Fluo 5n

The Ca²⁺ dye Fluo-5N binds Gd³⁺ and produce a fluorescence signal. The binding affinity of ProCA1 variants to Gd³⁺ was determined by a competition method using Gd³⁺ loaded Fluo-5N (Life Technologies). Fluo-5N emission spectrum was monitored from 500 nm to 600 nm when it was excited at 488 nm. The binding affinity of Fluo-5N to Gd³⁺, K_d1, was determined by a Gd³⁺ titration in Gd³⁺ buffer system which used 1 mM nitrilotriacetic acid (NTA, Sigma) to control the concentration of free Gd³⁺. To calculate the Gd³⁺ affinity to ProCA1 variant, Fluo-5N was mixed with Gd³⁺ at 1:1 ratio. ProCA1 variants were gradually added into the system to compete Fluo-5N binding with Gd³⁺. An apparent dissociation constant, K_app, was estimated by fitting the fluorescence emission intensity of Fluo-5N at 520 nm with different ProCA1 variant concentrations as a 1:1 binding model. Gd^{3 +} binding affinities of ProCA1 variants, Kd₂, were calculated with the equation 1:

The CLC of frozen HEK293F-derived mitochondria was determined using a method based on that previously described for fresh rat liver mitochondria (23 (link)). Briefly, a buffer (130 mM Sucrose, 37.54 mM KCl, 2.5 mM KH₂PO₄, 5 mM HEPES) containing 0.5 mM Ca²⁺ and supplemented with 15 mM succinate, 2.5 μM rotenone and 0.15 μg/ml cell impermeant Fluo 5N (Life Technologies), was added to each well of a 384 well microplate (control wells also contained 0.24 μl/well DMSO or 3 μM cyclosporine). Thawed mitochondria suspended at 0.6 mg/ml were added to all wells to initiate the assay, and the fluorescence measured every 5 minutes at 520 nm in a PHERAstar FS microplate reader (BMG LabTech, Aylesbury, UK), (25 reads over 2 hours). Area under the curve (AUC) values for the first 10 reads were exported and the concentration-response relationship was modelled using the following formula: y=((B-A)/(1+(10^X/10^C)^D)+A where B=max, A=min, C=IC50, D=slope. The fitted curves were then used to calculate a pXC₅₀ value.

POPC, DOPC, 1,2-dioleoyl-sn-glycero-3-phospho-(1’-myo-inositol-4’,5’-bisphosphate) (di18:1 PI(4,5)P₂), 1-stearoyl-2-arachidonoyl-sn-glycero-3-phospho-(1’-myo-inositol-4’,5’-bisphosphate) (18:0 20:4 PI(4,5)P₂), 1-oleoyl-2-{6-[4-(dipyrrometheneboron difluoride)butanoyl]amino}hexanoyl-sn-glycero-3-phosphoinositol-4,5-bisphosphate (TopFluor PI(4,5)P₂), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (biotinylated DOPE) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). 1,2-Dipalmitoyl-sn-glycero-3-phospho-(1’-myo-inositol-4’,5’-bisphosphate) (di16:0 PI(4,5)P2) was from Echelon Biosciences (Salt Lake City, UT, USA). Lipid stock solutions were prepared in chloroform, with the exception of the phosphoinositides, which were prepared in chloroform:methanol (MeOH) 2:1 (v/v). Both solvents were obtained from Merck (Darmstadt, Germany) and were of spectroscopic grade. 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), ethanol (EtOH), NaCl, Sucrose, EDTA, glucose, and CaCl₂ were from Sigma-Aldrich (St. Louis, MO, USA). TMA-DPH, tPnA, Rhodamine 110, and Fluo-5N were from Molecular Probes, Invitrogen (Eugene, OR, USA).