Abstract
Cd2+ causes damages to several human tissues. Although the toxicological and carcinogenetic mechanisms of Cd2+ have been previously established, some basic questions on this toxicant remain unclear. In this study, we constructed Met-cad 1.57, a new fluorescent resonance energy transfer (FRET)-based Cd2+ indicator, which contains a portion of a Cd2+-binding protein (CadR) obtained from Pseudomonas putida as the Cd2+ sensing key. We produced a human embryonic kidney cell line HEK-MCD157 which stably expresses the Met-cad 1.57 for further investigations. Both fluorescence spectroscopy and FRET microscopic ratio imaging were used to monitor the Cd2+ concentration within the living HEK-MCD157 cells. The dissociation constant of Met-cad 1.57 was approximately 271 nM. The function of Ca2+ channels as a potential Cd2+ entry gateway was further confirmed in the HEK-MCD157 cells. The organelle-targeted property of the protein-based Cd2+ indicator directly reveals the nucleus accumulation phenomena. In summary, a human kidney cell line that stably expresses the FRET-based Cd2+ indicator Met-cad 1.57 was constructed for reliable and convenient investigations to determine the Cd2+ concentration within living cells, including the identification of the entry pathway of Cd2+ and sub-cellular sequestration.
Highlights
In contrast to ions that are important to life, heavy metal/ metalloid ions such as Pb2+, Cd2+, Hg2+, and As3+ are extremely toxic and not beneficial to living organisms [1]
We proposed that a certain part of MerR-like protein is a possible metal ion sensing toolbox that produces an identical metal ion indicator, such as Met-lead with PbrR [13]
The proposed dimer structure of a fluorescent resonance energy transfer (FRET)-based Cd2+ indicator is shown in Figure 1A according to previous report about the structure property of CadR [12]
Summary
In contrast to ions that are important to life, heavy metal/ metalloid ions such as Pb2+, Cd2+, Hg2+, and As3+ are extremely toxic and not beneficial to living organisms [1]. The subcellular sequestration and accumulation of Cd2+ have been observed in several organelles such as the mitochondria or nucleus [9], but have not been completely elucidated. Conclusions for these issues cannot be obtained partially because of the lack of suitable live-cell tools that are specific for cytosolic and subcellular Cd2+ sensing [10,11]. To obtain accurate information on the succeeding sequestration and the bio-magnification of Cd2+ after cytotoxic entry, appropriate live-cell sensing approaches for intra/sub-cellular Cd2+ monitoring have been attempted.
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