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Engineering of the FRET‐based biosensor LIBRA for improved pH‐stability and increased affinity for inositol 1,4,5‐trisphosphate
Author(s) -
Tanimura Akihiko,
Takao Morita,
Akiko Shitara,
Akihiro Nezu,
Yosuke Tojyo
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a978
Subject(s) - inositol , inositol phosphate , inositol trisphosphate receptor , biophysics , mutant , inositol trisphosphate , chemistry , förster resonance energy transfer , amino acid , yellow fluorescent protein , receptor , biochemistry , biology , fluorescence , gene , physics , quantum mechanics
LIBRA, a ratiometric biosensor for inositol 1,4,5‐trisphosphate (IP 3 ), consists of the IP 3 binding domain of the rat type 3 IP 3 receptor fused between the FRET pair ECFP and EYFP, and preceded by a membrane‐targeted signal, GAP43. The original LIBRA was sensitive to pH, which constituted a potential problem when monitoring agonist induced changes in IP3 in certain cell types. To improve the pH‐stability, we replaced the FRET accepter EYFP with the brighter and more pH‐stable mutant Venus containing defined amino acids substitutions. The resulting variant LIBRAv was less pH‐sensitive than the original LIBRA, and additionally had a 1.3‐fold larger emission ratio between zero and the saturating IP 3 concentration (Rmax). We next engineered the IP 3 ‐binding domain of LIBRAv. Mutation of a critical amino acid within the IP 3 ‐binding domain (K508A) attenuated its response to IP3, whereas a R440Q mutation increased by ~2‐fold its affinity for IP 3 . The K508A mutant would be ideal as a negative control for IP 3 measurements. We also constructed another high affinity LIBRA construct using the IP 3 ‐binding domain of type 2 IP 3 R with a R440Q mutation, which exhibited a ~10‐fold increase in affinity for IP 3 and a ~2‐fold increase in the Rmax. The simultaneous monitoring of IP 3 and Ca 2+ using LIBRA variants and fura‐2 indicated a monophasic increase in IP 3 levels during oscillatory changes in Ca 2+ concentrations with 1–3μM ATP in COS7 cells. We will also discuss the feedback effect of Ca 2+ on agonist‐induced IP 3 dynamics.