| Assay Method Information | |
| | Cell-based assay of NaP2b activity |
| Description: | The rate of phosphate (Pi) uptake into cells was measured using a modification of a literature method (see Mohrmann et al. Am. J. Phys. 250(3 pt 1):G323-30, 1986). Briefly, HEK293 cells were transiently transfected with an expression clone encoding either rat or human NaP2b. The next day, transfected cells were treated with a pharmacological agent to minimize endogenous PiT-mediated phosphate transport activity, such that the only remaining sodium-dependent phosphate transport activity is that which was bestowed by introduction of the NaP2b gene. Cells were incubated with radioactive inorganic phosphate in the presence or absence of varying concentrations of test compound. After a short time, cells were washed, harvested, and the amount of hot phosphate taken up in the cells determined by liquid scintillation counting.HEK293 cells were obtained from the American Type Culture collection and propagated per their instructions. Expression clones for rat and human NaP2b (SLC34A2) were obtained from Open Biosystems (Catalog numbers MRN1768-9510282, and MHS1010-99823026, respectively). There are two putative splice variants of human NaP2b, designated as isoform a and isoform b (NCBI Reference Sequences: NP_006415.2 and NP 001171470.1, respectively). The sequence of the open reading from in MHS1010-99823026 corresponds to isoform b; transfection with this construct was found to confer only very low levels of nonendogenous Pi transport activity. The cDNA was therefore mutated to correspond with isoform a; transfection with this sequence conferred Pi transport significantly over background. Thus, studies of the inhibition of human NaP2b used isoform a exclusively.Cells were seeded into 96-well plates at 25,000 cells/well and cultured overnight. Lipofectamine 2000 (Invitrogen) was used to introduce the NaP2b cDNA, and the cells were allowed to approach confluence during a second overnight incubation. Medium was aspirated from the cultures, and the cells were washed once with choline uptake buffer (14 mM Tris, 137 mM choline chloride, 5.4 mM KCl, 2.8 mM CaCl2, 1.2 mM MgSO4, 100 uM KH2PO4, 1 mg/mL Bovine Serum Albumin, pH 7.4). Cells were then overlayed with either choline uptake buffer or sodium uptake buffer (14 mM Tris, 137 mM sodium chloride, 5.4 mM KCl, 2.8 mM CaCl2, 1.2 mM MgSO4, 100 uM KH2PO4, PiT-silencing agent, 1 mg/mL Bovine Serum Albumin, pH 7.4) containing 6-9 uCi/mL 33P orthophosphoric acid (Perkin Elmer) and test compound. Each compound was tested at twelve concentrations ranging from 0.1 nM to 30 uM. Assays were run in duplicate and compounds of interest were tested multiple times. After incubation for 23 minutes at room temperature, assay mixtures were removed, and the cells were washed twice with ice cold stop solution (137 mM sodium chloride, 14 mM Tris, pH 7.4). Cells were lysed by addition of 20 μL 0.1% Tween 80 followed by 100 μL scintillation fluid, and counted using a TopCount (Perkin Elmer). The pIC50 (the negative log of the IC50) values of the test compounds were calculated using GraphPad Prism. Preliminary studies showed that under these conditions, sodium-dependent Pi uptake was linear for at least 30 minutes and tolerated 0.6% (v/v) DMSO without deleterious effects. |
| Affinity data for this assay | |
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