Detection of Na_v1.5 conformational change in mammalian cells using the non-canonical amino acid ANAP

TY - JOUR

T1 - Detection of Nav1.5 conformational change in mammalian cells using the non-canonical amino acid ANAP

AU - Shandell, Mia Ann

AU - Quejada, Jose R.

AU - Yazawa, Masayuki

AU - Cornish, Virginia W.

AU - Kass, Robert S.

N1 - © 2019 Biophysical Society. This is an author-produced version of the published paper. Uploaded in accordance with the publisher’s self-archiving policy.

PY - 2019/8/29

Y1 - 2019/8/29

N2 - 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.

AB - 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.

U2 - 10.1016/j.bpj.2019.08.028

DO - 10.1016/j.bpj.2019.08.028

M3 - Article

SN - 0006-3495

JO - Biophysical Journal

JF - Biophysical Journal

ER -