Figure 1.
Store-operated Ca2+ influx in lung epithelia.
A, Following store depletion with thapsigargin (2 µM) in Ca2+-free solution, readmission of 2 mM external Ca2+ resulted in Ca2+ influx. Ba2+ and Sr2+ were permeable too. B, Aggregate data (normalised to the rate of Ca2+ influx) is summarised. Each bar represents between 40 and 55 cells. C-H, Store-operated Ca2+ influx is inhibited by the CRAC channel blockers Synta66 (C and D; aggregate data shown represent 53 control cells and 44 cells in the Synta66 group); BTP2 (E and F; aggregate data shown represent 44 control cells and 36 cells in the BTP2 group); 2-APB (G and H; aggregate data shown represent 41 control cells and 59 cells in the 2-APB group). Inhibitors were applied 5 minutes before stimulation with thapsigargin.
Figure 2.
La3+ blocks store-operated Ca2+ entry in 16HBE cells.
A, Ca2+ influx is inhibited in a dose-dependent manner by La3+ in RBL-1 cells. B, The inhibition of Ca2+ influx by La3+ in epithelial cells is shown, taken from identical conditions to that in panel A and used on the same days. C, Dose-inhibition curves for the two cell types are compared. Data are fitted with a Hill-type equation of the form: % Inhibition = [La3+]n/([La3+]n+Xn) where n is the Hill coefficient and X is the IC50. The Y-axis denotes the normalised rate of Ca2+ entry, in the presence of different concentrations of La3+. A value of 1.0 represents the entry rate in the absence of La3+.
Figure 3.
Whole cell patch clamp recordings demonstrate the presence of CRAC channels.
A, Passive store depletion by inclusion of 10 mM EGTA in the pipette activates a slowly developing inward current that is inhibited by 10 µM Synta66. B, Current-voltage-relationship taken at 270 seconds from panel A. C, Aggregate data are compared. Current was measured at −80 mV. Control bar is from 7 cells and Synta66 group is from 5 cells. D, Fast inactivation of the CRAC current in epithelia is shown. Upper panel depicts the voltage protocol and lower panel shows the current during the pulse to −120 mV. Pipette solution was the same as in panel A. E, Aggregate data is compared between RBL-1 and 16HBE cells. Holding potential was 0 mV. Each point is the average of between 3 and 6 cells.
Figure 4.
STIM1 and Orai1 are expressed and functional in 16HBE cells.
A–B, Western blots show the presence of STIM1 (A) and STIM2 (B) in 16HBE lysates. Aggregate data from 3 independent experiments are shown in B. C, RT-PCR reveals the presence of Orai transcripts. D, Averaged data are compared. E, Western blot shows the presence of Orai1 protein and that siRNA construct to knock down Orai1 (labelled Orai1 KD) reduces protein expression. F, The histogram summarises the extent of Orai1 knockdown from 3 independent experiments. G, Ca2+ measurements show that knock down of Orai1 reduces the rate and extent of Ca2+ influx. H, Aggregate data are summarised. Control group is the average of 63 cells and Orai1 KD group is 81 cells. I, Western blot shows that siRNA against STIM1 reduces protein expression. J, Aggregate data from 4 independent experiments are compared. K, Store-operated Ca2+ influx is reduced after knockdown of STIM1. L, Histogram compares the rate of Ca2+ influx for the two conditions. Control group is 45 cells, STIM1 KD group is 59 cells.
Figure 5.
CRAC channels activate gene expression in 16HBE cells.
A, C-fos mRNA is increased by thapsigargin. B, Aggregate data from 4 experiments are summarised. C-F, c-fos expression induced by thapsigargin is reduced by Synta66 (C and D) and BTP2 (E and F). G, RT-PCR reveals the presence of mRNA for NFAT1 and NFAT4. H, Relative levels of NFAT are compared. I, NFAT-dependent GFP reporter gene expression is compared for the different conditions. J, Aggregate data from 3 independent experiments are compared. Basal denotes untreated cells.
Figure 6.
CRAC channels increase EGF expression.
A, EGF mRNA levels are increased by thapsigargin. B, Aggregate data from 4 experiments are compared. C, The increase in EGF mRNA after thapsigargin stimulation is reduced following knockdown of Orai1 D, Aggregate data from 3 experiments are compared. KD here denotes knock down of Orai1. E, STIM1 knockdown reduces EGF mRNA levels. Basal denotes untreated. F, Aggregate data from 3 experiments are compared. KD denotes STIM1 knockdown.
Figure 7.
Exposure to cold transiently increases store-operated Ca2+ influx in 16HBE cells.
A, Ca2+ entry was increased in cells pre-exposed to a cold shock. B, Effect of cold shock on store-operated Ca2+ entry in HEK293 cells. C, Rate of Ca2+ influx is compared between the different conditions. Control denotes cells not exposed to a cold shock. Recovery refers to cells exposed to a cold shock but allowed to recover at 37°C overnight hours. Each bar is the average of >88 cells. D, RT-PCR measurements of cfos mRNA, U denotes untreated. D, Aggregate data from 4 independent experiments compares the increase in c-fos mRNA.
Figure 8.
CRAC channel activity is required for 16HBE cell migration in a scrape wound assay.
A, transillumination images of cells prior to a scrape wound are shown. After scraping, images are shown at the times indicated for control cells and those continually exposed to Synta66. B, Aggregate data from three independent scrape would assays are compared. Several snapshots were taken per culture dish and the averaged number of cells per image are plotted.