Figure 3.

Functional and molecular evidence against endogenous Ca²⁺-activated adenylyl cyclase 8 in HEK cells. (A) FRET measurements show no rise in cAMP following Ca²⁺ readmission (2 mM; arrow) to cells bathed in thapsigargin (2 μm) in Ca²⁺-free solution. Stimulation with forskolin (50 μm) gave a substantial FRET signal. Upward deflection indicates an increase in cAMP. (B) Aggregate data are compared from experiments as in Panel A. Basal denotes FRET signal under non-stimulated conditions, averaged for 60 s prior to stimulation. Thapsigargin data were measured after readmission of external Ca²⁺. (C) Forskolin evokes a dose-dependent increase in cAMP, measured using an ELISA-based system. (D) Thapsigargin fails to increase cAMP, detected using ELISA. IBMX was present prior to and during thapsigargin stimulation. Thapsigargin was applied at 2 μm for 10 min. The thapsigargin response is compared with the effect seen with a sub-maximal dose of forskolin (16 nM). (E) Protein kinase A activity is compared for the conditions shown. Basal denotes activity in the absence of stimulation (cells exposed to DMSO solvent control), thapsigargin (2 μm), forskolin (50 μm), and ionomycin (5 μm) were all applied in 2 mM Ca²⁺-containing external solution for 10 min prior to measurement of enzyme activity. F, RT-PCR reveals expression of AC8 in the DAOY neuronal cell line, but not in either wild type HEK293 cells or HEK293 cells in which all three Orai genes had been knocked out.¹³ (G) Aggregate data from three independent experiments are summarised in the bar chart. (H) qPCR was used to detect AC8 in the various cell types shown.