フルタ　トシアキ   Furuta Toshiaki   古田　寿昭    所属   東邦大学  理学部 生物分子科学科    職種   教授
論文種別	原著
言語種別	英語
査読の有無	査読あり
表題	Red fluorescent cAMP indicator with increased affinity and expanded dynamic range
掲載誌名	正式名：Scientific Reports
掲載区分	国外
巻・号・頁	8,pp.1866
総ページ数	9
著者・共著者	Y. Ohta, T. Furuta, T. Nagai and K. Horikawa
発行年月	2018/01/30
概要	cAMP is one of the most important second messengers in biological processes. Cellular dynamics of cAMP have been investigated using a series of fluorescent indicators; however, their sensitivity was suboptimal for detecting cAMP dynamics at a low concentration range, due to a low ligand affinity and/or poor dynamic range. Seeking an indicator with improved detection sensitivity, we performed insertion screening of circularly permuted mApple, a red fluorescent protein, into the cAMP-binding motif of PKA regulatory subunit I alpha and developed an improved cAMP indicator named R-FlincA (Red Fluorescent indicator for cAMP). Its increased affinity (K-d = 0.3 mu M) and expanded dynamic range (860% at pH 7.2) allowed the detection of subtle changes in the cellular cAMP dynamics at sub-mu M concentrations, which could not be easily observed with existing indicators. Increased detection sensitivity also strengthened the advantages of using R-FlincA as a red fluorescent indicator, as it permits a series of applications, including multi-channel/function imaging of multiple second messengers and combinatorial imaging with photo-manipulation. These results strongly suggest that R-FlincA is a promising tool that accelerates cAMP research by revealing unobserved cAMP dynamics at a low concentration range.