Shandell, Mia Ann orcid.org/0000-0002-7864-758X, Quejada, Jose R., Yazawa, Masayuki et al. (2 more authors) (2019) Detection of Nav1.5 conformational change in mammalian cells using the non-canonical amino acid ANAP. Biophysical Journal. ISSN 0006-3495
Abstract
Nav1.5 inactivation is necessary for healthy conduction of the cardiac action potential. Genetic mutations of Nav1.5 perturb inactivation and cause potentially fatal arrhythmias associated with long QT syndrome type 3. The exact structural dynamics of the inactivation complex is unknown. To sense inactivation gate conformational change in live mammalian cells, we incorporated the solvatochromic fluorescent non-canonical amino acid ANAP into single sites in the Nav1.5 inactivation gate. ANAP was incorporated in full-length and C-terminally truncated Nav1.5 channels using mammalian cell synthetase-tRNA technology. ANAP-incorporated channels were expressed in mammalian cells and they exhibited pathophysiological function. A spectral imaging potassium-depolarization assay was designed to detect ANAP emission shifts associated with Nav1.5 conformational change. Site-specific intracellular ANAP incorporation affords live-cell imaging and detection of Nav1.5 inactivation gate conformational change in mammalian cells.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2019 Biophysical Society. This is an author-produced version of the published paper. Uploaded in accordance with the publisher’s self-archiving policy. |
Dates: |
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Institution: | The University of York |
Academic Units: | The University of York > Faculty of Sciences (York) > Centre for Immunology and Infection (CII) (York) |
Depositing User: | Pure (York) |
Date Deposited: | 30 Aug 2019 08:00 |
Last Modified: | 16 Oct 2024 15:58 |
Published Version: | https://doi.org/10.1016/j.bpj.2019.08.028 |
Status: | Published |
Refereed: | Yes |
Identification Number: | 10.1016/j.bpj.2019.08.028 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:150217 |
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