Semi Simultaneous Imaging of Ca2+ and Ca2+ Dependent Calmodulin Interactions by FRET

Calcium dynamics and its linked molecular interactions contribute to a variety of biological responses, thus it is essential to exploit techniques for detecting both concurrently. Here we describe a new method to image Ca2+ and protein-protein interaction on semi-simultaneous base through combined application of Fura-2 fluorescence imaging and Fluorescence or F[[Unsupported Character - Orster Resonance Energy Transfer (FRET) with genetically encoded fluorescence proteins in single living cells. By applying Ca2+-dependent corrections for Fura-2 fluorescence superposition on FRET images, we could semi-simultaneously analyze in vivo Fura-2 and FRET signals with high quantitative fidelity.Furthermore, by producing gradual stepwise increases in [Ca2+]i in HEK cells, we could obtain quantitative and synchronous dose-response relationships for [Ca2+]i versus protein-protein interaction between Ca2+ binding protein ‘calmodulin’ (CaM) and its binding domain from an enzyme, which were fused with fluorophores CFP and YFP, respectively. The same approach applied to voltage-gated Na+, Ca2+ and TRP channels revealed their distinct sensitivities to basal Ca2+ through CaM binding.This semi-simultaneous imaging system could be applicable to the identification of Ca2+-dependent protein interactions with further potential application to elucidating the interrelations between Ca2+ signals and cellular functions such as enzyme activities, signal transductions, and membrane protein regulations in various living cells.