Biochemical, single-channel, whole-cell patch clamp, and pharmacological analyses of endogenous TRPM4 channels in HEK293 cells

Amarouch, Mohamed Yassine; Syam, Ninda; Abriel, Hugues

doi:10.1016/j.neulet.2013.02.011

Cited by 36 publications

(43 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…9-phenanthrol was applied to the bath solution 10 min after glutamate application when near-maximal glutamate-induced currents were obtained. Within 2 minutes of application of 9-phenanthrol (100 μM), the glutamate-induced currents were inhibited by 82.8 ± 2.9%, which is similar to the reported percentage of TRPM4 inhibition by 9-phenanthrol [[37]]. In addition, in the presence of 9-phenanthrol, glutamate failed to activate TRPM4b-mediated currents in HT-22 cells (see Additional file 3: Figure S3B).…”

Section: Resultssupporting

confidence: 83%

Depletion of 14-3-3γ reduces the surface expression of Transient Receptor Potential Melastatin 4b (TRPM4b) Channels and attenuates TRPM4b-mediated glutamate-induced neuronal cell death

et al. 2014

View full text Add to dashboard Cite

BackgroundTRPM4 channels are Ca2+-activated nonselective cation channels which are deeply involved in physiological and pathological conditions. However, their trafficking mechanism and binding partners are still elusive.ResultsWe have found the 14-3-3γ as a binding partner for TRPM4b using its N-terminal fragment from the yeast-two hybrid screening. Ser88 at the N-terminus of TRPM4b is critical for 14-3-3γ binding by showing GST pull-down and co-immunoprecipitation. Heterologous overexpression of 14-3-3γ in HEK293T cells increased TRPM4b expression on the plasma membrane which was measured by whole-cell recordings and cell surface biotinylation experiment. Surface expression of TRPM4b was greatly reduced by short hairpin RNA (shRNA) against 14-3-3γ. Next, endogenous TRPM4b-mediated currents were electrophysiologically characterized by application of glutamate and 9-phenanthrol, a TRPM4b specific antagonist in HT-22 cells which originated from mouse hippocampal neurons. Glutamate-induced TRPM4b currents were significantly attenuated by shRNAs against 14-3-3γ or TRPM4b in these cells. Finally, glutamate-induced cell death was greatly prevented by treatment of 9-phenanthrol or 14-3-3γ shRNA.ConclusionThese results showed that the cell surface expression of TRPM4 channels is mediated by 14-3-3γ binding, and the specific inhibition of this trafficking process can be a potential therapeutic target for glutamate-induced neuronal cell death.

show abstract

Section: Resultssupporting

confidence: 83%

Depletion of 14-3-3γ reduces the surface expression of Transient Receptor Potential Melastatin 4b (TRPM4b) Channels and attenuates TRPM4b-mediated glutamate-induced neuronal cell death

et al. 2014

View full text Add to dashboard Cite

show abstract

“…First, NAADPinduced currents display the same biophysical features, i.e. linear I-V relationship, E rev close to 0 mV, permeability to the monovalent cations Na + and Cs + , but not to Ca 2+ , as TRPM4-mediated currents both in naïve cells [64][65][66] and in heterologous expression systems [67]. Second, the pharmacological profile of NAADP-elicited currents is fully compatible with that of TRPM4 [46,47].…”

Section: Discussionmentioning

confidence: 69%

A novel Ca2+-mediated cross-talk between endoplasmic reticulum and acidic organelles: Implications for NAADP-dependent Ca2+ signalling

et al. 2015

View full text Add to dashboard Cite

“…Nowadays, the expression of voltage-gated ion channels and transmembrane receptors from cultured HEK293 cells for electrophysiological recording is an ubiquitous research strategy. HEK293 cells possess low expression of native channels and are similar to early developing neurons for the expression of voltage-and non-voltage-gated channels [Thomas and Smart, 2005;Amarouch et al, 2013;Ooi et al, 2016]. Besides, HEK293 cells also exhibit high transfection efficiency with high fidelity of the recombinant proteins produced.…”

Section: Hek293 Cells For Electrophysiologymentioning

confidence: 99%

“…Many protocols are available for the expression of ion channels in HEK293 cells for whole-cell patch clamp recordings [Senatore et al, 2011;Ooi et al, 2016]. Over the years, numerous ion channels or mem- , and K + channels, which play a significant role in new drug discoveries [Amarouch et al, 2013;Yu et al, 2016].…”

Section: Hek293 Cells For Electrophysiologymentioning

confidence: 99%

“…(d) HEK293 cells are a feasible tool for biotherapeutics production, because of their high growth rate, high cell density in shake flasks, efficient and flexible metabolism, and genetic manipulation [Román et al, 2016]. (e) HEK293 cells are sensitive human embryonic cells with characteristics similar to early differentiating neurons, which are more susceptible to environmental factors in vitro and commonly used as a model for new drug discovery and toxicity testing [Eglen and Reisine, 2011;Amarouch et al, 2013;Mesnage et al, 2013].…”

mentioning

confidence: 99%

See 1 more Smart Citation

Human Embryonic Kidney 293 Cells: A Vehicle for Biopharmaceutical Manufacturing, Structural Biology, and Electrophysiology

Han

et al. 2018

Cells Tissues Organs

View full text Add to dashboard Cite

Mammalian cells, e.g., CHO, BHK, HEK293, HT-1080, and NS0 cells, represent important manufacturing platforms in bioengineering. They are widely used for the production of recombinant therapeutic proteins, vaccines, anticancer agents, and other clinically relevant drugs. HEK293 (human embryonic kidney 293) cells and their derived cell lines provide an attractive heterologous system for the development of recombinant proteins or adenovirus productions, not least due to their human-like posttranslational modification of protein molecules to provide the desired biological activity. Secondly, they also exhibit high transfection efficiency yielding high-quality recombinant proteins. They are easy to maintain and express with high fidelity membrane proteins, such as ion channels and transporters, and thus are attractive for structural biology and electrophysiology studies. In this article, we review the literature on HEK293 cells regarding their origins but also stress their advancements into the different cell lines engineered and discuss some significant aspects which make them versatile systems for biopharmaceutical manufacturing, drug screening, structural biology research, and electrophysiology applications.

show abstract

Biochemical, single-channel, whole-cell patch clamp, and pharmacological analyses of endogenous TRPM4 channels in HEK293 cells

Cited by 36 publications

References 24 publications

Depletion of 14-3-3γ reduces the surface expression of Transient Receptor Potential Melastatin 4b (TRPM4b) Channels and attenuates TRPM4b-mediated glutamate-induced neuronal cell death

Depletion of 14-3-3γ reduces the surface expression of Transient Receptor Potential Melastatin 4b (TRPM4b) Channels and attenuates TRPM4b-mediated glutamate-induced neuronal cell death

A novel Ca2+-mediated cross-talk between endoplasmic reticulum and acidic organelles: Implications for NAADP-dependent Ca2+ signalling

Human Embryonic Kidney 293 Cells: A Vehicle for Biopharmaceutical Manufacturing, Structural Biology, and Electrophysiology

Contact Info

Product

Resources

About