Intracellular Pb2+ content monitoring using a protein-based Pb2+ indicator

Lead ion (Pb(2+)) is one of the most hazardous heavy metals to almost all life forms. The components of store-operated Ca(2+) entry as a molecular gateway have been previously found to participate in the cytotoxic entry of Pb(2+). However, the safe levels of intracellular Pb(2+) hiding in blood Pb(2+) levels are still not determined with full certainty. The present study aimed to construct protein-based Pb(2+) indicators to help establish a reliable setting for the content monitoring of intracellular Pb(2+). A series of Pb(2+) indicators based on fluorescence resonance energy transfer, Met-leads, were developed. The Pb(2+)-binding protein PbrR (from Cupriavidus metallidurans CH34) was applied between the fluorescent protein pair ECFP(ΔC11) and cp173Venus. The spectral patterns and sensing ranges of all Met-leads were characterized in vitro. Among these constructs, Met-lead 1.59 had relatively high ion selectivity and broad dynamic range (3.3-5.7). Consequently, this Met-lead was adopted in the cellular Pb(2+) biosensing. The intracellular Pb(2+) content in human embryonic kidney cells was successfully monitored using Met-lead 1.59 under both short- and long-term treatments. The existence of intracellular Pb(2+) can be significantly sensed using Met-lead 1.59 after 3 h 0.5μM (10 μg/dl) exposure, which is 200 times more improved than previous live-cell indicators. In summary, a new Pb(2+) indicator, Met-lead 1.59, was successfully developed for advanced research on Pb(2+) toxicology.