Flim of Physiological Free CU(II) in Living Cells with a Protein-Based Biosensor

Abstract

Recently, the biology of copper ion has excited substantial interest not only in the biomedical field but in oceanography and the environment as well. Rae, et al., famously predicted (Science 284, 805 (1999))that ordinarily no free copper ion would be found in resting cells based on the femtomolar affinity of a yeast copper chaperone. We have developed a biosensor that employs variants of human carbonic anhydrase II (CA II) with very high affinity and selectivity to quantitate free Cu(II) by changes in fluorescence lifetime. Using a membrane-penetrant CA II variant with much higher selectivity for Cu(II) over Zn (II) than wild type, we measured free Cu(II) levels by confocal fluorescence lifetime imaging microscopy in resting PC-12 cells in vitro. We found that the indicator system could be successfully calibrated in the cell, and that the resting level was very low, near the detection limit of ∼10 femtomolar. The implications of the findings are discussed. The authors thank NIH for support (RO1- EB003924-05 [RBT, CAF, HHZ, and BJM] and RC1-GM091081-01 [JRL, HS, and RBT]) and ISS (Champaign, IL) for the use of the Alba confocal FLIM.View Large Image | View Hi-Res Image | Download PowerPoint Slide