Electrophysiological properties of mouse and epitope-tagged human cardiac sodium channel Nav1.5 expressed in HEK293 cells

Q: What are the key findings of this study?

Adding a FLAG-tag to human Nav1.5 significantly decreased peak sodium current to 57 pA/pF vs 240 pA/pF in untagged channels (P<0.01), whereas N-terminal GFP tagging did not alter channel properties.

Q: What is the clinical evidence from this study?

Population: Genetically determined cardiac arrhythmias / Cardiac sodium channel (Nav1.5) electrophysiology (n=76). Intervention: FLAG-tagged human Nav1.5 (pcDNA3.1-FLAG(299/300)-hNav1.5) vs. Untagged wild-type human Nav1.5 (pcDNA3.1-hNav1.5). Primary outcome: Peak sodium current density (Imax, pA/pF) (p=<0.01).

Q: What does this research mean for the field?

Adding a FLAG-tag or N-terminal YFP to human Nav1.5 significantly reduces peak sodium current and alters activation kinetics, whereas N-terminal GFP tagging preserves baseline biophysical channel properties. Novelty: ClaimNovelty.INCREMENTAL. Consensus alignment: ConsensusAlignment.NEUTRAL.

Q: What question did this study set out to answer?

To compare the biophysical properties of mouse and human Nav1.5 channels and examine whether adding specific epitope tags to human Nav1.5 alters its electrophysiological function.

Katja Reinhard; Jean‐Sébastien Rougier; Jakob Ogrodnik; Hugues Abriel

doi:10.12688/f1000research.2-48.v2

April 5, 2013F1000ResearchOpen Access

Electrophysiological properties of mouse and epitope-tagged human cardiac sodium channel Nav1.5 expressed in HEK293 cells

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Key result

FLAG-tagging human Nav1.5 cuts peak sodium current ~76% vs untagged channels.

P<0.01
n=76

Why the study?

Generating genetically-modified mice with high-affinity epitopes in Nav1.5 can help identify in situ interacting proteins, requiring evaluation of mouse versus human Nav1.5 biophysical properties and potential alterations caused by epitope modification.

Key Points

To compare the biophysical properties of mouse and human Nav1.5 channels and examine whether adding specific epitope tags to human Nav1.5 alters its electrophysiological function.
Transiently expressed untagged human Nav1.5, mouse Nav1.5, and epitope-tagged human Nav1.5 constructs in HEK293 cells.
Tested epitope additions including N-terminal GFP, N-terminal YFP, and an extracellular FLAG tag inserted between residues Leu-299 and Val-300.
Applied whole-cell voltage-clamp protocols to measure current-voltage activation, steady-state inactivation, recovery from inactivation, and onset of slow inactivation.
Mouse and human Nav1.5 showed similar biophysical properties, differing only in reversal potential (56.6 mV vs. 51.0 mV, P<0.01) and inactivation curve slope (6.67 mV/e-fold vs. 5.95 mV/e-fold, P<0.01).
Tagging human Nav1.5 with FLAG or YFP significantly reduced peak current compared to wild-type (FLAG: 57 pA/pF, P<0.01; YFP: 120 pA/pF, P<0.05; wild-type: 240 pA/pF) and produced shallower activation slopes (FLAG: 6.96 vs. 5.91 mV/e-fold; YFP: 6.87 vs. 5.91 mV/e-fold, P<0.05).
Adding GFP to the N-terminus did not alter any of the measured biophysical properties of human Nav1.5.

PICO

P

Population

76 transiently transfected HEK293 cells expressing mouse Nav1.5, wild-type human Nav1.5, or epitope-tagged human Nav1.5 constructs evaluated by whole-cell voltage-clamp electrophysiology.

I

Intervention / Comparator

FLAG-tagged human Nav1.5 (pcDNA3.1-FLAG(299/300)-hNav1.5) vs Untagged wild-type human Nav1.5 (pcDNA3.1-hNav1.5) (1 µg plasmid DNA)

O

Primary Outcome

Peak sodium current density (Imax, pA/pF), p=<0.01

Main Result

Absolute Event Rate: 57% vs 240%

p-value: p=<0.01

Limitations

Mammalian heterologous expression systems (HEK293 cells) have limitations when studying the trafficking properties of ion channels compared to native cardiomyocytes or in vivo models.
A different vector was used for the YFP-hNav1.5 construct, which might partially explain the smaller changes found in its biophysical properties.
Experiments were performed in HEK293 cells without co-expressing other subunits and regulating proteins.

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Implication

Reveals that mouse and human Nav1.5 share similar biophysics while specific epitope tags impair sodium current, highlighting design considerations for modified animal models.

Abstract

BACKGROUND: The pore-forming subunit of the cardiac sodium channel, Na v1.5, has been previously found to be mutated in genetically determined arrhythmias. Na v1.5 associates with many proteins that regulate its function and cellular localisation. In order to identify more in situ Na v1.5 interacting proteins, genetically-modified mice with a high-affinity epitope in the sequence of Na v1.5 can be generated. METHODS: In this short study, we (1) compared the biophysical properties of the sodium current (I Na) generated by the mouse Na v1.5 (mNa v1.5) and human Na v1.5 (hNa v1.5) constructs that were expressed in HEK293 cells, and (2) investigated the possible alterations of the biophysical properties of the human Na v1.5 construct that was modified with specific epitopes. RESULTS: The biophysical properties of mNa v1.5 were similar to the human homolog. Addition of epitopes either up-stream of the N-terminus of hNa v1.5 or in the extracellular loop between the S5 and S6 transmembrane segments of domain 1, significantly decreased the amount of I Na and slightly altered its biophysical properties. Adding green fluorescent protein (GFP) to the N-terminus did not modify any of the measured biophysical properties of hNa v1.5. CONCLUSIONS: These findings have to be taken into account when planning to generate genetically-modified mouse models that harbour specific epitopes in the gene encoding mNa v1.5.

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Cite This Study

Reinhard et al. (2013) studied Genetically determined cardiac arrhythmias / Cardiac sodium channel (Nav1.5) electrophysiology (n=76). FLAG-tagged human Nav1.5 (pcDNA3.1-FLAG(299/300)-hNav1.5) vs. Untagged wild-type human Nav1.5 (pcDNA3.1-hNav1.5) was evaluated on Peak sodium current density (Imax, pA/pF) (p=<0.01). Adding a FLAG-tag to human Nav1.5 significantly decreased peak sodium current to 57 pA/pF vs 240 pA/pF in untagged channels (P<0.01), whereas N-terminal GFP tagging did not alter channel properties.

synapsesocial.com/papers/6ae1fe7f88ecbc9d509f4543 https://doi.org/10.12688/f1000research.2-48.v2

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Electrophysiological properties of mouse and epitope-tagged human cardiac sodium channel Nav1.5 expressed in HEK293 cells

Key Points

PICO

Main Result

Limitations

Ask about this paper

Implication

Abstract

Discussion

Cite This Study

Electrophysiological properties of mouse and epitope-tagged human cardiac sodium channel Nav1.5 expressed in HEK293 cells

Key Points

PICO

Main Result

Limitations

Ask about this paper

Implication

Abstract

Discussion

Cite This Study

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