A novel Ca2+ indicator protein using FRET and calpain-sensitive linker.
Here, we report the properties of a FRET-based calcium indicator protein. We constructed a tandem fusion protein, named F2C, of ECFP and EYFP combined with calpain-sensitive sequences of alpha-spectrin, with N-terminal palmitoylation signal of GAP-43. It was previously reported that calpain cleaved a similar ECFP-EYFP fusion protein linked by a calpain-sensitive sequence of alpha-spectrin (fodrin). Unexpectedly, F2C was not cleaved by calpain, but demonstrated properties of a Ca(2+) indicator when transiently infected in Purkinje cells of rat primary cerebellar culture or in the brainstem neurons infected in vivo using Sindbis virus encoding F2C. The emission ratio of 480nm/535nm was repeatedly increased when the intracellular Ca(2+) concentration ([Ca(2+)](i)) was raised. F2C had a Ca(2+) sensitivity with an apparent dissociation constant (K(d) for Ca(2+)) of 150nM, and demonstrated kinetics that paralleled Fura-2 when [Ca(2+)](i) was measured simultaneously. These properties of F2C are useful to be a Ca(2+) indicator.
Takatsuka
Kenji
K
Department of Physiology, Faculty of Medicine, Kyoto University, Kyoto 606-8501, Japan.
Ishii
Takahiro M
TM
Ohmori
Harunori
H
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002154:Calpain / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D031541:Fluorescence Resonance Energy Transfer; D008969:Molecular Sequence Data; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D013050:Spectrometry, Fluorescence
D011506:Proteins; D002154:Calpain; D002118:Calcium