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 Optimization of insect odorant receptor trafficking and functional expression via transient transfection in HEK293 cells

2019 ◽  
Author(s):  
Fabio Miazzi ◽  
Carolin Hoyer ◽  
Silke Sachse ◽  
Markus Knaden ◽  
Dieter Wicher ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Odorant Receptor ◽  
Functional Expression ◽  
Transient Transfection ◽  
Hek293 Cells ◽  
Odorant Receptors ◽  
Expression Systems ◽  
Heterologous Expression Systems ◽  
High Throughput Imaging

AbstractInsect odorant receptors show a limited functional expression in various heterologous expression systems including insect and mammalian cells. This may be in part due to the absence of key components driving the release of these proteins from the endoplasmic reticulum and directing them to the plasma membrane. In order to mitigate this problem we took advantage of small export signals within the human HCN1 and Rhodopsin that have been shown to promote protein release from the endoplasmic reticulum and the trafficking of post-Golgi vesicles, respectively. Moreover, we designed a new vector based on a bidirectional expression cassette to drive the functional expression of the insect odorant receptor co-receptor (Orco) and an odor-binding odorant receptor, simultaneously. We show that this new method can be used to reliably express insect odorant receptors in HEK293 cells via transient transfection and that is highly suitable for downstream applications using automated and high-throughput imaging platforms.

scholarly journals Optimization of Insect Odorant Receptor Trafficking and Functional Expression Via Transient Transfection in HEK293 Cells

2019 ◽  
Vol 44 (9) ◽  
pp. 673-682 ◽  
Author(s):  
Fabio Miazzi ◽  
Carolin Hoyer ◽  
Silke Sachse ◽  
Markus Knaden ◽  
Dieter Wicher ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Odorant Receptor ◽  
Functional Expression ◽  
Transient Transfection ◽  
Hek293 Cells ◽  
Odorant Receptors ◽  
Expression Systems ◽  
Heterologous Expression Systems ◽  
High Throughput Imaging

Abstract Insect odorant receptors (ORs) show a limited functional expression in various heterologous expression systems including insect and mammalian cells. This may be in part due to the absence of key components driving the release of these proteins from the endoplasmic reticulum and directing them to the plasma membrane. In order to mitigate this problem, we took advantage of small export signals within the human HCN1 and Rhodopsin that have been shown to promote protein release from the endoplasmic reticulum and the trafficking of post-Golgi vesicles, respectively. Moreover, we designed a new vector based on a bidirectional expression cassette to drive the functional expression of the insect odorant receptor coreceptor (Orco) and an odor-binding OR, simultaneously. We show that this new method can be used to reliably express insect ORs in HEK293 cells via transient transfection and that is highly suitable for downstream applications using automated and high-throughput imaging platforms.

Abstract 1070: An Unconventional Vesicular Transport Pathway Regulates the Cell Surface Expression of hERG K + Channels

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Heather A Underkofler ◽  
Sadguna Balijepalli ◽  
Brooke M Moungey ◽  
Jessica K Slind ◽  
Jabe M Best ◽  
...  
Keyword(s):  
Cell Surface ◽  
Surface Membrane ◽  
Vesicular Transport ◽  
Surface Expression ◽  
Small Gtpases ◽  
Dominant Negative ◽  
Cell Surface Expression ◽  
Missense Mutations ◽  
Transport Pathway ◽  
Er Export

Approximately 35– 45% of patients that are genotype positive for congenital Long QT Syndrome (LQT) have mutations in the human Ether-a-go-go Related Gene ( hERG ). The purpose of this study was to elucidate the mechanisms that regulate ER export and cell surface expression of hERG channel protein, because these steps are impaired for ~90% of LQT-linked hERG missense mutations. The small GTPases Sar1 and Arf1 regulate the conventional vesicular transport (trafficking) for the ER export of proteins to the Golgi apparatus (Golgi). We generated dominant negative (DN) mutations for Sar1 and Arf1, and co-expressed these DN GTPases with hERG in HEK 293 cells. The trafficking of hERG through the Golgi can be visualized biochemically using Western blot analysis, because additional glycosylation of hERG in the Golgi (Golgi processing) increases the MW of hERG protein from 135kDa to 155kDa. Co-expression of hERG and DN-Sar1 inhibited Golgi processing, decreased hERG current (I hERG ) by 85% compared to control (n≥8 cells per group, p<0.05), and decreased the staining of hERG protein at the cell surface, while co-expression of hERG and DN-Arf1 showed no significant effect on Golgi processing or I hERG . This lack of an effect by DN-Arf1 was selective for hERG as it efficiently blocked the transport of previously reported proteins. Rab11 GTPases regulate the trafficking of proteins from endosomal compartments to the cell surface membrane and/or the Golgi. Rab11a is ubiquitously expressed, whereas Rab11b is expressed primarily in brain and heart. Co-expression of DN-Rab11a did not alter Golgi processing of hERG but reduced I hERG by 51% compared to control (n≥8 cells per group, p<0.05), whereas co-expression of DN-Rab11b inhibited Golgi processing of hERG and reduced I hERG by 79% compared to control (n=8 cells per group, p<0.05). Thus, Rab11a appears to regulate the trafficking of hERG to the cell surface after processing in the Golgi, whereas Rab11b regulates the trafficking of hERG prior to processing in the Golgi. These data suggest that hERG does not traffic via the conventional pathway from the ER to the Golgi, but rather in an unconventional pathway from the ER to endosomal compartments prior to Rab11b-mediated transport to the Golgi and subsequent delivery to the cell membrane.

scholarly journals Long QT Syndrome Type 2: Emerging Strategies for Correcting Class 2 KCNH2 (hERG) Mutations and Identifying New Patients

Biomolecules ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 1144
Author(s):  
Makoto Ono ◽  
Don E. Burgess ◽  
Elizabeth A. Schroder ◽  
Claude S. Elayi ◽  
Corey L. Anderson ◽  
...  
Keyword(s):  
Cell Surface ◽  
Long Qt Syndrome ◽  
Molecular Mechanisms ◽  
Surface Membrane ◽  
Long Qt ◽  
Cell Surface Membrane ◽  
Channel Protein ◽  
Channel Proteins ◽  
Qt Syndrome

Significant advances in our understanding of the molecular mechanisms that cause congenital long QT syndrome (LQTS) have been made. A wide variety of experimental approaches, including heterologous expression of mutant ion channel proteins and the use of inducible pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from LQTS patients offer insights into etiology and new therapeutic strategies. This review briefly discusses the major molecular mechanisms underlying LQTS type 2 (LQT2), which is caused by loss-of-function (LOF) mutations in the KCNH2 gene (also known as the human ether-à-go-go-related gene or hERG). Almost half of suspected LQT2-causing mutations are missense mutations, and functional studies suggest that about 90% of these mutations disrupt the intracellular transport, or trafficking, of the KCNH2-encoded Kv11.1 channel protein to the cell surface membrane. In this review, we discuss emerging strategies that improve the trafficking and functional expression of trafficking-deficient LQT2 Kv11.1 channel proteins to the cell surface membrane and how new insights into the structure of the Kv11.1 channel protein will lead to computational approaches that identify which KCNH2 missense variants confer a high-risk for LQT2.

scholarly journals TMEM16F activation by Ca2+triggers plasmalemma expansion and directs PD-1 trafficking

2018 ◽  
Author(s):  
Christopher Bricogne ◽  
Michael Fine ◽  
Pedro M. Pereira ◽  
Julia Sung ◽  
Maha Tijani ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Surface ◽  
T Cell Activation ◽  
Membrane Trafficking ◽  
Large Scale ◽  
Cell Activation ◽  
Transmembrane Domain ◽  
Surface Membrane ◽  
Hek293 Cells

AbstractTMEM16F, an ion channel gated by high cytoplasmic Ca2+, is required for cell surface phosphatidylserine exposure during platelet aggregation and T cell activation. Here we demonstrate in Jurkat T cells and HEK293 cells that TMEM16F activation triggers large-scale surface membrane expansion in parallel with lipid scrambling. Following TMEM16F mediated scrambling and surface expansion, cells undergo extensive membrane shedding. The membrane compartment that expands the cell surface does not involve endoplasmic reticulum or acidified lysosomes. Surprisingly, T cells lacking TMEM16F expression not only fail to expand surface membrane, but instead rapidly internalize membrane via massive endocytosis (MEND). The T cell co-receptor PD-1 is selectively shed when TMEM16F triggers membrane expansion, while it is selectively internalized in the absence of TMEM16F. Its participation in this trafficking is determined by its single transmembrane domain. Thus, we establish a fundamental role for TMEM16F as a regulator of Ca2+-activated membrane trafficking.

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 Missense Mutations in the Melanocortin 2 Receptor Accessory Protein That Lead to Late Onset Familial Glucocorticoid Deficiency Type 2

2010 ◽  
Vol 31 (3) ◽  
pp. 396-397
Author(s):  
C. R. Hughes ◽  
T. T. Chung ◽  
A. M. Habeb ◽  
F. Kelestimur ◽  
A. J. L. Clark ◽  
...  
Keyword(s):  
Dose Response ◽  
Late Onset ◽  
Hek293 Cells ◽  
Accessory Protein ◽  
Missense Mutations ◽  
Wild Type ◽  
Response Curves ◽  
Patient Reported ◽  
Glucocorticoid Deficiency

ABSTRACT Background Familial glucocorticoid deficiency (FGD) is an autosomal recessive disorder characterized by isolated glucocorticoid deficiency. Mutations in the ACTH receptor [melanocortin 2 receptor (MC2R)] or the MC2R accessory protein (MRAP) cause FGD types 1 and 2, respectively. Typically, type 2 patients present early (median age, 0.1 yr), and no patient reported to date has presented after 1.6 yr. Aim The aim of this study was to investigate the cause of disease in two families with late-onset FGD. Patients The proband in family 1 was diagnosed at age 4 yr. Family review revealed two older siblings with undiagnosed FGD. One sibling was well, whereas the second had cerebral palsy secondary to hypoglycemic seizures. The proband in family 2 was diagnosed at age 18 yr with symptoms of fatigue, weight loss, and depression. Methods The coding exons of MC2R and MRAP were sequenced. ACTH dose-response curves were generated for MC2R when transfected with wild-type or mutant MRAP constructs using HEK293 cells. MC2R trafficking with both mutant MRAPs was investigated using immunocytochemistry. Results MRAP gene analysis identified two novel homozygous missense mutations, c.175T&gt;G (pY59D) in family 1 and c.76T&gt;C (p.V26A) in family 2. In vitro analysis showed that the Y59D mutant had significant impairment of cAMP generation, and both mutants caused a shift in the dose-response curve to the right when compared to wild type. Immunocytochemistry showed normal trafficking of MC2R when transfected with both mutant MRAPs, indicating a probable signaling defect. Conclusion These results indicate that missense MRAP mutations present with a variable phenotype of ACTH resistance and can present late in life.

scholarly journals A 49-residue sequence motif in the C terminus of Nav1.9 regulates trafficking of the channel to the plasma membrane

2019 ◽  
Vol 295 (4) ◽  
pp. 1077-1090 ◽  
Author(s):  
Daria V. Sizova ◽  
Jianying Huang ◽  
Elizabeth J. Akin ◽  
Mark Estacion ◽  
Carolina Gomis-Perez ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Recombinant Expression ◽  
Critical Role ◽  
Functional Expression ◽  
Hek293 Cells ◽  
Sequence Motif ◽  
Functional Studies ◽  
C Terminus ◽  
Mechanistic Basis ◽  
Heterologous Expression Systems

Genetic and functional studies have confirmed an important role for the voltage-gated sodium channel Nav1.9 in human pain disorders. However, low functional expression of Nav1.9 in heterologous systems (e.g. in human embryonic kidney 293 (HEK293) cells) has hampered studies of its biophysical and pharmacological properties and the development of high-throughput assays for drug development targeting this channel. The mechanistic basis for the low level of Nav1.9 currents in heterologous expression systems is not understood. Here, we implemented a multidisciplinary approach to investigate the mechanisms that govern functional Nav1.9 expression. Recombinant expression of a series of Nav1.9-Nav1.7 C-terminal chimeras in HEK293 cells identified a 49-amino-acid-long motif in the C terminus of the two channels that regulates expression levels of these chimeras. We confirmed the critical role of this motif in the context of a full-length channel chimera, Nav1.9-Ct49aaNav1.7, which displayed significantly increased current density in HEK293 cells while largely retaining the characteristic Nav1.9-gating properties. High-resolution live microscopy indicated that the newly identified C-terminal motif dramatically increases the number of channels on the plasma membrane of HEK293 cells. Molecular modeling results suggested that this motif is exposed on the cytoplasmic face of the folded C terminus, where it might interact with other channel partners. These findings reveal that a 49-residue-long motif in Nav1.9 regulates channel trafficking to the plasma membrane.

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.

Levels of plasma membrane expression in progressive and benign mutations of the bile salt export pump (Bsep/Abcb11) correlate with severity of cholestatic diseases

2007 ◽  
Vol 293 (5) ◽  
pp. C1709-C1716 ◽  
Author(s):  
Ping Lam ◽  
Claire L. Pearson ◽  
Carol J. Soroka ◽  
Shuhua Xu ◽  
Albert Mennone ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Bile Salt ◽  
Intrahepatic Cholestasis ◽  
Surface Protein ◽  
Salt Transport ◽  
Hek293 Cells ◽  
Cell Protein ◽  
Membrane Expression

Human BSEP (ABCB11) mutations are the molecular basis for at least three clinical forms of liver disease, progressive familial intrahepatic cholestasis type 2 (PFIC2), benign recurrent intrahepatic cholestasis type 2 (BRIC2), and intrahepatic cholestasis of pregnancy (ICP). To better understand the pathobiology of these disease phenotypes, we hypothesized that different mutations may cause significant differences in protein defects. Therefore we compared the effect of two PFIC2 mutations (D482G, E297G) with two BRIC2 mutations (A570T and R1050C) and one ICP mutation (N591S) with regard to the subcellular localization, maturation, and function of the rat Bsep protein. Bile salt transport was retained in all but the E297G mutant. Mutant proteins were expressed at reduced levels on the plasma membrane of transfected HEK293 cells compared with wild-type (WT) Bsep in the following order: WT > N591S > R1050C ∼ A570T ∼ E297G >> D482G. Total cell protein and surface protein expression were reduced to the same extent, suggesting that trafficking of these mutants to the plasma membrane is not impaired. All Bsep mutants accumulate in perinuclear aggresome-like structures in the presence of the proteasome inhibitor MG-132, suggesting that mutations are associated with protein instability and ubiquitin-dependent degradation. Reduced temperature, sodium butyrate, and sodium 4-phenylbutyrate enhanced the expression of the mature and cell surface D482G protein in HEK293 cells. These results suggest that the clinical phenotypes of PFIC2, BRIC2, and ICP may directly correlate with the amount of mature protein that is expressed at the cell surface and that strategies to stabilize cell surface mutant protein may be therapeutic.

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