Optical Recording of Zn²⁺ Dynamics in the Mitochondrial Matrix and Intermembrane Space with the GZnP2 Sensor

The zinc ion (Zn²⁺) is emerging as an important signaling molecule. Here, we engineered an improved Zn²⁺ probe GZnP2 based on a previously developed fluorescent sensor GZnP1 to provide a higher fluorescent readout (2-fold higher) that is proportional to cellular labile Zn²⁺ concentrations. We further developed a set of GZnP2 derived imaging tools to determine the labile Zn²⁺ concentrations in the mitochondrial matrix, mitochondrial intermembrane space (IMS), and cytosol in four different cell lines (HeLa, Cos-7, HEK293, and INS-1). The labile Zn²⁺ concentration in the matrix was less than 1 pM, while the labile Zn²⁺ concentration in the IMS was comparable to the cytosol (∼100 pM). With these sensors, we showed that upon exposure to high Zn²⁺, only the cytosol and the IMS were overloaded with Zn²⁺, while the mitochondrial matrix was unable to sequester excess labile Zn²⁺ in depolarized INS-1 cells. This work highlighted the importance of distinguishing the labile Zn²⁺ concentrations and dynamics between the mitochondrial matrix and IMS.