Red fluorescent cAMP indicator with increased affinity and expanded dynamic range

Sci Rep. 2018 Jan 30;8(1):1866. doi: 10.1038/s41598-018-20251-1.

Abstract

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 sub-optimal 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 Iα and developed an improved cAMP indicator named R-FlincA (Red Fluorescent indicator for cAMP). Its increased affinity (Kd = 0.3 μM) and expanded dynamic range (860% at pH 7.2) allowed the detection of subtle changes in the cellular cAMP dynamics at sub-μM 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Calcium / metabolism
  • Cells, Cultured
  • Cyclic AMP / metabolism*
  • Dictyostelium / metabolism*
  • Fluorescent Dyes / chemistry*
  • Humans
  • Insulin-Secreting Cells / metabolism*
  • Luminescent Proteins / metabolism*
  • Molecular Imaging / methods*
  • Spectrometry, Fluorescence

Substances

  • Fluorescent Dyes
  • Luminescent Proteins
  • red fluorescent protein
  • Cyclic AMP
  • Calcium