Shandell Mia A, Quejada Jose R, Yazawa Masayuki, Cornish Virginia W, Kass Robert S
Department of Pharmacology, College of Physicians and Surgeons, Columbia University, New York, New York.
Department of Pharmacology, College of Physicians and Surgeons, Columbia University, New York, New York; Department of Rehabilitation and Regenerative Medicine, Columbia Stem Cell Initiative, Columbia University, New York, New York.
Biophys J. 2019 Oct 1;117(7):1352-1363. doi: 10.1016/j.bpj.2019.08.028. Epub 2019 Aug 29.
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.
Na1.5失活对于心脏动作电位的正常传导至关重要。Na1.5的基因突变会扰乱失活过程，并导致与3型长QT综合征相关的潜在致命性心律失常。失活复合物的确切结构动力学尚不清楚。为了检测活的哺乳动物细胞中失活门构象的变化，我们将溶剂化显色荧光非天然氨基酸3-((6-乙酰萘-2-基)氨基)-2-氨基丙酸(ANAP)引入到Na1.5失活门的单个位点。利用哺乳动物细胞合成酶-tRNA技术，将ANAP引入全长和C端截短的Na1.5通道。引入ANAP的通道在哺乳动物细胞中表达，并表现出病理生理功能。设计了一种光谱成像钾去极化测定法，以检测与Na1.5构象变化相关的ANAP发射位移。位点特异性细胞内ANAP的掺入为活细胞成像和检测哺乳动物细胞中Na1.5失活门构象变化提供了条件。
