Quantitative measurement of Ca2+-dependent calmodulin-target binding by Fura-2 and CFP and YFP FRET imaging in living cells

Masayuki X. Mori, Yuko Imai, Kyohei Itsuki, Ryuji Inoue

Research output: Contribution to journalArticlepeer-review

12 Citations (Scopus)


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 Ca2+ concentration ([Ca2+]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 [Ca2+]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 [Ca2+]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 [Ca2+]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 [Ca2+]i. Moreover, the CaM binding strength at basal or subbasal [Ca2+]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 Ca2+ and CaM changes rather than the local CaM-channel interface domain may be involved in Ca2+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 Ca2+-dependent functions.

Original languageEnglish
Pages (from-to)4685-4696
Number of pages12
Issue number21
Publication statusPublished - May 31 2011

All Science Journal Classification (ASJC) codes

  • Biochemistry


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