A ratiometric ER calcium sensor for quantitative comparisons across cell types and subcellular regions

The ER is an important regulator of Ca²⁺ in cells and dysregulation of ER Ca²⁺ homeostasis can lead to numerous pathologies. Understanding how various pharmacological and genetic perturbations of ER Ca²⁺ homeostasis impact cellular physiology would be facilitated by quantitative measurements of ER Ca²⁺ levels that allow for robust comparisons across conditions. To achieve this, we enhanced our original high dynamic range ER Ca²⁺ indicator, ER-GCaMP6-150, by fusing it to the HaloTag protein, which when bound to Janelia Fluor (JF) dyes creates a ratiometric ER Ca²⁺ probe. This probe (ER-Halo-GCaMP6-150) displayed minimal changes to the Ca²⁺-binding properties compared with our original ER Ca²⁺ probe as shown through in vitro and in cell Ca²⁺ calibrations. We describe a method to use this ratiometric probe for quantitative comparisons of ER Ca²⁺ concentrations and leverage this technique to compare ER Ca²⁺ levels across cell types and subcellular compartments. Using this approach, we show that the resting concentration of ER Ca²⁺ in primary dissociated neurons does not differ between excitatory and inhibitory subtypes nor between axonal and somatodendritic compartments. However, resting ER Ca²⁺ levels in neuronal somas are substantially lower than that measured in embryonic fibroblasts. The ER-Halo-GCaMP6-150 provides a robust tool to directly measure ER Ca²⁺ levels for studies of ER physiology across cell types and compartments.