Beta-subunit-eliminated eHAP expression (BeHAPe) cells reveal subunit regulation of the cardiac voltage-gated sodium channel

Minard, Annabel Y.; Clark, Colin J.; Ahern, Christopher A.; Piper, Robert C.

doi:10.1016/j.jbc.2023.105132

Cited by 2 publications

(7 citation statements)

References 63 publications

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“…We reasoned that directing diploidization in a controlled manner could provide an easy way to construct diploids of defined genotypes, such as heterozygous cells, or to introduce multiple GOIs into one cell line. In addition, we found that spontaneous diploids made from haploids, are larger and have different morphological characteristics that lend themselves better to certain experiments (3). These morphological differences were examined in detail by another group (17).…”

Section: Resultsmentioning

confidence: 87%

“…In our previous studies we optimized transient transfection protocols for eHAP cells, both by reverse-transfection with lipofectamine and by electroporation (see methods for protocols) (3). However, at best these methods achieved a transfection efficiency of 30%.…”

Section: Resultsmentioning

confidence: 99%

“…Previously, we introduced an FRT site into the eHAP genome by transducing eHAP cells with the pQCXIP FRT GFP-Neo vector by lentivirus (3). In this vector a CMV promoter drives expression of eGFP fused to a neomycin resistance gene (eGFP-Neo R ).…”

Section: Resultsmentioning

confidence: 99%

“…This includes to complement a phenotype by re-expression of a gene-of-interest (GOI), or to monitor a phenotype with reporter constructs. During our studies, we noted conventional strategies achieve poor transgene expression in eHAP cells (3). Notably, the efficiency of transient transfection was low, and stably integrated transgenes underwent silencing.…”

Section: Introductionmentioning

confidence: 82%

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InsulatedpiggyBacand FRT vectors for engineering transgenic homozygous and heterozygous eHAP cells

Minard,

Winistorfer,

Piper

2024

Preprint

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Transgene expression in eHAP cells, a haploid cell line popularly used to generate gene knockouts, is difficult owing to its low transfection efficiency and propensity for silencing integrated transgenes. To simplify transgene expression, we engineered insulated integrating plasmids that sustain high levels of transgene expression in eHAP cells, and that can be used in other cell lines. These vectors are compatible with FLP-FRT and piggyBac integration, they flank a gene-of interest bilaterally with tandem cHS4 core insulators, and co-express nuclear-localized blue fluorescent protein for identification of high expressing cells. We further demonstrate that transgenic haploid eHAP cells can be fused to form transgenic heterozygous diploid cells. This method creates diploid cells carrying the transgenic material of the haploid progenitors and could also be used to create heterozygous cells of defined genotypes. These tools expand the repertoire of experiments that can be performed in eHAP cells and other cultured cells.

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Section: Resultsmentioning

confidence: 87%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 82%

See 2 more Smart Citations

InsulatedpiggyBacand FRT vectors for engineering transgenic homozygous and heterozygous eHAP cells

Minard,

Winistorfer,

Piper

2024

Preprint

View full text Add to dashboard Cite

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“…Peak I Na density (pA/pF) previous studies reported conflicting results regarding the impact of Na v β1 on the function of Na v 1.5. While some groups detected changes of the peak current density and/or differences in the channel kinetics and gating properties, others like us did not observe any Na v β1-mediated changes of I Na [24][25][26][27][28][29][30][31]. In particular, cardiomyocytes of Scn1b null mice exhibited increased macroscopic I Na without any differences in voltage dependence or kinetics of the current [29].…”

Section: Activation Inactivationmentioning

confidence: 91%

The dispensability of 14-3-3 proteins for the regulation of human cardiac sodium channel Nav1.5

Iamshanova,

Hämmerli,

Ramaye

et al. 2024

PLoS ONE

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Background 14-3-3 proteins are ubiquitous proteins that play a role in cardiac physiology (e.g., metabolism, development, and cell cycle). Furthermore, 14-3-3 proteins were proposed to regulate the electrical function of the heart by interacting with several cardiac ion channels, including the voltage-gated sodium channel Nav1.5. Given the many cardiac arrhythmias associated with Nav1.5 dysfunction, understanding its regulation by the protein partners is crucial. Aims In this study, we aimed to investigate the role of 14-3-3 proteins in the regulation of the human cardiac sodium channel Nav1.5. Methods and results Amongst the seven 14-3-3 isoforms, only 14-3-3η (encoded by YWHAH gene) weakly co-immunoprecipitated with Nav1.5 when heterologously co-expressed in tsA201 cells. Total and cell surface expression of Nav1.5 was however not modified by 14-3-3η overexpression or inhibition with difopein, and 14-3-3η did not affect physical interaction between Nav1.5 α-α subunits. The current-voltage relationship and the amplitude of Nav1.5-mediated sodium peak current density were also not changed. Conclusions Our findings illustrate that the direct implication of 14-3-3 proteins in regulating Nav1.5 is not evident in a transformed human kidney cell line tsA201.

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Beta-subunit-eliminated eHAP expression (BeHAPe) cells reveal subunit regulation of the cardiac voltage-gated sodium channel

Cited by 2 publications

References 63 publications

InsulatedpiggyBacand FRT vectors for engineering transgenic homozygous and heterozygous eHAP cells

InsulatedpiggyBacand FRT vectors for engineering transgenic homozygous and heterozygous eHAP cells

The dispensability of 14-3-3 proteins for the regulation of human cardiac sodium channel Nav1.5

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