Targeting a low affinity GCaMP3 variant to the endoplasmic reticulum.

(a) Mutations to GCaMP3 (10.19 variant) creates a GECI with reduced affinity for calcium [33]. The EF-hand loops (yellow), mutated aspartic acid residues (red), and calcium ions (orange) are indicated on the GCaMP3 structure (PDB: 3SG7). (b) Ca²⁺ titration of purified GCaMP3 variant 10.19. K_d for Ca²⁺ ~ 400 μM, Hill slope ~ 1.9, ∆F_max/F₀ ~ 14. Points represent ∆F/F₀ (mean ± range, n = 2). (c) Schematic diagram of ER-targeting approach for GCaMP3 (10.19), creating GCaMPer. In vitro Ca²⁺ affinities for the untagged GCaMP3 variants are indicated. (d) Confocal microscopy of GCaMPer (10.19) localization in human neuroblastoma (SH-SY5Y) cells. Cells were transfected with pAAV-EF1α-GCaMPer (10.19 variant) and immunostained using GFP, PDI (ER resident chaperone), and RCAS1 (Golgi protein) antibodies. Nuclei were stained with DAPI. Scale bar = 10 microns. (e) Confocal microscopy of GCaMPer (10.19) localization in rat primary cortical neurons. Cells were transduced with AAV-hSYN1-GCaMPer (10.19) and immunostained using GFP, PDI, and RCAS1 antibodies. Nuclei were stained with DAPI. Scale bar = 10 microns. (f) GCaMPer (10.19) fluorescence in rat primary cortical neurons over a range of calcium concentrations after equilibrating calcium across cellular membranes using ionophores A23187 and ionomycin (mean ± SEM, n = 30 cells).