved in most, if not all, aspects of cardiomyocyte function. Palmitoylation dysregulation may contribute to pathological progression in hypertrophy leading to heart failure.

Introduction: Junctions between sarco/endoplasmic reticulum (SR/ER) and sarcolemma (SL), where Ca-induced Ca release (CICR) takes place, are important for myocardial contractility. JPH2 is the major SR/SL tether in cardiomyocytes. Hypothesis: JPH2 interacts with diverse proteins depending on its subcellular location. Furthermore, JPH2's distribution and relations with its interactors are subject to remodeling in diseased state. Methods: We studied spontaneously hypertensive rats: 2-3 months (SY, healthy) and ≥ 20 months (SO, in heart failure). We used immunoprecipitation (IP) and global unbiased search (proteomics) to identify JPH2's interactors in cardiomyocytes. We used high-resolution structured illumination microscopy (SIM) followed by deconvolution and object-based segmentation in 3 dimension (OBS3D) to quantify spatial relationships between JPH2 and interactors. Results: Based on proteomics JPH2's interactors are involved in: SR/ER function, vesicular transport, sub-sarcolemma scaffold, ion transporters/channels, intercalated disc (ICD) organization and mitochondrial function. We quantified spatial relationships between JPH2 and 6 interactors representing distinct subcellular compartments: RyR2 (junctional SR), Cav1.2 (t-tubules), dystrophin (peripheral SL), caveolin-3 (caveolae), connexin-43 (ICD), and prohibitin-1 (mitochondria). In SY myocytes, JPH2 clusters are organized along the z-lines and peripheral SL, consistent with close and reciprocal association in JPH2/RyR2 and JPH2/dystrophin. In SO myocytes, JPH2 is dispersed from z-lines but stays close to peripheral SL and clusters to ICD. This leads to reduced proximity between JPH2 and Cav1.2 or RyR2, but closer proximity to connexin-43. Conclusions: In failing heart, a portion of JPH2 shifts from t-tubule/SR junctions (where CICR occurs) to ICDs (where store-operated Ca entry 'SOCE' occurs), likely contributing to dysregulation of intracellular Ca handling.

Background In the heart, slow delayed rectifier channels provide outward currents (IKs) for action potential (AP) repolarization in a region- and context-dependent manner. In diseased hearts, chronic elevation of angiotensin II (Ang II) may remodel IKs in a region-dependent manner, contributing to atrial and ventricular arrhythmias of different mechanisms. Objective The purpose of this study was to study whether/how chronic in vivo Ang II administration remodels IKs in atrial and ventricular myocytes. Methods We used the guinea pig (GP) model whose myocytes express robust IKs. GPs were implanted with minipumps containing Ang II or vehicle. Treatment continued for 4–6 weeks. We used patch clamp, immunofluorescence/confocal microscopy, and immunoblots to evaluate changes in IKs function and to explore the underlying mechanisms. Results We confirmed the pathologic state of the heart after chronic Ang II treatment. IKs density was increased in atrial myocytes but decreased in ventricular myocytes in Ang II- vs vehicle-treated animals. The former was correlated with an increase in KCNQ1/KCNE1 colocalization in myocyte periphery, whereas the latter was correlated with a decrease in KCNQ1 protein level. Interestingly, these changes in IKs were not translated into expected alterations in AP duration or plateau voltage, indicating that other currents were involved. In atrial myocytes from Ang II–treated animals, the L-type Ca channel current was increased, contributing to AP plateau elevation and AP duration prolongation. Conclusion IKs is differentially modulated by chronic in vivo Ang II administration between atrial and ventricular myocytes. Other currents remodeled by Ang II treatment also contribute to changes in action potentials.

The cardiac potassium I(Ks) current is carried by a channel complex formed from α-subunits encoded by KCNQ1 and β-subunits encoded by KCNE1. Deleterious mutations in either gene are associated with hereditary long QT syndrome. Interactions between the transmembrane domains of the α- and β-subunits determine the activation kinetics of I(Ks). A physical and functional interaction between COOH termini of the proteins has also been identified that impacts deactivation rate and voltage dependence of activation. We sought to explore the specific physical interactions between the COOH termini of the subunits that confer such control. Hydrogen/deuterium exchange coupled to mass spectrometry narrowed down the region of interaction to KCNQ1 residues 352–374 and KCNE1 residues 70–81, and provided evidence of secondary structure within these segments. Key mutations of residues in these regions tended to shift voltage dependence of activation toward more depolarizing voltages. Double-mutant cycle analysis then revealed energetic coupling between KCNQ1-I368 and KCNE1-D76 during channel activation. Our results suggest that the proximal COOH-terminal regions of KCNQ1 and KCNE1 participate in a physical and functional interaction during channel opening that is sensitive to perturbation and may explain the clustering of long QT mutations in the region. NEW & NOTEWORTHY Interacting ion channel subunits KCNQ1 and KCNE1 have received intense investigation due to their critical importance to human cardiovascular health. This work uses physical (hydrogen/deuterium exchange with mass spectrometry) and functional (double-mutant cycle analyses) studies to elucidate precise and important areas of interaction between the two proteins in an area that has eluded structural definition of the complex. It highlights the importance of pathogenic mutations in these regions.

Background K channel interacting protein 2 (KChIP2), initially cloned as Kv4 channel modulator, is a multi-tasking protein. In addition to modulating several cardiac ion channels at the plasma membrane, it can also modulate microRNA transcription inside nuclei, and interact with presenilins to modulate Ca release through RyR2 in the cytoplasm. However, the mechanism regulating its subcellular distribution is not clear. Objective We tested whether palmitoylation drives KChIP2 trafficking and distribution in cells, and whether the distribution pattern of KChIP2 in cardiac myocytes is sensitive to cellular milieu. Method We conducted imaging and biochemical experiments on palmitoylatable and unpalmitoylatable KChIP2 variants expressed in COS-7 cells and in cardiomyocytes, and on native KChIP2 in myocytes. Results In COS-7 cells, palmitoylatable KChIP2 clustered to plasma membrane, while unpalmitoylatable KChIP2 exhibited higher cytoplasmic mobility and faster nuclear entry. The same differences in distribution and mobility were observed when these KChIP2 variants were expressed in cardiac myocytes, indicating that the palmitoylation-dependent distribution and trafficking are intrinsic properties of KChIP2. Importantly, acute stress in a rat model of cardiac arrest/resuscitation induced changes in native KChIP2 resembling those of KChIP2 depalmitoylation, promoting KChIP2 nuclear entry. Conclusion The palmitoylation status of KChIP2 determines its subcellular distribution in cardiac myocytes. Stress promotes nuclear entry of KChIP2, diverting it from ion channel modulation at the plasma membrane to other functions in the nuclear compartment.

Background In a canine model of premature ventricular contraction–induced cardiomyopathy (PVC-CM), Cav1.2 is downregulated and misplaced from transverse tubules (T tubules). Junctophilin-2 (JPH-2) is also downregulated. Objectives The objectives of this study were to understand the role of JPH-2 in PVC-CM and to probe changes in other proteins involved in dyad structure and function. Methods We quantify T-tubule contents (di-8-ANEPPS fluorescence in live myocytes), examine myocyte ultrastructures (electron microscopy), probe JPH-2–interacting proteins (co-immunoprecipitation), quantify dyad and nondyad protein levels (immunoblotting), and examine subcellular distributions of dyad proteins (immunofluorescence/confocal microscopy). We also test direct JPH-2 modulation of channel function (vs indirect modulation through dyad formation) using heterologous expression. Results PVC myocytes have reduced T-tubule contents but otherwise normal ultrastructures. Among 19 proteins examined, only JPH-2, bridging integrator-1 (BIN-1), and Cav1.2 are highly downregulated in PVC hearts. However, statistical analysis indicates a general reduction in dyad protein levels when JPH-2 is downregulated. Furthermore, several dyad proteins, including Na/Ca exchanger, are missing or shifted from dyads to the peripheral surface in PVC myocytes. JPH-2 directly or indirectly interacts with Ca i -handling proteins, Cav1.2 and KCNQ1, although not BIN-1 or other scaffolding proteins tested. Expression in mammalian cells that do not have dyads confirms direct JPH-2 modulation of the L-type Ca channel current (Cav1.2/voltage-gated Ca channel β subunit 2) and slow delayed rectifier current (KCNQ1/KCNE1). Conclusion JPH-2 is more than a "dyad glue": it can modulate Ca i handling and ion channel function in the dyad region. Downregulation of JPH-2, BIN-1, and Cav1.2 plays a deterministic role in PVC-CM. Dissecting the hierarchical relationship among the three is necessary for the design of therapeutic interventions to prevent the progression of PVC-CM.

Background— KCNQ1 and KCNE1 assemble to form the slow delayed rectifier ( I Ks ) channel critical for shortening ventricular action potentials during high β-adrenergic tone. However, too much I Ks under basal conditions poses an arrhythmogenic risk. Our objective is to understand how adult ventricular myocytes regulate the I Ks amplitudes under basal conditions and in response to stress. Methods and Results— We express fluorescently tagged KCNQ1 and KCNE1 in adult ventricular myocytes and follow their biogenesis and trafficking paths. We also study the distribution patterns of native KCNQ1 and KCNE1, and their relationship to I Ks amplitudes, in chronically stressed ventricular myocytes, and use COS-7 cell expression to probe the underlying mechanism. We show that KCNQ1 and KCNE1 are both translated in the perinuclear region but traffic by different routes, independent of each other, to their separate subcellular locations. KCNQ1 mainly resides in the jSR (junctional sarcoplasmic reticulum), whereas KCNE1 resides on the cell surface. Under basal conditions, only a small portion of KCNQ1 reaches the cell surface to support the I Ks function. However, in response to chronic stress, KCNQ1 traffics from jSR to the cell surface to boost the I Ks amplitude in a process depending on Ca binding to CaM (calmodulin). Conclusions— In adult ventricular myocytes, KCNE1 maintains a stable presence on the cell surface, whereas KCNQ1 is dynamic in its localization. KCNQ1 is largely in an intracellular reservoir under basal conditions but can traffic to the cell surface and boost the I Ks amplitude in response to stress.

KCNE2 functions as an auxiliary subunit in voltage-gated K and HCN channels in the heart. Genetic variations in KCNE2 have been linked to long QT syndrome. The underlying mechanisms are not entirely clear. One of the issues is whether KCNE2 protein is expressed in ventricles. We use adenovirus-mediated genetic manipulations of adult cardiac myocytes to validate two antibodies (termed Ab1 and Ab2) for their ability to detect native KCNE2 in the heart. Ab1 faithfully detects native KCNE2 proteins in spontaneously hypertensive rat and guinea pig hearts. In both cases, KCNE2 protein is more abundant in ventricles than in atria. In both ventricular and atrial myocytes, KCNE2 protein is preferentially distributed on the cell surface. Ab1 can detect a prominent KCNE2 band in human ventricular muscle from nonfailing hearts. The band intensity is much fainter in atria and in failing ventricles. Ab2 specifically detects S98 phosphorylated KCNE2. Through exploring the functional significance of S98 phosphorylation, we uncover a novel mechanism by which KCNE2 modulates the human ether-a-go-go related gene (hERG) current amplitude: by accelerating hERG protein degradation and thus reducing the hERG protein level on the cell surface. S98 phosphorylation appears to be required for this modulation, so that S98 dephosphorylation leads to an increase in hERG/rapid delayed rectifier current amplitude. Our data confirm that KCNE2 protein is expressed in the ventricles of human and animal models. Furthermore, KCNE2 can modulate its partner channel function not only by altering channel conductance and/or gating kinetics, but also by affecting protein stability.

KCNE1 associates with KCNQ1 to increase its current amplitude and slow the activation gating process, creating the slow delayed rectifier channel that functions as a “repolarization reserve” in human heart. The transmembrane domain (TMD) of KCNE1 plays a key role in modulating KCNQ1 pore conductance and gating kinetics, and the extracellular juxtamembrane (EJM) region plays a modulatory role by interacting with the extracellular surface of KCNQ1. KCNE2 is also expressed in human heart and can associate with KCNQ1 to suppress its current amplitude and slow the deactivation gating process. KCNE1 and KCNE2 share the transmembrane topology and a high degree of sequence homology in TMD and surrounding regions. The structural basis for their distinctly different effects on KCNQ1 is not clear. To address this question, we apply cysteine (Cys) scanning mutagenesis to TMDs and EJMs of KCNE1 and KCNE2. We analyze the patterns of functional perturbation to identify high impact positions, and probe disulfide formation between engineered Cys side chains on KCNE subunits and native Cys on KCNQ1. We also use methanethiosulfonate reagents to probe the relationship between EJMs of KCNE subunits and KCNQ1. Our data suggest that the TMDs of both KCNE subunits are at about the same location but interact differently with KCNQ1. In particular, the much closer contact of KCNE2 TMD with KCNQ1, relative to that of KCNE1, is expected to impact the allosteric modulation of KCNQ1 pore conductance and may explain their differential effects on the KCNQ1 current amplitude. KCNE1 and KCNE2 also differ in the relationship between their EJMs and KCNQ1. Although the EJM of KCNE1 makes intimate contacts with KCNQ1, there appears to be a crevice between KCNQ1 and KCNE2. This putative crevice may perturb the electrical field around the voltage-sensing domain of KCNQ1, contributing to the differential effects of KCNE2 versus KCNE1 on KCNQ1 gating kinetics.

KCNE1-KCNE5 are single membrane-spanning proteins that associate with voltage-gated potassium channels to diversify their function. Other than the KCNQ1/KCNE1 complex, little is known about how KCNE proteins work. We focus on KCNE2, which associates with KCNQ1 to form K channels critical for gastric acid secretion in parietal cells. We use cysteine (Cys)-scanning mutagenesis to probe the functional role of residues along the KCNE2 transmembrane domain (TMD) in modulating KCNQ1 function. There is an alpha-helical periodicity in how Cys substitutions along the KCNE2 TMD perturb KCNQ1 pore conductance/ion selectivity. However, positions where Cys substitutions perturb KCNQ1 gating kinetics cluster to the extracellular end and cytoplasmic half of the KCNE2 TMD. This is the first systematic perturbation analysis of a KCNE TMD. We propose that the KCNE2 TMD adopts an alpha-helical secondary structure with one face making intimate contact with the KCNQ1 pore domain, while the contacts with the KCNQ1 voltage-sensing domain appear more dynamic.

Background Y81C is a new long QT-5 (LQT5)-related KCNE1 mutation, which is located in the post-transmembrane domain (post-TMD) region in close proximity to three other LQT5 mutations (S74L, D76N, and W87R). Objective We examine the effects of Y81C on the function and drug sensitivity of the slow delayed rectifier channel (I Ks ) formed by KCNE1 with pore-forming KCNQ1 subunits. We also infer a structural basis for the detrimental effects of Y81C on I Ks function. Methods Wild-type (WT) and mutant (harboring Y81C) I Ks channels are expressed in oocytes or COS-7 cells. Channel function and KCNQ1 protein expression/subcellular distribution are studied by techniques of electrophysiology, biochemistry, and immunocytochemistry. Ab initio structure predictions of KCNE1 cytoplasmic domain are performed by the Robetta server. Results Relative to WT KCNE1, Y81C reduces I Ks current amplitude and shifts the voltage range of activation to a more positive range. Y81C does not reduce whole-cell KCNQ1 protein level or interfere with KCNQ1 trafficking to cell surface. Thus, its effects are mediated by altered KCNQ1/KCNE1 interactions in cell surface channels. Importantly, Y81C potentiates the effects of an I Ks activator. Preserving the aromatic or hydroxyl side chain at position 81 (Y81F or Y81T) does not prevent the detrimental effects of Y81C. Structure predictions suggest that the post-TMD region of KCNE1 may adopt a helical secondary structure. Conclusion We propose that the post-TMD region of KCNE1 interacts with the KCNQ1 channel to modulate I Ks current amplitude and gating kinetics. Other LQT5 mutations in this region share the Y81C phenotype and probably affect the I Ks channel function by a similar mechanism.

Objectives The function of Kv4.3 (KCND3) channels, which underlie the transient outward current I to in human heart, can be modulated by several accessory subunits such as KChIP2 and KCNE1–KCNE5. Here we aimed to determine the regional expression of Kv4.3, KChIP2, and KCNE mRNAs in non-failing and failing human hearts and to investigate the functional consequences of subunit coexpression in heterologous expression systems. Methods We quantified mRNA levels for two Kv4.3 isoforms, Kv4.3-S and Kv4.3-L, and for KChIP2 as well as KCNE1–KCNE5 with real-time RT-PCR. We also studied the effects of KCNEs on Kv4.3+KChIP2 current characteristics in CHO cells with the whole-cell voltage-clamp method. Results: In non-failing hearts, low expression was found for KCNE1, KCNE3, and KCNE5, three times higher expression for KCNE2, and 60 times higher for KCNE4. Transmural gradients were detected only for KChIP2 in left and right ventricles. Compared to non-failing tissue, failing hearts showed higher expression of Kv4.3-L and KCNE1 and lower of Kv4.3-S, KChIP2, KCNE4, and KCNE5. In CHO cells, Kv4.3+KChIP2 currents were differentially modified by co-expressed KCNEs: time constants of inactivation were shorter with KCNE1 and KCNE3-5 while time-to-peak was decreased, and V 0.5 of steady-state inactivation was shifted to more negative potentials by all KCNE subunits. Importantly, KCNE2 induced a unique and prominent 'overshoot' of peak current during recovery from inactivation similar to that described for human I to while other KCNE subunits induced little (KCNE4,5) or no overshoot. Conclusions: All KCNEs are expressed in the human heart at the transcript level. Compared to I to in native human myocytes, none of the combination of KChIP2 and KCNE produced an ideal congruency in current characteristics, suggesting that additional factors contribute to the regulation of the native I to channel.

The present study was designed to characterize pharmacological, biophysical and electrophysiological properties of the recombinant human cardiac I Ks (KCNQ1/KCNE1) channels at physiological temperature. Human cardiac KCNQ1 and KCNE1 genes were cotransfected into HEK 293 cells, and a cell clone stably expressing both genes was selected. Membrane currents were recorded using a perforated patch-clamp technique. The typical I Ks was slowly activated upon depolarization voltages in HEK 293 cells stably expressing human cardiac KCNQ1 and KCNE1 genes, and the current was inhibited by I Ks blockers HMR 1556 and chromanol 293B, with 50% inhibitory concentrations (IC50s) of 83.8 nM and 9.2 μM, respectively. I Ks showed a significant temperature-dependent increase in its magnitude upon elevating bath temperature to 36°C from room temperature (21°C). The current was upregulated by the β-adrenoceptor agonist isoproterenol, and the effect was reversed by H89. In addition, I Ks was inhibited by Ba2+ in a concentration-dependent manner (IC50 = 1.4 mM). Action potential clamp revealed a “bell-shaped” time course of I Ks during the action potential, and maximal peak current was seen at the plateau of the action potential. A significant use- and frequency-dependent increase of I Ks was observed during a train of action potential clamp. These results indicate that the recombinant human cardiac I Ks stably expressed in HEK 293 cells is similar to native I Ks in drug sensitivity and regulated by Ba2+ and β-adrenoceptor via the cyclic adenosine monophosphate/protein kinase A pathway. Importantly, the current exhibits significant temperature dependence, a bell-shaped time course during action potential and prominent use- or frequency-dependent accumulation during a train of action potentials.

The hERG channel has an unusually long ‘S5–P linker’ (residues 571–613) that lines the outer mouth of the pore. Previously, we have shown that residues along this S5–P linker are critical for the fast-inactivation process and K+ selectivity of the hERG channel. Here we used several approaches to probe the structure of this S5–P linker and its interactions with other domains of the hERG channel. Circular dichroism and NMR analysis of a synthetic hERG S5–P linker peptide suggested that this linker is quite dynamic: its central region (positions 583–593) can be unstructured or helical, depending on whether it is immersed in an aqueous phase or in contact with a hydrophobic environment. Cysteine introduced into positions 583–597 of the S5–P linker can form intersubunit disulphide bonds, and at least four of them (at 584, 585, 588 and 589) can form disulphide bonds with counterparts from neighbouring subunits. We propose that the four S5–P linkers in a hERG channel can engage in dynamic conformational changes during channel gating, and interactions between S5–P linkers from neighbouring subunits contribute importantly to channel inactivation.

K-channel-interacting-proteins (KChIPs) are EF-hand-motif-bearing Ca sensors that serve diverse functions. KChIP2 is the major KChIP expressed in the heart, that can modulate key cardiac channels (Itof, Nav, CaV and IKur) by directly interacting with their pore-forming subunits (Kv4, Nav1.5, Cav1.2, and Kv1.5, respectively) on the cell surface. More recent data suggest that KChIP2 can traffic to nuclei and exert transcriptional activity. Nine KChIP2 splice variants have been identified, that share C-terminal EF-hand-motifs but diverge in N-termini. Based on N-terminal sequences, KChIP2 splice variants can be broadly divided as palmitoylation-capable and palmitoylation-incapable. Questions: (1) Does palmitoylation impact on KChIP2 distribution and function?(2) Is KChIP2 splicing pattern altered in diseased heart? Methods: (1) Tag KChIP2a and 2c (palmitoylation-capable and -incapable) with mCherry, (2) Express KChIP2 isoforms in COS-7 cells alone or with GFP-Kv4.3, (3) Track KChIP2a/c distribution and relationship with Kv4.3 with confocal microscopy, (4) Quantify Itof channel function and subcellular distribution with patch clamp and subcellular fractionation/immunoblots, (5) Quantify native KChIP2 and Kv4 proteins and Itof channel function in ventricular myocytes from young and old spontaneously hypertensive rats (SY and SO, 4-6 and 22-24 months respectively; the latter suffers from severe hypertrophy/heart failure). Results: (1) When expressed alone, KChIP2a is strong in vesicles, Golgi and plasma membrane (PM), while KChIP2c is strong in nuclei and weak in PM. Pharmacological inhibition of palmitoylation directs KChIP2a from PM to nuclei, but does not alter nuclei localization of KChIP2c. (2) Kv4.3 coexpression leads to Kv4.3/KChIP2a overlap in Golgi, post-Golgi vesicles and PM. KChIP2c and Kv4.3 overlap on PM. While both KChIP2a and KChIP2c accelerate Kv4.3 recovery from inactivation, KChIP2a but not KChIP2c increases the current amplitude. (3) Relative to SY, Itof density is markedly reduced in epicardial myocytes (Epi VMs) of SO hearts. Kv4.2 and Kv4.3 proteins are modestly or not lowered in Epi VMs. While KChIP2a protein is markedly reduced, KChIP2c protein is significantly increased. Conclusions: Reversible palmitoylation confers dynamic KChIP2a localization between PM and nuclei, while KChIP2c has a more static distribution. Chronic hypertension/hypertrophy differentially impacts the expression of KChIP2a and 2c.

Background— Mutations in KCNE2 have been linked to long-QT syndrome (LQT6), yet KCNE2 protein expression in the ventricle and its functional role in native channels are not clear. Methods and Results— We detected KCNE2 protein in human, dog, and rat ventricles in Western blot experiments. Immunocytochemistry confirmed KCNE2 protein expression in ventricular myocytes. To explore the functional role of KCNE2, we studied how its expression was altered in 2 models of cardiac pathology and whether these alterations could help explain observed changes in the function of native channels, for which KCNE2 is a putative auxiliary (β) subunit. In canine ventricle injured by coronary microembolizations, the rapid delayed rectifier current ( I Kr ) density was increased. Although the protein level of ERG ( I Kr pore-forming, α, subunit) was not altered, the KCNE2 protein level was markedly reduced. These data are consistent with the effect of heterologously expressed KCNE2 on ERG and suggest that in canine ventricle, KCNE2 may associate with ERG and suppress its current amplitude. In aging rat ventricle, the pacemaker current ( I f ) density was increased. There was a significant increase in the KCNE2 protein level, whereas changes in the α-subunit (HCN2) were not significant. These data are consistent with the effect of heterologously expressed KCNE2 on HCN2 and suggest that in aging rat ventricle, KCNE2 may associate with HCN2 and enhance its current amplitude. Conclusions— KCNE2 protein is expressed in ventricles, and it can play diverse roles in ventricular electrical activity under (patho)physiological conditions.

The hERG channel has a relatively slow activation process but an extremely fast and voltage-sensitive inactivation process. Direct measurement of hERG's gating current (Piper, D.R., A. Varghese, M.C. Sanguinetti, and M. Tristani-Firouzi. 2003. PNAS. 100:10534–10539) reveals two kinetic components of gating charge transfer that may originate from two channel domains. This study is designed to address three questions: (1) which of the six positive charges in hERG's major voltage sensor, S4, are responsible for gating charge transfer during activation, (2) whether a negative charge in the cytoplasmic half of S2 (D466) also contributes to gating charge transfer, and (3) whether S4 serves as the sole voltage sensor for hERG inactivation. We individually mutate S4's positive charges and D466 to cysteine, and examine (a) effects of mutations on the number of equivalent gating charges transferred during activation (za) and inactivation (zi), and (b) sidedness and state dependence of accessibility of introduced cysteine side chains to a membrane-impermeable thiol-modifying reagent (MTSET). Neutralizing the outer three positive charges in S4 and D466 in S2 reduces za, and cysteine side chains introduced into these positions experience state-dependent changes in MTSET accessibility. On the other hand, neutralizing the inner three positive charges in S4 does not affect za. None of the charge mutations affect zi. We propose that the scheme of gating charge transfer during hERG's activation process is similar to that described for the Shaker channel, although hERG has less gating charge in its S4 than in Shaker. Furthermore, channel domain other than S4 contributes to gating charge involved in hERG's inactivation process.

The transmembrane domains of HERG (S1–S3) contain six negative charges: three are conserved in all voltage-gated K channels (D456 and D466 in S2, D501 in S3) and three are unique to the EAG family (D411 in S1, D460 in S2, and D509 in S3). We infer the functional role of these aspartates by studying how substituting them with cysteine, one at a time, affects the channel function. D456C is not functional, suggesting that this negative charge may play a critical role in channel protein folding during biogenesis, as has been shown for its counterpart in the Shaker channel. Data from the other five functional mutants suggest that D411 can stabilize the HERG channel in the closed state, while D460 and D509 have the opposite effect. D466 and D501 both may contribute to voltage-sensing during the activation process. On the other hand, all five aspartates work in a concerted fashion in contributing to the slow deactivation process of the HERG channel. Accessibility tests of the introduced thiol groups to extracellular MTS reagents indicate that water-filled crevices penetrate deep into the HERG protein core, reaching the cytoplasmic halves of S1 and S2. At these deep locations, accessibility of 411C and 466C to the extracellular aqueous phase is voltage dependent, suggesting that conformational changes occur in S1 and S2 or the surrounding crevices during gating. Increasing extracellular [H+] accelerates HERG deactivation. This effect is suppressed by substituting the aspartates with cysteine, suggesting that protonation of these aspartates may contribute to the signaling pathway whereby external [H+] influences conformational changes in the channel's cytoplasmic domains (where deactivation takes place). There is no evidence for a metal ion binding site coordinated by negative charges in the transmembrane domains of HERG, as the one described for the EAG channel.

Peptide toxins with disulfide-stabilized structures have been used as molecular calipers to probe the outer vestibule structure of K channels. We want to apply this approach to the human ether-a-go-go-related gene (HERG) channel, whose outer vestibule is unique in structure and function among voltage-gated K channels. Our focus here is BeKm-1, a HERG-specific peptide toxin that can suppress HERG in the low nM concentration range. Although BeKm-1 shares the three-dimensional scaffold with the well-studied charybdotoxin, the two use different mechanisms in suppressing currents through their target K channels. BeKm-1 binds near, but not inside, the HERG pore, and it is possible that BeKm-1-bound HERG channels can conduct currents although with markedly altered voltage-dependence and kinetics of gating. BeKm-1 and ErgTx1 differ in three-dimensional scaffold, but the two share mechanism of action and have overlapping binding sites on the HERG channel. For both, residues in the middle of the S5-P linker (the putative 583–597 helix) and residues at the pore entrance are critical for binding, although specific contact points vary between the two. Toxin foot printing using BeKm-1 and ErgTx1 will likely provide complementary information about the unique outer vestibule structure of the HERG channel.

none 7 si Abstract: The hERG potassium channel is currently emerging as a potential target for the treatment of some forms of arrhythmias or to contrast an unintentional channel block caused by drugs. Despite its therapeutic relevance, so far only few compounds are described as able to enhance channel function by potentiating hERG currents. This gap is also related to the lack of hERG crystal structure which strongly limits the possibility to employ structure-based techniques in the search and design of novel activators. To overcome this limitation, in the present work, a ligand-based virtual screening was performed using as separate search queries two conformations of NS1643, the most deeply investigated and better characterized hERG activator. The library of compounds resulting from the virtual screening was then clustered based on recurring chemical features, and 5 hits were selected to be evaluated for their ability to enhance hERG current in vitro. Compound 3 showed a good activating effect, also displaying a mechanism of action similar to that of NS1643. Moreover, the most interesting compounds were further investigated by synthesizing in a parallel fashion some analogs, with the aim to get insights about structure-activity relationships. Giacomini, E.; Buonfiglio, R.; Masetti, M.; Wang, Y.; Tseng, G.-N.; Roberti, M.; Recanatini, M. Giacomini, E.; Buonfiglio, R.; Masetti, M.; Wang, Y.; Tseng, G.-N.; Roberti, M.; Recanatini, M.

C-type inactivation in the HERG channel is unique among voltage-gated K channels in having extremely fast kinetics and strong voltage sensitivity. This suggests that HERG may have a unique outer mouth structure (where conformational changes underlie C-type inactivation), and/or a unique communication between the outer mouth and the voltage sensor. We use cysteine-scanning mutagenesis and thiol-modifying reagents to probe the structural and functional role of the S5-P (residues 571-613) and P-S6 (residues 631-638) linkers of HERG that line the outer vestibule of the channel. Disulfide formation involving introduced cysteine side chains or modification of side chain properties at "high-impact" positions produces a common mutant phenotype: disruption of C-type inactivation, reduction of K+ selectivity, and hyperpolarizing shift in the voltage-dependence of activation. In particular, we identify 15 consecutive positions in the middle of the S5-P linker (583-597) where side chain modification has marked impact on channel function. Analysis of the degrees of mutation-induced perturbation in channel function along 583-597 reveals an alpha-helical periodicity. Furthermore, the effects of MTS modification suggest that the NH2-terminal of this segment (position 584) may be very close to the pore entrance. We propose a structural model for the outer vestibule of the HERG channel, in which the 583-597 segment forms an alpha-helix. With the NH2 terminus of this helix sitting at the edge of the pore entrance, the length of the helix (approximately 20 A) allows its other end to reach and interact with the voltage-sensing domain. Therefore, the "583-597 helix" in the S5-P linker of the HERG channel serves as a bridge of communication between the outer mouth and the voltage sensor, that may make important contribution to the unique C-type inactivation phenotype.

The goals of this study are to investigate the mechanism and site of action whereby a human ether-a-go-go-related gene (HERG)-specific scorpion peptide toxin, ErgTx, suppresses HERG current. We apply cysteine-scanning mutagenesis to the S5-P and P-S6 linkers of HERG and examine the resulting changes in ErgTx potency. Data are compared with the characteristics of charybdotoxin (ChTx, or its analogs) binding to the Shaker channel. ErgTx binds to the outer vestibule of HERG but may not physically occlude the pore. In contrast to ChTx. Shaker interaction, elevating [K](o) (from 2 to 98 mm) does not affect ErgTx potency, and through-solution electrostatic forces only play a minor role in influencing ErgTx.HERG interaction. Cysteine mutations of three positions in S5-P linker (Trp-585, Gly-590, and Ile-593) and 1 position in P-S6 linker (Pro-632) induce profound changes in ErgTx binding (DeltaDeltaG2 kcal/mol). We propose that the long S5-P linker of the HERG channel forms an amphipathic alpha-helix that, together with the P-S6 linker, forms a hydrophobic ErgTx binding site. This study paves the way for future mutant cycle analysis of interacting residues in the ErgTx.HERG complex, which, in conjunction with NMR determination of the ErgTx solution structure, will yield information about the topology of HERG's outer vestibule.

The slow delayed rectifier (IKs) channel is composed of two main components: a pore-forming KCNQ1 channel and regulatory KCNE1 subunits. Due to its slow rate of activation, IKs makes little contribution to ventricular action potential repolarization under the ‘basal’ conditions1. However, when the β-adrenergic tone is high or when other repolarizing currents are compromised (e.g. acquired LQT syndrome), IKs becomes larger and activates faster, assuming a more important role in repolarizing action potentials1. There are at least two mechanisms for the increase in IKs under stressful conditions. First, high β-adrenergic tone accelerates the heart rate. The shortened diastolic intervals allow IKs channels to accumulate in the activated state, producing an increase in IKs amplitude during the early plateau phase of action potentials2. Second, β-adrenergic stimulation leads to cAMP production and PKA activation. PKA phoshphorylation of KCNQ1 and the associated AKAP9 leads to an increase in IKs amplitude and a left shift in its voltage-dependence of activation. People carrying loss-of-function mutations in KCNQ1 (LQT1) have a higher risk for ventricular arrhythmia during exercise or under emotional stress. LQT1-related mutations in KCNQ1 can impair the IKs channel function by a number of mechanisms: (a) trafficking defects, (b) lower PIP2 binding affinity, (c) reduced responsiveness to β-adrenergic stimulation, and (d) gating kinetic defects. Some of the LQT1 mutations occur in the pore-loop region, which presumably can reduce the IKs channel conductance. The design of ‘personalized medicine’ to treat LQT1 patients needs to consider the molecular mechanisms of IKs defects. For example, an IKs activator that works by enhancing the IKs single channel conductance will not be effective if there are very few or no IKs channels on the cell surface to respond to the drug. In this issue of Heart Rhythm, David Fedida’s group reports the ‘microscopic mechanisms’ for LQT1 revealed by single channel analysis of IKs3. They studied four LQT1-related mutations (D202H, I204F, V205M, and S209F) that occur in the S3 segment of the KCNQ1 channel. S3 is the third transmembrane helix in KCNQ1’s voltage-sensing domain. It helps stabilizing the main voltage-sensor, the S4 segment, in the lipid bilayer environment. S3 also participates in the formation of a ‘gating pore’ through which the arginine residues on S4 move in response to changes in the membrane voltage, triggering the opening or closing of the activation gate. A previous report from the same group used the whole cell (macroscopic) current recording to characterize these S3 mutations2. They found that except S209F, the other S3 mutations shifted the voltage-dependence of IKs activation in the depolarizing direction, making it harder for the IKs channels to activate in the physiological plateau voltage range. The D202H mutation also accelerated IKs deactivation, thus reducing the degree of IKs accumulation in the activated state during tachycardia. S209F had the opposite phenotype: it stabilized the IKs channel in the activated state. S209F caused a loss of IKs function by reducing the channel’s ability to traffic to the cell surface. With the IKs channel defects associated with these S3 mutations well characterized at the macroscopic current level, what more information can one gain by studying these mutant channels at the single channel level? Macroscopic currents are the ensembles of stochastic opening and closing of single channels. Direct observations of channel behaviors at the single molecule level can reveal details about how the channel behaves. A good example is the gating behavior of Na channels. During membrane depolarization, macroscopic Na channel currents exhibit a fast rising phase followed by a slower decaying phase. This time course can be equally well explained by two opposite scenarios at the single channel level: fast activation followed by slow inactivation (as in the Hodgkin-Huxley formalism), or slow activation followed by fast inactivation. Single Na channel recordings supported the latter scenario4. Given the value of single channel recordings in revealing the fundamental properties of channel gating behavior, what questions about the IKs channel can be better addressed by single channel than whole cell current recordings? One of the most intriguing and important question is: how does KCNE1 slow the KCNQ1 gating transitions so dramatically? The Fedida group has taken a meticulous approach to record IKs at the single channel level, and their data have provided new insights into how the IKs channels behave5. They showed that single IKs channel first opens to sub-conductance levels and then gradually reaches the fully open level. They argued that the sub-conductance states represent partial S6 gate opening before all four S4 voltage-sensors have reached the fully activated state. This argument was supported by their kinetic modeling5. The IKs gating scheme appears to be in conflict with the IKs gating scheme proposed by another group: the S6 gate of the IKs channel can open only after all four S4 voltage-sensors have reached the activated state6. However, this difference in opinion may be explained by the different modes of IKs recording, single channel5 vs whole cell current6. In the current study, the Fedida group showed that at the single channel level, the S3 mutations displayed diverse kinetic defects in IKs gating3. D202H shortened the single channel open time and prolonged the closed times, without affecting the latency to first opening. I204F shortened the single channel open time and prolonged the latency to first opening, without affecting the closed time distribution. V205M was similar to I204F in the effects on gating kinetics. Furthermore, V205M accentuated the sub-conductance levels, preventing the IKs channel from reaching the fully open state. S209F prolonged the open time and shortened the latency to first opening, producing a marked increase in single channel open probability. Do these sophisticated single channel recordings help us understand why the S3 mutations induce such diverse gating defects? Single channel recordings alone, despite the ability to reveal fundamental channel behavior, cannot tell us how the mutations affect the channel function or how the channel works. To gain this information, a more comprehensive, ‘global’, approach is necessary, that should include structural predictions. There is no KCNQ1 or KCNQ1/KCNE1 crystal structure. Even if such crystal structures are available, their static nature will not tell us how live channels change their conformations in response to the membrane voltage, or how they open their pores to allow K+ ions through. Given the current knowledge and technologies, we suggest that the best strategy to move forward is to combine structural modeling (based on exiting Kv channel crystal structures7 and KCNE1 NMR structure8) with careful experimental validation, different methods of molecular dynamics simulations, and detailed computational analysis9. We are mindful of the many (potential) caveats in this process, e.g. the available Kv channel crystal structures may not be good templates for KCNQ1 homology modeling, and the currently achievable time scale of molecular dynamics simulations (hundreds of microseconds)10 is at least 104 fold shorter than the time scale of IKs gating. This process will be work-in-progress in the foreseeable future, during which the models need to be updated periodically as major new experimental data become available. After we have sufficient understanding of the structure and function of this important yet mysterious channel, can we begin to explain, or even predict, how KCNQ1 mutations lead to LQT1.

Background Frequent premature ventricular contractions (PVCs) are associated with increased risk of sudden cardiac death and can cause secondary cardiomyopathy. Objective We sought to determine the mechanism(s) responsible for prolonged refractory period and left ventricular (LV) dysfunction demonstrated in our canine model of PVC-induced cardiomyopathy. Methods Single myocytes were isolated from LV free wall of PVC and control canines and used for patch-clamp recording, intracellular Ca 2+ measurements, and immunocytochemistry/confocal microscopy. LV tissues adjacent to the area of myocyte isolation were used for the immunoblot quantification of protein expression. Results In the PVC group, LV ejection fraction decreased from 57.6% ± 1.5% to 30.4% ± 3.1% after ≥4 months of ventricular bigeminy. Compared to control myocytes, PVC myocytes had decreased densities of both outward (transient outward current [I to ] and inward rectifier current [I K1 ]) and inward (L-type Ca current [I CaL ]) currents, but no consistent changes in rapid or slow delayed rectifier currents. The reduction in I to , I K1 , and I CaL was accompanied by decreased protein levels of their channel subunits. The extent of reduction in I to , I K1 , and I CaL varied among PVC myocytes, creating marked heterogeneity in action potential configurations and durations. PVC myocytes showed impaired Ca-induced Ca release from the sarcoplasmic reticulum (SR), without increase in SR Ca leak or decrease in SR Ca store. This was accompanied by a decrease in dyad scaffolding protein, junctophilin-2, and loss of Cav1.2 registry with Ca-releasing channels (ryanodine receptor 2). Conclusion PVCs increase dispersion of action potential configuration/duration, a risk factor for sudden cardiac death, because of the heterogeneous reduction in I to , I K1 , and I CaL . The excitation-contraction coupling is impaired because of the decrease in I CaL and Cav1.2 misalignment with respect to ryanodine receptor 2.

The fast-inactivation process in the hERG channel can be affected by mutations in the pore or S6 domain, similar to the C-type inactivation in the Shaker channel. However, differences in the kinetics and voltage dependence of inactivation between these two channels suggest that different structural determinants may be involved. To explore this possibility, we mutated a serine in the outer mouth region of hERG (S631) to residues of different physicochemical properties and compared the resulting changes in the channel's inactivation process with those resulting from mutations of an equivalent position in the Shaker channel (T449). The most dramatic differences are seen when this position is occupied by a charged residue: S631K and S631E disrupted C-type inactivation in hERG, whereas T449K and T449E facilitate C-type inactivation in Shaker. S631K and S631E also disrupted the K selectivity of hERG pore, a change not seen in T449K or T449E of Shaker. To further study why there are such differences, we replaced S631 with cysteine. This allowed us to manipulate the properties of thiol groups at position 631 and correlate side-chain properties here with changes in channel function. S631C behaved like the wild-type channel when the thiol groups were in the reduced state. Oxidizing thiol groups with H2O2 or modifying them with MTSET or MTSES disrupted C-type inactivation and K selectivity, similar to the phenotype of S631K and S631E. The same thiol-modifying maneuvers did not affect the wild-type channel function. Our results suggest differences in the outer mouth structure between hERG and Shaker, and we propose a “molecular spring” hypothesis to explain these differences.

Mechanisms of Bupivacaine's Actions on K Channels. Introduction: We examined the effects of a nonspecific ion channel blocker, bupivacaine, on K channels encoded by hERG, rKvl.4, rKv4.3, and hKvLQTI along with hIsK. Their native counterparts in the heart are important for the function of IKr, Ito, and IKs and, thus, play an important role in repolarization. Methods and Results: To elucidate the mechanisms and sites of bupivacaine's actions, we correlated the voltage and time dependencies of drug effects with those of channel gating. We also studied the effects of altering the C-type (hERG) or N-type (rKvl.4) inactivation process or the subunit composition (hKvLQT1 with or without hIsK) on bupivacaine's actions. The results suggest that, except for hKvLQT1 co-expressed with hIsK, bupivacaine binding occurred at depolarized voltages coinciding with channel activation. With hKvLQT1 co-expressed with hIsK, bupivacaine bound preferentially at negative voltages when channels were in the closed state, and unbound at depolarized voltages when channels opened. The C-type inactivation of hERG enhanced, whereas the N-type inactivation of rKv1.4 hindered, bupivacaine's effects. Conclusion: We propose that bupivacaine's actions on these K channels can be described as a nonspecific pore blockade in the inner mouth region. However, the apparent binding affinity and voltage dependence of binding can be differentially influenced by the inactivation processes occurring at two ends of the pore (C-type inactivation at the outer end and N-type inactivation at the inner end), or by the interaction between hIsK and hKvLQT1 subunits.

It has been suggested that the cardiac slow delayed rectifier channel is formed by the association of two subunits: IsK (also called minK) and KvLQT1. N-terminal splice variants of the human KvLQT1 gene have been identified, but the functional roles of different KvLQT1 isoforms are not clear. Using the nested 5′-rapid amplification of cDNA ends technique, we obtained a truncated isoform of KvLQT1 (termed tKvLQT1) that lacks the N-terminal cytoplasmic domain and the initial one-third of the first transmembrane domain. The function of tKvLQT1 was tested by oocyte expression, alone or together with the full-length KvLQT1 or a human IsK clone (hIsK). tKvLQT1 alone did not generate functional channels. However, it suppressed the KvLQT1 current when coexpressed with the full-length isoform. It also suppressed the slow delayed rectifier current induced by hIsK, probably by competing with the KvLQT1 subunit endogenous toXenopus oocytes in coassembly with the hIsK subunit. On the other hand, tKvLQT1 did not suppress the expression of Kv1.4, Kv4.3, or hERG. Using the reverse transcription-polymerase chain reaction technique, we further show that the truncated and full-length isoforms are coexpressed in different regions of human heart. Therefore, tKvLQT1 may modulate the function of IKs in human cardiac myocytes.

To explore the role of sulfhydryl (SH) groups in the function of cardiac slow delayed rectifier channels, we tested the effects of extracellular thimerosal (TMS, a hydrophilic SH modifier) on slow delayed rectifier current (IKs) induced by human IsK (hIsK) in oocytes and on the native IKs in canine ventricular myocytes. TMS (25 or 50 microM) had similar effects on the two currents: current amplitude increased, and there was an acceleration of activation and a slowing of deactivation. These effects showed little or no reversal after washout of TMS. The effects did not depend on intracellular Ca release or protein kinase activities but could be suppressed by dithiothreitol pretreatment. According to the current model of transmembrane topology, there is no cystein in the extracellular domain of hIsK. A likely candidate for TMS modification is the SH group on another subunit in oocyte cell membrane that interacts with IsK to form a functional channel. To explore the domain of hIsK involved in the interaction, extracellular serines of hIsK were mutated to cysteines at three locations: S37C (close to the transmembrane domain), S4C (close to the NH2-terminus), and S28C (in between). S37C and S28C mutations did not affect channel properties or hIsK response to TMS. On the other hand, S4C mutation reduced current expression even when S4C cRNA was injected at a quantity 50-fold higher than that of the other three proteins. Importantly, the response to TMS was markedly reduced in S4C compared with the other three proteins. Therefore, the NH2-terminus of hIsK may be involved in hIsK interaction with the SH-bearing subunit, and this interaction modulates slow delayed rectifier channel function.

alpha1-Adrenoceptors participate in the regulation of inotropy and chronotropy in the heart. Modulation of cardiac K-channel function plays an important role in these alpha1-adrenergic functions. Studies of the mechanisms of K-channel modulation by alpha1-adrenoceptors are hampered by the coexistence of multiple receptor and channel subtypes in the heart. We therefore used a model system of coexpressing a specific receptor (human alpha1c-adrenoceptor) and a K-channel clone (hIsK, rKv1.2, or rKv1.4) in oocytes. alpha1c-Adrenoceptor stimulation caused a rapid upregulation of hIsK by elevating the intracellular Ca concentration. At least part of this effect was due to an activation of calmodulin and Ca/calmodulin-dependent protein kinase II. On the other hand, alpha1c-adrenoceptor stimulation caused a slow downregulation of rKv1.2 and rKvl.4 by activating protein kinase C. The differential modulation of K channels by alpha1c-adrenoceptors demonstrated in our experiments corroborates the complexity of alpha1-adrenergic functions in the heart. Our results indicate that the oocyte model system can be a useful approach in studying alpha1-adrenergic modulation of ion-channel function and signal transduction.

Our goals are to simultaneously determine the three-dimensional distribution patterns of KCNQ1 and KCNE1 in cardiac myocytes and to study the mechanism and functional implications for variations in KCNQ1/KCNE1 colocalization in myocytes. We monitored the distribution patterns of KCNQ1, KCNE1, and markers for subcellular compartments/organelles using immunofluorescence/confocal microscopy and confirmed the findings in ventricular myocytes by directly observing fluorescently tagged KCNQ1-GFP and KCNE1-dsRed expressed in these cells. We also monitored the effects of stress on KCNQ1-GFP and endoplasmic reticulum (ER) remodeling during live cell imaging. The data showed that 1) KCNE1 maintained a stable cell surface localization, whereas KCNQ1 exhibited variations in the cytosolic compartment (striations versus vesicles) and the degree of presence on the cell surface; 2) the degree of cell surface KCNQ1/KCNE1 colocalization was positively correlated with slow delayed rectifier (IKs) current density; 3) KCNQ1 and calnexin (an ER marker) shared a cytosolic compartment; and 4) in response to stress ([Ca(2+)]i elevation, oxidative overload, or AT1R stimulation), KCNQ1 exited the cytosolic compartment and trafficked to the cell periphery in vesicles. This was accompanied by partial ER fragmentation. We conclude that the cellular milieu regulates KCNQ1 distribution in cardiac myocytes and that stressful conditions can increase IKs by inducing KCNQ1 movement to the cell surface. This represents a hitherto unrecognized mechanism by which IKs fulfills its function as a repolarization reserve in ventricular myocytes.

The slow delayed rectifier (IKs) channel is composed of KCNQ1 (pore-forming) and KCNE1 (auxiliary) subunits, and functions as a repolarization reserve in the human heart. Design of IKs-targeting anti-arrhythmic drugs requires detailed three-dimensional structures of the KCNQ1/KCNE1 complex, a task made possible by Kv channel crystal structures (templates for KCNQ1 homology-modeling) and KCNE1 NMR structures. Our goal was to build KCNQ1/KCNE1 models and extract mechanistic information about their interactions by molecular-dynamics simulations in an explicit lipid/solvent environment. We validated our models by confirming two sets of model-generated predictions that were independent from the spatial restraints used in model-building. Detailed analysis of the molecular-dynamics trajectories revealed previously unrecognized KCNQ1/KCNE1 interactions, whose relevance in IKs channel function was confirmed by voltage-clamp experiments. Our models and analyses suggest three mechanisms by which KCNE1 slows KCNQ1 activation: by promoting S6 bending at the Pro hinge that closes the activation gate; by promoting a downward movement of gating charge on S4; and by establishing a network of electrostatic interactions with KCNQ1 on the extracellular surface that stabilizes the channel in a pre-open activated state. Our data also suggest how KCNE1 may affect the KCNQ1 pore conductance.

KCNE1 and KCNE2 are both expressed in human heart and can associate with KCNQ1. KCNE1 and KCNE2 share transmembrane topology and sequence homology, yet they differ in KCNQ1 modulation (KCNE1 slows KCNQ1 Closed-to-Open transition, while KCNE2 slows KCNQ1 Open-to-Closed transition) and pharmacology (KCNQ1/KCNE2 is 5 and 10 fold less sensitive than KCNQ1/KCNE1 to niflumic acid, IKs activator, and azimilide, IKs suppressor). Previous work has shown that the extracellular juxtamembrane (EJM) region of KCNE1 interacts with the extracellular surface of KCNQ1 to modulate gating kinetics and to form IKs activator binding sites. This prompts us to compare the EJMs of the two KCNE subunits in terms of KCNQ1 interaction. We apply cysteine (Cys) scanning mutagenesis to EJMs of KCNE1 and KCNE2 and analyze the patterns of functional perturbation when coexpressed with KCNQ1. We use methanethiosulfonate (MTS) reagents to probe the relationship between EJMs of KCNE subunits and KCNQ1. Finally we probe disulfide formation between Cys engineered into the EJMs of KCNE subunits and those engineered into extracellular surface of KCNQ1. The EJM of KCNE1 makes frequent contacts with KCNQ1, so that MTS modification of exposed Cys side chains can affect channel gating. KCNQ1/KCNE2 is largely indifferent to MTS modification of exposed Cys side chains engineered to EJM of KCNE2. However, MTS can slowly access 7 consecutive hydrophobic positions in the beginning of KCNE2 transmembrane domain, as if there is a crevice between KCNQ1 and KCNE2. Disulfide trapping experiments suggest that KCNE2 is leaning more toward KCNQ1 S2 than KCNE1. We propose that KCNE2 interferes with S2-S4 interactions during KCNQ1 Open-to-Closed transition and thus slows deactivation. This weakens sensitivity to niflumic acid and azimilide by affecting binding site directly or by an allosteric mechanism.

We examined the state-, voltage-, and time dependences of interaction between 4-AP and a mammalian A-type K channel clone (rKv1.4) expressed in Xenopus oocytes using whole-cell and single-channel recordings. 4-AP blocked rKv1.4 from the cytoplasmic side of the membrane. The development of block required channel opening. Block was potentiated by removing the fast inactivation gate of the channel (deletion mutant termed "Del A"). A short-pulse train that activated rKv1.4 without inactivation induced more block by 4-AP than a long pulse that activated and then inactivated the channel. These observations suggest that both activation and inactivation gates limit the binding of 4-AP to the channel. Unblock of 4-AP also occurred during channel opening, because unblock required depolarization and was accelerated by more frequent or longer depolarization pulses (use-dependent unblock). Analysis of the concentration dependence of rate of block development indicated that 4-AP blocked rKv1.4 with slow kinetics (at -20 mV, binding and unbinding rate constants were 3.2 mM-1 s-1 and 4.3 s-1). This was consistent with single-channel recordings: 4-AP induced little or no changes in the fast kinetics of opening and closing within bursts, but shortened the mean burst duration and, more importantly, reduced the probability of channel opening by depolarization. Depolarization might decrease the affinity of 4-AP binding site in the open channel, because stronger depolarization reduced the degree of steady-state block by 4-AP. Furthermore, after 4-AP block had been established at a depolarized holding voltage, further depolarization induced a time-dependent unblock. Our data suggest that 4-AP binds to and unbinds from open rKv1.4 channels with slow kinetics, with the binding site accessibility controlled by the channel gating apparatus and binding site affinity modulated by membrane voltage.

It has been shown for a Shaker channel (H-4) that its NH2-terminal cytoplasmic domain may form a "ball and chain" structure, with the "chain" tethering the "ball" to the channel while the "ball" capable of binding to the channel in its open state and causing inactivation. Equivalent structures have not been identified in mammalian Shaker homologues. We studied the functional role of the NH2-terminal region of a fast-inactivating mammalian K channel, RHK1 (Kv1.4), by deleting different domains in this region and examining the resultant changes in channel properties at whole cell and single channel levels. Deleting the NH2-terminal hydrophobic domain (domain A) or the subsequent positive charges (domain I) from RHK1 greatly slowed the decay of whole cell currents, suggesting the existence of a ball-like structure in RHK1 similar to that in the Shaker channel. The function of the ball appeared to be abolished by deleting domain A, while modified but maintained by deleting domain I. In the latter case, the data suggest that the positive charges needed for the function of the ball can be replaced by amino acids from a following region (domain III) that has a high positive charge density. Deleting multiple domains from the NH2 terminus of RHK1 corresponding to the chain in Shaker H-4 did not induce expected changes in channel properties that might result from a shortening of a chain. A comparison of single channel kinetics of selected mutant channels with those of the wild-type channel indicated that these deletion mutations slowed whole cell currents by prolonging burst durations and by increasing the probability of reopening during depolarization. There were no changes in single channel current amplitude or latency to first opening. In conclusion, our observations indicate that the inactivation mechanism of RHK1 is similar to that of Shaker H-4 in that a positively charged cytoplasmic domain is important for such a process. The NH2-terminal domain is not involved in channel activation or ion permeation process.

Cardiac slow delayed rectifier (I(Ks)) channel complex consists of KCNQ1 channel and KCNE1 auxiliary subunits. The extracellular juxtamembranous region of KCNE1 is an unstructured loop that contacts multiple KCNQ1 positions in a gating-state-dependent manner. Congenital arrhythmia-related mutations have been identified in the extracellular S1-S2 linker of KCNQ1. These mutations manifest abnormal phenotypes only when coexpressed with KCNE1, pointing to the importance of proper KCNQ1/KCNE1 interactions here in I(Ks) channel function. We investigate the interactions between the KCNE1 loop (positions 36-47) and KCNQ1 S1-S2 linker (positions 140-148) by means of disulfide trapping and voltage clamp techniques. During transitions among the resting-state conformations, KCNE1 positions 36-43 make contacts with KCNQ1 positions 144, 145, and 147 in a parallel fashion. During conformational changes in the activated state, KCNE1 position 40 can make contacts with all three KCNQ1 positions, while the neighboring KCNE1 positions (36, 38, 39, and 41) can make contact with KCNQ1 position 147. Furthermore, KCNQ1 positions 143 and 146 are high-impact positions that cannot tolerate cysteine substitution. To maintain the proper I(Ks) channel function, position 143 requires a small side chain with a hydroxyl group, and position 146 requires a negatively charged side chain. These data and the proposed molecular motions provide insights into the mechanisms by which mutations in the extracellular juxtamembranous region of the I(Ks) channel impair its function.

Cardiac cell swelling occurs under abnormal conditions. Currents through volume-sensitive channels, if present in heart, will affect the cardiac electrical activity. Single canine ventricular myocytes were voltage clamped under conditions that largely suppressed Na, K, and Ca channel currents and currents generated by electrogenic transport systems. Cell width and membrane conductance were monitored continuously. Swelling was induced by increasing the osmolarity of the pipette solution or by decreasing the osmolarity of the external solution. During cell swelling, the cell widened and membrane conductance increased. This increase in membrane conductance was sensitive to Cl channel blockers and to external Cl removal, suggesting that a major component was provided by a Cl channel. The current-voltage relationship of the swelling-induced current displayed an outward rectification, with an average zero-current voltage of -60 mV. The activation of the swelling-induced current did not seem to depend on external or internal Ca and was not sensitive to a protein kinase inhibitor (H-8). Shape-altering agents chlorpromazine decreased while dipyridamole and trinitrophenol increased the membrane conductance without osmotic perturbations, suggesting that changes in tension in the cell membrane may play a role in opening and closing of the swelling-induced channels.

The cardiac I(Ks) current is involved in action potential repolarization, where its primary function is to limit action potential prolongation during sympathetic stimulation. The I(Ks) channel is mainly composed of K(V)7.1 ion channels associated with KCNE1 auxiliary subunits. The availability of KCNE1 solution structure by nuclear magnetic resonance spectroscopy in conjunction with biochemical assays addressing K(V)7.1-KCNE1 residue interactions has provided new insights into the structural basis for K(V)7.1 modulation by KCNE1. Recent evidence further suggests that KCNE2 may associate with the K(V)7.1-KCNE1 channel complex and modulate its current amplitude. Here we review recent studies in this area and discuss potential roles for multiple KCNE(x) subunits in I(Ks) generation and modulation as well as the clinical relevance of the new information.

Cardiac slow delayed rectifier (IKs) channel is composed of KCNQ1 (pore-forming) and KCNE1 (auxiliary) subunits. Although KCNE1 is an obligate IKs component that confers the uniquely slow gating kinetics, KCNE2 is also expressed in human heart. In vitro experiments suggest that KCNE2 can associate with the KCNQ1-KCNE1 complex to suppress the current amplitude without altering the slow gating kinetics. Our goal here is to test the role of KCNE2 in cardiac IKs channel function. Pulse-chase experiments in COS-7 cells show that there is a KCNE1 turnover in the KCNQ1-KCNE1 complex, supporting the possibility that KCNE1 in the IKs channel complex can be substituted by KCNE2 when the latter is available. Biotinylation experiments in COS-7 cells show that although KCNE1 relies on KCNQ1 coassembly for more efficient cell surface expression, KCNE2 can independently traffic to the cell surface, thus becoming available for substituting KCNE1 in the IKs channel complex. Injecting vesicles carrying KCNE1 or KCNE2 into KCNQ1-expressing oocytes leads to KCNQ1 modulation in the same manner as KCNQ1+KCNEx (where x = 1 or 2) cRNA coinjection. Thus, free KCNEx peptides delivered to the cell membrane can associate with existing KCNQ1 channels to modulate their function. Finally, adenovirus-mediated KCNE2 expression in adult guinea pig ventricular myocytes exhibited colocalization with native KCNQ1 protein and reduces the native IKs current density. We propose that in cardiac myocytes the IKs current amplitude is under dynamic control by the availability of KCNE2 subunits in the cell membrane.

We studied the effects of changing the intracellular Ca level ([Ca]i) and activating protein kinase C on the cardiac T and L Ca channels in single canine ventricular and Purkinje cells. Lowering [Ca]i increased the L current but decreased the T current, whereas elevating [Ca]i caused opposite changes. In ventricular cells, isoproterenol (1 microM) increased the amplitude of not only the L but also the T currents; the latter effect probably was secondary to a rise in [Ca]i following the augmentation of the L current. 12-O-tetradecanoylphorbol-13-acetate (TPA, 100 nM) decreased the T current but first increased and then decreased the L current. The TPA effects on the T and L currents were not mimicked by a phorbol ester that does not activate PKC (4 alpha-phorbol 12,13-didecanoate) and were prevented by a protein kinase inhibitor (H-8), confirming the involvement of PKC activity in these modulatory processes. We conclude that elevating [Ca]i and activating PKC have opposite effects on the T and L Ca currents in canine cardiac cells. The extent and time course of the changes in these two intracellular messengers will most likely determine the effects on the two cardiac Ca currents of neurotransmitters and hormones that can activate phospholipase C.