Authors
Jan L. Vinkenborg, Elisa A. Bellomo, Tamara J. Nicolson, Melissa S. Koay, Maarten Merkx, and Guy A. Rutter
Summary
Zinc plays a critical role in many fundamental cellular processes, acting as a Lewis acid catalyst in numerous enzymes, having a structural function in DNA binding proteins and acting as a modulator in neurotransmission1-3. At the same time, low nanomolar concentrations of free Zn2+ can be cytotoxic, rendering zinc homeostasis a delicate balance that is not well understood. We developed a toolbox of genetically-encoded Forster Resonance Energy Transfer (FRET)-based sensors that allow monitoring of fluctuations in intracellular free zinc levels. These sensors, called eCALWY-1, -2, -3, -4, -5 and -6, display a large decrease in energy transfer upon Zn2+ binding and have affinities that span the pico- to nanomolar range (Table 1). They were successfully used to determine the cytosolic free Zn2+ concentrations of HEK293 and INS-1(832/13) cells, which were both found to be 0.4 nM. In addition, the sensors can be targeted to subcellular organelles, as was shown for secretory granules in pancreatic beta-cells4. An important issue when measuring cytosolic free zinc concentrations is whether the presence of micromolar concentrations of sensor is not affecting the free zinc level. The availability of a toolbox of sensors spanning a range of affinities allows addressing this issue. Each sensor can be intracellularly saturated with and depleted from zinc, after which the individual sensor occupancies at steady-state zinc levels can be calculated. When combining these occupancies with the in vitro determined Kd, the steady-state cytosolic free zinc concentration can be accurately determined. Here we describe the complete protocol to quantify changes in intracellular free Zn2+, based on a protocol described by Palmer et al for genetically encoded Ca2+ sensors5. Next, we explain how to use the toolbox of sensors to accurately determine the steady-state cytosolic free zinc concentration.Further details
The protocol was published on Protocol Exchange on 22 September 2009. To see the entire protocol, click on the source link.Advertisement
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