Functional Expression of Ion Channels in Mammalian Systems

Clare, Jeffrey J.

doi:10.1002/9783527610754.tr06

Cited by 6 publications

(3 citation statements)

References 97 publications

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“…Purification of the protein from guinea pig heart also revealed that this protein was glutathionylated in response to oxidative stress, and the associated constitutive activity was thought to contribute to the pathology of the heart disease (Tang et al, 2011), but progress on characterizing the regulation of this class of transporters has been slow. One of the reasons for the relatively slow progress in studies on these channels has been the toxicity and instability of their cDNAs when expressed in E. coli (Clare, 2008). The yeast homolog is also toxic in E. coli, and thus in this work with Cch1p, the different mutants and constructs were created using the more arduous in vivo gap repair strategy which involves homologous recombination in yeast (Iida et al, 2004;Vu et al, 2009).…”

Section: Discussionmentioning

confidence: 99%

Redox regulation of the yeast voltage-gated Ca2+ channel homolog Cch1p by glutathionylation of specific cysteine residues

Chandel

Bachhawat

2017

Journal of Cell Science

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Cch1p, the yeast homolog of the pore-forming subunit α 1 of the mammalian voltage-gated Ca 2+ channel (VGCC), is located on the plasma membrane and mediates the redox-dependent influx of Ca 2+ . Cch1p is known to undergo both rapid activation (after oxidative stress and or a change to high pH) and slow activation (after ER stress and mating pheromone activation), but the mechanism of activation is not known. We demonstrate here that both the fast activation (exposure to pH 8-8.5 or treatment with H 2 O 2 ) and the slow activation (treatment with tunicamycin or α-factor) are mediated through a common redoxdependent mechanism. Furthermore, through mutational analysis of all 18 exposed cysteine residues in the Cch1p protein, we show that the four mutants C587A, C606A, C636A and C642A, which are clustered together in a common cytoplasmic loop region, were functionally defective for both fast and slow activations, and also showed reduced glutathionylation. These four cysteine residues are also conserved across phyla, suggesting a conserved mechanism of activation. Investigations into the enzymes involved in the activation reveal that the yeast glutathione S-transferase Gtt1p is involved in the glutathionylation of Cch1p, while the thioredoxin Trx2p plays a role in the Cch1p deglutathionylation.

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Section: Discussionmentioning

confidence: 99%

Redox regulation of the yeast voltage-gated Ca2+ channel homolog Cch1p by glutathionylation of specific cysteine residues

Chandel

Bachhawat

2017

Journal of Cell Science

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“…High-level expression of ion channels can also be difficult because the overexpression can sometimes result in toxicity to cells, precluding the generation of stable cell lines. 56 It is possible to address some of these concerns by using methods that enhance detection of ion channels or that result in transient, regulated expression of the ion channels. To improve detection of ion channels, a wide range of strategies have been used to introduce epitope tags into ion channel sequences to facilitate detection using methods such as flow cytometry.…”

Section: Antigen and Immunogen Formatsmentioning

confidence: 99%

Discovery of Functional Antibodies Targeting Ion Channels

2015

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Ion channels play critical roles in physiology and disease by modulation of cellular functions such as electrical excitability, secretion, cell migration, and gene transcription. Ion channels represent an important target class for drug discovery that has been largely addressed, to date, using small-molecule approaches. A significant opportunity exists to target these channels with antibodies and alternative formats of biologics. Antibodies display high specificity and affinity for their target antigen, and they have the potential to target ion channels very selectively. Nevertheless, isolating antibodies to this target class is challenging due to the difficulties in expression and purification of ion channels in a format suitable for antibody drug discovery in addition to the complexity of screening for function. In this article, we will review the current state of ion channel biologics discovery and the progress that has been made. We will also highlight the challenges in isolating functional antibodies to these targets and how these challenges may be addressed. Finally, we also illustrate successful approaches to isolating functional monoclonal antibodies targeting ion channels by way of a number of case studies drawn from recent publications.

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“…Use of inducible promoters in stable cell lines can sometimes overcome this difficulty, with transduction by BacMam to induce short‐term expression of a gene product being an equivalent approach. Indeed, BacMam viruses have allowed the creation of robust cell‐based assays for two ion channels, K ATP (Pfohl et al, ) and NMDA‐NR2b glutamate receptor (Clare, ), for which stable cell line generation was unsuccessful. Transient expression with these viruses has also made possible the analysis of several membrane transporters (Hassan et al, ).…”

Section: Commentarymentioning

confidence: 99%

Pharmacological Applications of Baculovirus‐Mediated Protein Expression in Mammalian Cells

Condreay

Watson

2010

CP Pharmacology

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The development of cell-based assays for cellular receptors, ion channels, and transporters requires the delivery and expression of transgenes. Viral-mediated gene delivery is a particularly attractive approach for this purpose because of its efficiency and potential to deliver genes to a wide variety of cell types. Recombinant baculoviruses, long used to deliver genes to insect cells for overexpression, also effectively transfer genes to mammalian cells. The only required modification to the virus for this purpose is the addition of transgene expression cassettes controlled by mammalian cell-active promoters. These so-called BacMam viruses are useful for developing mammalian cell-based assays for investigating the function of recombinant proteins and for assessing the action of pharmacological modulators of their function. The use of such viruses is gaining popularity because of the ease of optimizing assay conditions, the ability to deliver multiple gene products, and of their flexibility in terms of host cells and levels of transgene expression. BacMam-mediated assays may be used for studying a wide variety of target proteins and assay technologies. Described in this unit is an example of BacMam-mediated gene delivery to configure a cell-based assay for pharmacological assessment of a G protein-coupled receptor. A protocol is also provided describing the use of a GFP-expressing BacMam to assess the susceptibility of new cell lines to transduction by the virus.

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Functional Expression of Ion Channels in Mammalian Systems

Cited by 6 publications

References 97 publications

Redox regulation of the yeast voltage-gated Ca2+ channel homolog Cch1p by glutathionylation of specific cysteine residues

Redox regulation of the yeast voltage-gated Ca2+ channel homolog Cch1p by glutathionylation of specific cysteine residues

Discovery of Functional Antibodies Targeting Ion Channels

Pharmacological Applications of Baculovirus‐Mediated Protein Expression in Mammalian Cells

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