New sensors for quantitative measurement of mitochondrial Zn(2+).

Zinc (Zn(2+)) homeostasis plays a vital role in cell function, and the dysregulation of intracellular Zn(2+) is associated with mitochondrial dysfunction. Few tools exist to quantitatively monitor the buffered, free Zn(2+) concentration in mitochondria of living cells (Zn(2+)). We have validated three high dynamic range, ratiometric, genetically encoded, fluorescent Zn(2+) sensors that we have successfully used to precisely measure and monitor Zn(2+) in several cell types. Using one of these sensors, called mito-ZapCY1, we report observations that free Zn(2+) is buffered at concentrations about 3 orders of magnitude lower in mitochondria than in the cytosol and that HeLa cells expressing mito-ZapCY1 have an average Zn(2+) of 0.14 pM, which differs significantly from other cell types. These optimized mitochondrial Zn(2+) sensors could improve our understanding of the relationship between Zn(2+) homeostasis and mitochondrial function.