摘要
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 ZnT72 R,which has an approximate K_d that varies from 6.07±0.28 μmol/L to 7.84±0.42μmol/L in vitro and a cytosol-calibrated K_d of approximately 55.56±4.59μmol/L in HEK293T cells.To pinpoint the free zinc concentration of insulin-storing vesicles,we initially targeted ZnT72R to beta-cell vesicles by fu sing 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±1.33μmol/L) was significantly lower than that of control mice(41.46±3.53μmol/L).
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 ZnT72 R,which has an approximate K_d that varies from 6.07±0.28 μmol/L to 7.84±0.42μmol/L in vitro and a cytosol-calibrated K_d of approximately 55.56±4.59μmol/L in HEK293T cells.To pinpoint the free zinc concentration of insulin-storing vesicles,we initially targeted ZnT72R to beta-cell vesicles by fu sing 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±1.33μmol/L) was significantly lower than that of control mice(41.46±3.53μmol/L).
基金
supported by a China Postdoctoral Science Foundation-funded project(No.2017M612440)。
