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.

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.

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 Inhibitors of Mammalian Aquaporin Water Channels

2019 ◽  
Vol 20 (7) ◽  
pp. 1589 ◽  
Author(s):  
Mohammed Abir-Awan ◽  
Philip Kitchen ◽  
Mootaz Salman ◽  
Matthew Conner ◽  
Alex Conner ◽  
...  
Keyword(s):  
Structural Biology ◽  
Mammalian Cells ◽  
Tumor Metastasis ◽  
Traumatic Injury ◽  
Water Channel ◽  
Experimental System ◽  
Expression Systems ◽  
Novel Treatments ◽  
Health And Disease ◽  
Heterologous Expression Systems

Aquaporins (AQPs) are water channel proteins that are essential to life, being expressed in all kingdoms. In humans, there are 13 AQPs, at least one of which is found in every organ system. The structural biology of the AQP family is well-established and many functions for AQPs have been reported in health and disease. AQP expression is linked to numerous pathologies including tumor metastasis, fluid dysregulation, and traumatic injury. The targeted modulation of AQPs therefore presents an opportunity to develop novel treatments for diverse conditions. Various techniques such as video microscopy, light scattering and fluorescence quenching have been used to test putative AQP inhibitors in both AQP-expressing mammalian cells and heterologous expression systems. The inherent variability within these methods has caused discrepancy and many molecules that are inhibitory in one experimental system (such as tetraethylammonium, acetazolamide, and anti-epileptic drugs) have no activity in others. Some heavy metal ions (that would not be suitable for therapeutic use) and the compound, TGN-020, have been shown to inhibit some AQPs. Clinical trials for neuromyelitis optica treatments using anti-AQP4 IgG are in progress. However, these antibodies have no effect on water transport. More research to standardize high-throughput assays is required to identify AQP modulators for which there is an urgent and unmet clinical need.

Isozymes of the Na-K-ATPase: heterogeneity in structure, diversity in function

1998 ◽  
Vol 275 (5) ◽  
pp. F633-F650 ◽  
Author(s):  
Gustavo Blanco ◽  
Robert W. Mercer
Keyword(s):  
Mammalian Cells ◽  
Molecular Heterogeneity ◽  
Kinetic Characteristics ◽  
Expression Systems ◽  
Na Pump ◽  
Physiological Relevance ◽  
Intracellular Messengers ◽  
Heterologous Expression Systems ◽  
The Individual ◽  
Structure Diversity

The Na-K-ATPase is characterized by a complex molecular heterogeneity that results from the expression and differential association of multiple isoforms of both its α- and β-subunits. At present, as many as four different α-polypeptides (α1, α2, α3, and α4) and three distinct β-isoforms (β1, β2, and β3) have been identified in mammalian cells. The stringent constraints on the structure of the Na pump isozymes during evolution and their tissue-specific and developmental pattern of expression suggests that the different Na-K-ATPases have evolved distinct properties to respond to cellular requirements. This review focuses on the functional properties, regulation, and possible physiological relevance of the Na pump isozymes. The coexistence of multiple α- and β-isoforms in most cells has hindered the understanding of the roles of the individual polypeptides. The use of heterologous expression systems has helped circumvent this problem. The kinetic characteristics of different Na-K-ATPase isozymes to the activating cations (Na+ and K+), the substrate ATP, and the inhibitors Ca2+ and ouabain demonstrate that each isoform has distinct properties. In addition, intracellular messengers differentially regulate the activity of the individual Na-K-ATPase isozymes. Thus the regulation of specific Na pump isozymes gives cells the ability to precisely coordinate Na-K-ATPase activity to their physiological requirements.

scholarly journals A variant of the bovine noradrenaline transporter reveals the importance of the C-terminal region for correct targeting to the membrane and functional expression

1998 ◽  
Vol 330 (2) ◽  
pp. 909-914 ◽  
Author(s):  
D. Lucille BURTON ◽  
G. Andree KIPPENBERGER ◽  
Bettina LINGEN ◽  
Michael BRÜSS ◽  
Heinz BÖNISCH ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Functional Expression ◽  
Hek293 Cells ◽  
Cdna Clones ◽  
Similar Treatment ◽  
Noradrenaline Transporter ◽  
C Terminus ◽  
Golgi Region ◽  
Uptake Activity ◽  
High Level

We have characterized a cDNA clone which encodes a variant (bNAT2) of the bovine noradrenaline transporter. This cDNA differs from the previously identified bovine noradrenaline transporter (bNAT1) in the sequence encoding part of the cytoplasmic-facing C-terminus and the 3ʹ-untranslated region. The bNAT1 and bNAT2 cDNA clones are encoded by a 5.8 and 3.6 kb mRNA species respectively. The bNAT1 and bNAT2 proteins, which are identical apart from their C-terminal 31 and 18 residues, were stably expressed in HEK293 cells. Cells expressing bNAT1 showed a high level of desipramine-sensitive [3H]noradrenaline uptake activity, whereas no activity was present in bNAT2 cells. The bNAT1 and bNAT2 proteins were present as major 80 and 50 kDa species respectively. Cells expressing bNAT1 showed strong immunostaining of the plasma membrane, whereas bNAT2 was present in the endoplasmic reticulum/Golgi region. Treatment of membrane samples from bNAT1 cells with peptide N-glycosidase F resulted in the formation of a predominantly 50 kDa species, but little effect was observed after similar treatment of bNAT2 cell membranes. These results indicate that bNAT2 is retained in the endoplasmic reticulum and that the glycosylation of this variant differs from that of bNAT1. The characterization of bNAT2 and its comparison with bNAT1 highlight the importance of the cytoplasmic-facing C-terminus for the intracellular trafficking of neurotransmitter transporters.

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 Functional Expression of Odorant Receptors of the Zebrafish Danio rerio and of the Nematode C. elegans in HEK293 Cells

1997 ◽  
Vol 22 (4) ◽  
pp. 467-476 ◽  
Author(s):  
Ch. Wellerdieck ◽  
M. Oles ◽  
L. Pott ◽  
S. Korsching ◽  
G. Gisselmann ◽  
...  
Keyword(s):  
Danio Rerio ◽  
Functional Expression ◽  
Hek293 Cells ◽  
Odorant Receptors ◽  
C Elegans

scholarly journals SAP97 interacts with Kv1.5 in heterologous expression systems

2001 ◽  
Vol 281 (6) ◽  
pp. H2575-H2584 ◽  
Author(s):  
Mitsunobu Murata ◽  
Peter D. Buckett ◽  
Jun Zhou ◽  
Michael Brunner ◽  
Eduardo Folco ◽  
...  
Keyword(s):  
Cardiac Myocytes ◽  
Xenopus Oocytes ◽  
Pdz Domain ◽  
Transient Transfection ◽  
Amino Acid Residues ◽  
Expression Systems ◽  
Terminal Amino Acid ◽  
Terminal Amino ◽  
Heterologous Expression Systems ◽  
Functional Analyses

PDZ domain-containing proteins such as SAP97 and ZO-1 have been implicated in the targeting and clustering of ion channels. We have explored the interactions of these polypeptides with a cardiac voltage-gated potassium channel. Immunocytochemistry in cardiac myocytes revealed colocalization of SAP97 and Kv1.5, both at the intercalated disks and the lateral membranes. Transient transfection experiments in COS-7 cells revealed that SAP97 and Kv1.5 polypeptides formed perinuclear clustered complexes that could be coimmunoprecipitated. Mutation of the three COOH-terminal amino acid residues of Kv1.5 (T-D-L to A-A-A) abolished these interactions. Whereas in most COS-7 cells the SAP97-Kv1.5 complexes were retained in the ER, functional analyses in Xenopus oocytes showed that Kv1.5-encoded outward potassium currents were augmented by coexpression with SAP97. By contrast, cotransfected ZO-1 and Kv1.5 polypeptides in COS-7 cells could not be coprecipitated nor did the coinjection of ZO-1 augment the Kv1.5-encoded currents in oocytes. Collectively, our results suggest that SAP97 may play an important role in the modulation of Kv1.5 channel function in cardiac myocytes.

scholarly journals The state of the art of odorant receptor deorphanization: A report from the orphanage

2014 ◽  
Vol 143 (5) ◽  
pp. 527-542 ◽  
Author(s):  
Zita Peterlin ◽  
Stuart Firestein ◽  
Matthew E. Rogers
Keyword(s):  
State Of The Art ◽  
Odorant Receptor ◽  
Functional Expression ◽  
The State ◽  
Odorant Receptors ◽  
Volatile Chemicals ◽  
Tiered Approach ◽  
The World ◽  
Initial Discovery

The odorant receptors (ORs) provide our main gateway to sensing the world of volatile chemicals. This involves a complex encoding process in which multiple ORs, each of which detects its own set of odorants, work as an ensemble to produce a distributed activation code that is presumably unique to each odorant. One marked challenge to decoding the olfactory code is OR deorphanization, the identification of a set of activating odorants for a particular receptor. Here, we survey various methods used to try to express defined ORs of interest. We also suggest strategies for selecting odorants for test panels to evaluate the functional expression of an OR. Integrating these tools, while retaining awareness of their idiosyncratic limitations, can provide a multi-tiered approach to OR deorphanization, spanning the initial discovery of a ligand to vetting that ligand in a physiologically relevant setting.

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