title: Quantitative measurement of Ca(2+)-dependent calmodulin-target binding by Fura-2 and CFP and YFP FRET imaging in living cells.
abstract: Calcium dynamics and its linked molecular interactions cause a variety of biological responses; thus, exploiting techniques for detecting both concurrently is essential. Here we describe a method for measuring the cytosolic Ca(2+) concentration ([Ca(2+)](i)) and protein-protein interactions within the same cell, using Fura-2 and superenhanced cyan and yellow fluorescence protein (seCFP and seYFP, respectively) FRET imaging techniques. Concentration-independent corrections for bleed-through of Fura-2 into FRET cubes across different time points and [Ca(2+)](i) values allowed for an effective separation of Fura-2 cross-talk signals and seCFP and seYFP cross-talk signals, permitting calculation of [Ca(2+)](i) and FRET with high fidelity. This correction approach was particularly effective at lower [Ca(2+)](i) levels, eliminating bleed-through signals that resulted in an artificial enhancement of FRET. By adopting this correction approach combined with stepwise [Ca(2+)](i) increases produced in living cells, we successfully elucidated steady-state relationships between [Ca(2+)](i) and FRET derived from the interaction of seCFP-tagged calmodulin (CaM) and the seYFP-fused CaM binding domain of myosin light chain kinase. The [Ca(2+)](i) versus FRET relationship for voltage-gated sodium, calcium, and TRPC6 channel CaM binding domains (IQ domain or CBD) revealed distinct sensitivities for [Ca(2+)](i). Moreover, the CaM binding strength at basal or subbasal [Ca(2+)](i) levels provided evidence of CaM tethering or apoCaM binding in living cells. Of the ion channel studies, apoCaM binding was weakest for the TRPC6 channel, suggesting that more global Ca(2+) and CaM changes rather than the local CaM-channel interface domain may be involved in Ca(2+)CaM-mediated regulation of this channel. This simultaneous Fura-2 and CFP- and YFP-based FRET imaging system will thus serve as a simple but powerful means of quantitatively elucidating cellular events associated with Ca(2+)-dependent functions.
authors.affiliation: Department of Physiology, School of Medicine, Fukuoka University, 7-45-1 Nanakuma, Fukuoka, Japan. mxmori@fukuoka-u.ac.jp
authors.forename: Masayuki X
authors.initials: MX
authors.lastname: Mori
authors.forename: Yuko
authors.initials: Y
authors.lastname: Imai
authors.forename: Kyohei
authors.initials: K
authors.lastname: Itsuki
authors.forename: Ryuji
authors.initials: R
authors.lastname: Inoue
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002460:Cell Line; D031541:Fluorescence Resonance Energy Transfer; D016257:Fura-2 / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D001426:Bacterial Proteins; D002147:Calmodulin; C485182:Cyan Fluorescent Protein; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D002118:Calcium; D016257:Fura-2