Sub-nanomolar sensitive GZnP3 reveals TRPML1-mediated neuronal Zn²⁺ signals

Although numerous fluorescent Zn²⁺ sensors have been reported, it is unclear whether and how Zn²⁺ can be released from the intracellular compartments into the cytosol due to a lack of probes that can detect physiological dynamics of cytosolic Zn²⁺. Here, we create a genetically encoded sensor, GZnP3, which demonstrates unprecedented sensitivity for Zn²⁺ at sub-nanomolar concentrations. Using GZnP3 as well as GZnP3-derived vesicular targeted probes, we provide the first direct evidence that Zn²⁺ can be released from endolysosomal vesicles to the cytosol in primary hippocampal neurons through the TRPML1 channel. Such TRPML1-mediated Zn²⁺ signals are distinct from Ca²⁺ in that they are selectively present in neurons, sustain longer, and are significantly higher in neurites as compared to the soma. Together, our work not only creates highly sensitive probes for investigating sub-nanomolar Zn²⁺ dynamics, but also reveals new pools of Zn²⁺ signals that can play critical roles in neuronal function.