Detection of Na1.5 Conformational Change in Mammalian Cells Using the Noncanonical Amino Acid ANAP

Overview

Journal Biophys J

Publisher Cell Press

Specialty Biophysics

Date 2019 Sep 16

PMID 31521331

Citations 8

Authors

Mia A Shandell

Jose R Quejada

Masayuki Yazawa

Virginia W Cornish

Robert S Kass

Affiliations

Soon will be listed here.

Abstract

Na1.5 inactivation is necessary for healthy conduction of the cardiac action potential. Genetic mutations of Na1.5 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 noncanonical amino acid 3-((6-acetylnaphthalen-2-yl)amino)-2-aminopropanoic acid (ANAP) into single sites in the Na1.5 inactivation gate. ANAP was incorporated in full-length and C-terminally truncated Na1.5 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 Na1.5 conformational change. Site-specific intracellular ANAP incorporation affords live-cell imaging and detection of Na1.5 inactivation gate conformational change in mammalian cells.

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