A FRET biosensor reveals free zinc deficiency in diabetic beta-cell vesicles

The concentration of free zinc within insulin-storing vesicles is important for vesicle maturity and therefore requires accurate measurement. However, common small-molecule intensity-based probes and most available genetically encoded FOrster resonance energy transfer (FRET)-based sensors for zinc are unsuitable for estimating the free zinc concentration in insulin-storing vesicles. Therefore, we have developed a novel FRET-based zinc sensor based on the RING motif of TRIM72, referred to as ZnT72R, which has an approximate K-d that varies from 6.07 +/- 0.28 mu mol/L to 7.84 +/- 0.42 mu mol/L in vitro and a cytosol-calibrated K-d of approximately 55.56 +/- 4.59 mu mol/L in HEK293 T cells. To pinpoint the free zinc concentration of insulin-storing vesicles, we initially targeted ZnT72R to beta-cell vesicles by fusing them to NPY (neuropeptide Y). Following NPY-ZnT72R labeling, the FRET intensity ratios of vesicles were quantified. We found that the free zinc concentration in insulin-storing vesicles of diabetic db/db mice (28.30 +/- 1.33 mu mol/L) was significantly lower than that of control mice (41.46 +/- 3.53 mu mol/L). (C) 2019 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences. Published by Elsevier B.V. All rights reserved.