Fig. S5.

(Related to Fig. 5) Characterization of genetically encoded Zn²⁺ sensors. (A) Vesicular and nuclear Zn²⁺ were monitored concurrently in a mixture of HEK293 cells expressing VIP36-eCALWY4 or ZapCmR2-NLS and titrated with increasing Zn²⁺ concentrations in 20 µM pyrithione. (B) VIP36-eCALWY4 FRET was monitored in HEK293 cells before and after neutralization of vesicular pH with NH₄Cl. (C) HEK293 cells expressing intravesicular ZnGreen1 (in the TRPM7 S1/2 loop) were incubated with 50 µM TPEN or 50 µM Zn²⁺/20 µM pyrithione. (D) Cytosolic and vesicular ZnGreen1 sensors were saturated (indicated by the axis break) 20 min after loading with 500 µM extracellular Zn²⁺ at room temperature. (E and F) Cytosolic (E) and vesicular (F) ZnGreen1 were titrated with increasing concentrations of Zn²⁺ in 20 µM pyrithione to determine Zn²⁺ affinities of the sensors. The K_ds of the TRPM7-conjugated sensors (21–2.9 nM) indicate higher affinity than that reported for the naked sensor (633 nM) (51). Note: We have inverted eCALWY-4 FRET changes to overlay the calibration curve of eCALWY-4 and that of a conventional FRET-based sensor (ZapCmR2) in Fig. S5A. To keep the panels consistent, intensity changes of ZnGreen1 were also inverted.