Fluorescent indicators for Ca²⁺based on green fluorescent proteins and calmodulin

Abstract

Important Ca²⁺ signals in the cytosol and organelles are often extremely localized and hard to measure. To overcome this problem we have constructed new fluorescent indicators for Ca²⁺ that are genetically encoded without cofactors and are targetable to specific intracellular locations. We have dubbed these fluorescent indicators ‘cameleons’. They consist of tandem fusions of a blue- or cyan-emitting mutant of the green fluorescent protein (GFP)¹,², calmodulin^3,4,5, the calmodulin-binding peptide M13 (ref. 6), and an enhanced green- or yellow-emitting GFP^7,8,9. Binding of Ca²⁺ makes calmodulin wrap around the M13 domain, increasing the fluorescence resonance energy transfer (FRET) between the flanking GFPs². Calmodulin mutations can tune the Ca²⁺ affinities to measure free Ca²⁺ concentrations in the range 10⁻⁸ to 10⁻² M. We have visualized free Ca²⁺ dynamics in the cytosol, nucleus and endoplasmic reticulum of single HeLa cells transfected with complementary DNAs encoding chimaeras bearing appropriate localization signals. Ca²⁺ concentration in the endoplasmic reticulum of individual cells ranged from 60 to 400 µM at rest, and 1 to 50 µM after Ca²⁺ mobilization. FRET is also an indicator of the reversible intermolecular association of cyan-GFP-labelled calmodulin with yellow-GFP-labelled M13. Thus FRET between GFP mutants can monitor localized Ca²⁺ signals and protein heterodimerization in individual live cells.