scholarly journals Pharmacological correction of long QT-linked mutations in KCNH2 (hERG) increases the trafficking of Kv11.1 channels stored in the transitional endoplasmic reticulum

2013 ◽  
Vol 305 (9) ◽  
pp. C919-C930 ◽  
Author(s):  
Jennifer L. Smith ◽  
Allison R. Reloj ◽  
Parvathi S. Nataraj ◽  
Daniel C. Bartos ◽  
Elizabeth A. Schroder ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Therapeutic Potential ◽  
Functional Expression ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Delayed Rectifier ◽  
Protein Synthesis Inhibitor ◽  
Missense Mutations ◽  
Long Qt ◽  
Pharmacological Correction

KCNH2 encodes Kv11.1 and underlies the rapidly activating delayed rectifier K+ current ( IKr) in the heart. Loss-of-function KCNH2 mutations cause the type 2 long QT syndrome (LQT2), and most LQT2-linked missense mutations inhibit the trafficking of Kv11.1 channels. Drugs that bind to Kv11.1 and block IKr (e.g., E-4031) can act as pharmacological chaperones to increase the trafficking and functional expression for most LQT2 channels (pharmacological correction). We previously showed that LQT2 channels are selectively stored in a microtubule-dependent compartment within the endoplasmic reticulum (ER). We tested the hypothesis that pharmacological correction promotes the trafficking of LQT2 channels stored in this compartment. Confocal analyses of cells expressing the trafficking-deficient LQT2 channel G601S showed that the microtubule-dependent ER compartment is the transitional ER. Experiments with E-4031 and the protein synthesis inhibitor cycloheximide suggested that pharmacological correction promotes the trafficking of G601S stored in this compartment. Treating cells in E-4031 or ranolazine (a drug that blocks IKr and has a short half-life) for 30 min was sufficient to cause pharmacological correction. Moreover, the increased functional expression of G601S persisted 4–5 h after drug washout. Coexpression studies with a dominant-negative form of Rab11B, a small GTPase that regulates Kv11.1 trafficking, prevented the pharmacological correction of G601S trafficking from the transitional ER. These data suggest that pharmacological correction quickly increases the trafficking of LQT2 channels stored in the transitional ER via a Rab11B-dependent pathway, and we conclude that the pharmacological chaperone activity of drugs like ranolazine might have therapeutic potential.

scholarly journals Trafficking-deficient hERG K+ channels linked to long QT syndrome are regulated by a microtubule-dependent quality control compartment in the ER

2011 ◽  
Vol 301 (1) ◽  
pp. C75-C85 ◽  
Author(s):  
Jennifer L. Smith ◽  
Christie M. McBride ◽  
Parvathi S. Nataraj ◽  
Daniel C. Bartos ◽  
Craig T. January ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
Long Qt Syndrome ◽  
Functional Expression ◽  
Delayed Rectifier ◽  
Temperature Sensitive ◽  
Missense Mutations ◽  
Long Qt ◽  
Delayed Rectifier Current ◽  
Golgi Compartment ◽  
Qt Syndrome

The human ether-a-go-go related gene ( hERG) encodes the voltage-gated K+ channel that underlies the rapidly activating delayed-rectifier current in cardiac myocytes. hERG is synthesized in the endoplasmic reticulum (ER) as an “immature” N-linked glycoprotein and is terminally glycosylated in the Golgi apparatus. Most hERG missense mutations linked to long QT syndrome type 2 (LQT2) reduce the terminal glycosylation and functional expression. We tested the hypothesis that a distinct pre-Golgi compartment negatively regulates the trafficking of some LQT2 mutations to the Golgi apparatus. We found that treating cells in nocodazole, a microtubule depolymerizing agent, altered the subcellular localization, functional expression, and glycosylation of the LQT2 mutation G601S-hERG differently from wild-type hERG (WT-hERG). G601S-hERG quickly redistributed to peripheral compartments that partially colocalized with KDEL (Lys-Asp-Glu-Leu) chaperones but not calnexin, Sec31, or the ER golgi intermediate compartment (ERGIC). Treating cells in E-4031, a drug that increases the functional expression of G601S-hERG, prevented the accumulation of G601S-hERG to the peripheral compartments and increased G601S-hERG colocalization with the ERGIC. Coexpressing the temperature-sensitive mutant G protein from vesicular stomatitis virus, a mutant N-linked glycoprotein that is retained in the ER, showed it was not restricted to the same peripheral compartments as G601S-hERG at nonpermissive temperatures. We conclude that the trafficking of G601S-hERG is negatively regulated by a microtubule-dependent compartment within the ER. Identifying mechanisms that prevent the sorting or promote the release of LQT2 channels from this compartment may represent a novel therapeutic strategy for LQT2.

scholarly journals Balance Between Rapid Delayed Rectifier K + Current and Late Na + Current on Ventricular Repolarization

2020 ◽  
Vol 13 (4) ◽  
Author(s):  
Bence Hegyi ◽  
Ye Chen-Izu ◽  
Leighton T. Izu ◽  
Sridharan Rajamani ◽  
Luiz Belardinelli ◽  
...  
Keyword(s):  
Heart Failure ◽  
Long Qt Syndrome ◽  
Therapeutic Potential ◽  
Heart Diseases ◽  
Ionic Currents ◽  
Close Correlation ◽  
Delayed Rectifier ◽  
Long Qt ◽  
Comparable Amount ◽  
Qt Syndrome

Background: Rapid delayed rectifier K + current (I Kr ) and late Na + current (I NaL ) significantly shape the cardiac action potential (AP). Changes in their magnitudes can cause either long or short QT syndromes associated with malignant ventricular arrhythmias and sudden cardiac death. Methods: Physiological self AP-clamp was used to measure I NaL and I Kr during the AP in rabbit and porcine ventricular cardiomyocytes to test our hypothesis that the balance between I Kr and I NaL affects repolarization stability in health and disease conditions. Results: We found comparable amount of net charge carried by I Kr and I NaL during the physiological AP, suggesting that outward K + current via I Kr and inward Na + current via I NaL are in balance during physiological repolarization. Remarkably, I Kr and I NaL integrals in each control myocyte were highly correlated in both healthy rabbit and pig myocytes, despite high overall cell-to-cell variability. This close correlation was lost in heart failure myocytes from both species. Pretreatment with E-4031 to block I Kr (mimicking long QT syndrome 2) or with sea anemone toxin II to impair Na + channel inactivation (mimicking long QT syndrome 3) prolonged AP duration (APD); however, using GS-967 to inhibit I NaL sufficiently restored APD to control in both cases. Importantly, I NaL inhibition significantly reduced the beat-to-beat and short-term variabilities of APD. Moreover, I NaL inhibition also restored APD and repolarization stability in heart failure. Conversely, pretreatment with GS-967 shortened APD (mimicking short QT syndrome), and E-4031 reverted APD shortening. Furthermore, the amplitude of AP alternans occurring at high pacing frequency was decreased by I NaL inhibition, increased by I Kr inhibition, and restored by combined I NaL and I Kr inhibitions. Conclusions: Our data demonstrate that I Kr and I NaL are counterbalancing currents during the physiological ventricular AP and their integrals covary in individual myocytes. Targeting these ionic currents to normalize their balance may have significant therapeutic potential in heart diseases with repolarization abnormalities. Visual Overview: A visual overview is available for this article.

scholarly journals Epilepsy and neurobehavioral abnormalities in mice with a KCNB1 pathogenic variant that alters conducting and non-conducting functions of KV2.1

2019 ◽  
Author(s):  
Nicole A. Hawkins ◽  
Sunita N. Misra ◽  
Manuel Jurado ◽  
Nicholas C. Vierra ◽  
Kimberly Nguyen ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
De Novo ◽  
Pathogenic Variant ◽  
Repetitive Behaviors ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Delayed Rectifier ◽  
Eeg Abnormalities ◽  
Surface Ecg

AbstractDevelopmental and epileptic encephalopathies (DEE) are a group of severe epilepsies that usually present with intractable seizures, developmental delay and are at a higher risk for premature mortality. Numerous genes have been identified as a monogenic cause of DEE, including KCNB1. The voltage-gated potassium channel KV2.1, encoded by KCNB1, is primarily responsible for delayed rectifier potassium currents that are important regulators of excitability in electrically excitable cells, including neurons and cardiomyocytes. The de novo pathogenic variant KCNB1-p.G379R was identified in an infant with epileptic spasms, atonic, focal and tonic-clonic seizures that were refractory to treatment with standard antiepileptic drugs. Previous work demonstrated deficits in potassium conductance, but did not assess non-conducting functions. To determine if the G379R variant affected clustering at endoplasmic reticulum-plasma membrane junctions KV2.1-G379R was expressed in HEK293T cells. KV2.1-G379R expression did not induce formation of endoplasmic reticulum-plasma membrane junctions, and co-expression of KV2.1-G379R with KV2.1-WT lowered induction of these structures relative to KV2.1-WT alone, suggesting a dominant negative effect. To model this variant in vivo, we introduced Kcnb1G379R into mice using CRISPR/Cas9 genome editing. We characterized neurological and neurobehavioral phenotypes of Kcnb1G379R/+ (Kcnb1R/+) and Kcnb1G379R/G379R (Kcnb1R/R) mice, and screened for cardiac abnormalities. Immunohistochemistry studies on brains from Kcnb1+/+ (WT), Kcnb1R/+ and Kcnb1R/R mice revealed genotype-dependent differences in the levels and subcellular localization of KV2.1, with reduced plasma membrane expression of the KV2.1-G379R protein, consistent with in vitro data. Kcnb1R/+ and Kcnb1R/R mice displayed profound hyperactivity, repetitive behaviors, impulsivity and reduced anxiety. In addition, both Kcnb1R/+ and Kcnb1R/R mice exhibited abnormal interictal EEG abnormalities, including isolated spike and slow waves. Spontaneous seizure events were observed in Kcnb1R/R mice during exposure to novel environments and/or handling, while both Kcnb1R/+ and Kcnb1R/R mutants were more susceptible to induced seizures. Kcnb1R/+ and Kcnb1R/R mice exhibited prolonged rate-corrected QT interval on surface ECG recording. Overall, the Kcnb1G379R mice recapitulate many features observed in individuals with DEE due to pathogenic variants in KCNB1. This new mouse model of KCNB1 associated DEE will be valuable for improving the understanding of the underlying pathophysiology and will provide a valuable tool for the development of therapies to treat this pharmacoresistant DEE.

Retention in the Endoplasmic Reticulum as a Mechanism of Dominant-negative Current Suppression in Human Long QT Syndrome

2000 ◽  
Vol 32 (12) ◽  
pp. 2327-2337 ◽  
Author(s):  
Eckhard Ficker ◽  
Adrienne T Dennis ◽  
Carlos A Obejero-Paz ◽  
Pasqualina Castaldo ◽  
Maurizio Taglialatela ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Long Qt Syndrome ◽  
Dominant Negative ◽  
Long Qt ◽  
Negative Current ◽  
Qt Syndrome

Mechanisms of I Ks suppression in LQT1 mutants

2000 ◽  
Vol 279 (6) ◽  
pp. H3003-H3011 ◽  
Author(s):  
Laura Bianchi ◽  
Silvia G. Priori ◽  
Carlo Napolitano ◽  
Krystyna A. Surewicz ◽  
Adrienne T. Dennis ◽  
...  
Keyword(s):  
Cell Surface ◽  
Dominant Negative ◽  
Delayed Rectifier ◽  
Poor Correlation ◽  
Type I ◽  
Missense Mutations ◽  
Wild Type ◽  
Negative Effects ◽  
Clinical Phenotypes ◽  
First Time

Mutations in the cardiac potassium ion channel gene KCNQ1 (voltage-gated K+ channel subtype KvLQT1) cause LQT1, the most common type of hereditary long Q-T syndrome. KvLQT1 mutations prolong Q-T by reducing the repolarizing cardiac current [slow delayed rectifier K+ current ( I Ks )], but, for reasons that are not well understood, the clinical phenotypes may vary considerably even for carriers of the same mutation, perhaps explaining the mode of inheritance. At present, only currents expressed by LQT1 mutants have been studied, and it is unknown whether abnormal subunits are transported to the cell surface. Here, we have examined for the first time trafficking of KvLQT1 mutations and correlated the results with the I Ks currents that were expressed. Two missense mutations, S225L and A300T, produced abnormal currents, and two others, Y281C and Y315C, produced no currents. However, all four KvLQT1 mutations were detected at the cell surface. S225L, Y281C, and Y315C produced dominant negative effects on wild-type I Ks current, whereas the mutant with the mildest dysfunction, A300T, did not. We examined trafficking of a severe insertion deletion mutant Δ544 and detected this protein at the cell surface as well. We compared the cellular and clinical phenotypes and found a poor correlation for the severely dysfunctional mutations.

Compound heterozygosity for mutations Asp611→Tyr in KCNQ1 and Asp609→Gly in KCNH2 associated with severe long QT syndrome

2005 ◽  
Vol 108 (2) ◽  
pp. 143-150 ◽  
Author(s):  
Masato YAMAGUCHI ◽  
Masami SHIMIZU ◽  
Hidekazu INO ◽  
Hidenobu TERAI ◽  
Kenshi HAYASHI ◽  
...  
Keyword(s):  
Long Qt Syndrome ◽  
Clinical Manifestations ◽  
Torsades De Pointes ◽  
Dominant Negative ◽  
Missense Mutations ◽  
Wild Type ◽  
Long Qt ◽  
Mild Phenotype ◽  
Electrophysiological Properties ◽  
Qt Syndrome

LQTS (long QT syndrome) is an inherited cardiac disorder characterized by prolongation of QT interval, torsades de pointes and sudden death. We have identified two heterozygous missense mutations in the KCNQ1 and KCNH2 (also known as HERG) genes [Asp611→Tyr (D611Y) in KCNQ1 and Asp609→Gly (D609G) in KCNH2] in a 2-year-old boy with LQTS. The aim of the present study was to characterize the contributions of the mutations in the KCNQ1 and KCNH2 genes relative to the clinical manifestations and electrophysiological properties of LQTS. Six of 11 carriers of D611Y in KCNQ1 had long QT intervals. D609G in KCNH2 was detected only in the proband. Studies on the electrophysiological alterations due to the two missense mutations revealed that the D611Y mutation in KCNQ1 did not show a significant suppression of the currents compared with wild-type, but the time constants of current activation in the mutants were increased compared with that in the wild-type. In contrast, the D609G mutation in KCNH2 showed a dominant-negative suppression. Our results suggest that the mild phenotype produced by the D611Y mutation in KCNQ1 became more serious by addition of the D609G mutation in KCNH2 in the proband.

scholarly journals Properties of WT and mutant hERG K+ channels expressed in neonatal mouse cardiomyocytes

2010 ◽  
Vol 298 (6) ◽  
pp. H1842-H1849 ◽  
Author(s):  
Eric C. Lin ◽  
Katherine M. Holzem ◽  
Blake D. Anson ◽  
Brooke M. Moungey ◽  
Sadguna Y. Balijepalli ◽  
...  
Keyword(s):  
Protein Trafficking ◽  
Neonatal Mouse ◽  
Delayed Rectifier ◽  
Missense Mutations ◽  
Expression Systems ◽  
Hek 293 ◽  
Deactivation Kinetics ◽  
Pharmacological Correction ◽  
Cell Expression ◽  
Herg Channels

Mutations in human ether-a-go-go-related gene 1 ( hERG) are linked to long QT syndrome type 2 (LQT2). hERG encodes the pore-forming α-subunits that coassemble to form rapidly activating delayed rectifier K+ current in the heart. LQT2-linked missense mutations have been extensively studied in noncardiac heterologous expression systems, where biogenic (protein trafficking) and biophysical (gating and permeation) abnormalities have been postulated to underlie the loss-of-function phenotype associated with LQT2 channels. Little is known about the properties of LQT2-linked hERG channel proteins in native cardiomyocyte systems. In this study, we expressed wild-type (WT) hERG and three LQT2-linked mutations in neonatal mouse cardiomyocytes and studied their electrophysiological and biochemical properties. Compared with WT hERG channels, the LQT2 missense mutations G601S and N470D hERG exhibited altered protein trafficking and underwent pharmacological correction, and N470D hERG channels gated at more negative voltages. The ΔY475 hERG deletion mutation trafficked similar to WT hERG channels, gated at more negative voltages, and had rapid deactivation kinetics, and these properties were confirmed in both neonatal mouse cardiomyocyte and human embryonic kidney (HEK)-293 cell expression systems. Differences between the cardiomyocytes and HEK-293 cell expression systems were that hERG current densities were reduced 10-fold and deactivation kinetics were accelerated 1.5- to 2-fold in neonatal mouse cardiomyocytes. An important finding of this work is that pharmacological correction of trafficking-deficient LQT2 mutations, as a potential innovative approach to therapy, is possible in native cardiac tissue.

Abstract 1071: Expression and Pharmacological Correction of a LQT2 Mutant (hERG) Channel in Native Cardiomyocytes

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Katherine M Holzem ◽  
Ravi C Balijepalli ◽  
Eric C Lin ◽  
Jing Wang ◽  
Timothy J Kamp ◽  
...  
Keyword(s):  
Cell Surface ◽  
Surface Membrane ◽  
Functional Expression ◽  
Hek293 Cells ◽  
Herg Channel ◽  
Missense Mutations ◽  
Expression Systems ◽  
Pharmacological Correction ◽  
Heterologous Expression Systems

Mutations within the human Ether-a-go-go Related Gene (hERG) encoding for the hERG K + channel can lead to long QT syndrome type 2 (LQT2). Previous investigations have been performed using non-cardiac heterologous expression systems, and in mammalian systems most LQT2-linked missense mutations are trafficking deficient, with the channel protein failing to express at the cell surface membrane. Furthermore, the majority of these mutations can be induced to traffic to the cell surface by incubation in drugs (pharmacological correction), which has suggested that pharmacological correction might be developed as a clinical therapy. To further establish the feasibility of pharmacological correction in the heart, we demonstrated it in cardiomyocytes. We studied expression of WT-hERG and the trafficking-deficient LQT2-linked N470D-hERG mutation in HEK293 cells (using Superfect) or cardiomyocytes (using electroporation) isolated from 2-day-old mice. Using the patch clamp method to measure I hERG , peak tail current amplitude for N470D-hERG was reduced by 74% compared to WT-hERG in HEK293 cells (n=7–13 cells, p<0.05) and 94% in cardiomyocytes (n=6 –9 cells, p<0.05). HEK293 cells or cardiomyocytes expressing N470D-hERG were incubated with or without E-4031 (10 μM for 24 hrs) and E-4031 was washed out prior to recording I hERG . Incubation in E-4031 increased I hERG by 447% in HEK293 cells (n=9 –13 cells, p<0.05) and 1300% in cardomyocytes (n=6 –9 cells, p<0.05). We also measured the activation properties for I hERG . The potentials for half-maximal activation (V 1/2 ) of WT-hERG expressed in HEK293 cells or cardiomyocytes were −7.5±1.8 and −12.9±1.1mV with slope factors of 6.7±0.2 and 6.2±0.3mV/ e -foldΔ, respectively (n=5 cells each). The V 1/2 for N470D-hERG after pharmacological correction in E-4031 was -38.1±2.4mV for HEK293 cells and -50.6±5.3mV for cardiomyocytes with slope factors of 7.1±0.3 and 9.9±1.1 mV/ e -foldΔ, respectively (n=5 cells each). Our results demonstrate functional expression of WT- and N470D-hERG in a native cardiomyocyte system and that hERG channel properties are similar between expression systems. These are the first data to show pharmacological correction in cardiomyocytes.

scholarly journals Coptisine Attenuates Diabetes—Associated Endothelial Dysfunction through Inhibition of Endoplasmic Reticulum Stress and Oxidative Stress

Molecules ◽  
2021 ◽  
Vol 26 (14) ◽  
pp. 4210
Author(s):  
Yan Zhou ◽  
Chunxiu Zhou ◽  
Xutao Zhang ◽  
Chi Teng Vong ◽  
Yitao Wang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Risk Factors ◽  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Vascular Function ◽  
Inflammatory Responses ◽  
Therapeutic Potential ◽  
Ex Vivo ◽  
Diabetic Mice ◽  
And Oxidative Stress

Coptisine is the major bioactive protoberberine alkaloid found in Rhizoma Coptidis. Coptisine reduces inflammatory responses and improves glucose tolerance; nevertheless, whether coptisine has vasoprotective effect in diabetes is not fully characterized. Conduit arteries including aortas and carotid arteries were obtained from male C57BL/6J mice for ex vivo treatment with risk factors (high glucose or tunicamycin) and coptisine. Some arterial rings were obtained from diabetic mice, which were induced by high-fat diet (45% kcal% fat) feeding for 6 weeks combined with a low-dose intraperitoneal injection of streptozotocin (120 mg/kg). Functional studies showed that coptisine protected endothelium-dependent relaxation in aortas against risk factors and from diabetic mice. Coptisine increased phosphorylations of AMPK and eNOS and downregulated the endoplasmic reticulum (ER) stress markers as determined by Western blotting. Coptisine elevates NO bioavailability and decreases reactive oxygen species level. The results indicate that coptisine improves vascular function in diabetes through suppression of ER stress and oxidative stress, implying the therapeutic potential of coptisine to treat diabetic vasculopathy.

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