A, representative Ca²⁺ imaging experiment (fura-5f) showing the effects of RNAi against STIM1 on endogenous SOCE evoked by store depletion with the SERCA pump inhibitor, thapsigargin (TG; 2 μm). B, bar graph showing STIM1 siRNA treatment significantly reduces (unpaired t test; P= 0.00047) endogenous SOCE in HEK293 cells. C, experiments carried out as in A; however, TRPC5 was transiently expressed in these cells (see Methods) and 200 μm carbachol was used instead of thapsigargin in order to activate TRPC5. Note also the presence of 5 μm Gd³⁺ in these experiments to inhibit endogenous SOCE. D, bar graph showing no effect (unpaired t test) of STIM1 knockdown on TRPC5-evoked Ca²⁺ entry after stimulation with carbachol. Three to four coverslips with at least 25 cells for each coverslip were carried out for each condition.

Representative confocal images of live HEK293 cells expressing eYFP-STIM1 (A) and labelled for the lipid raft marker, GM1 (B) before (Control) and after (+TG) 15 min treatment with the SERCA pump inhibitor, thapsigargin. C, merge of the eYFP-STIM1 (green) and GM1 (red) images before and after TG treatment showing the lack of co-localization between STIM1 and GM1. Results are shown from an experiment utilizing a cross-linking antibody directed against cholera toxin; similar results were obtained in the absence of the cross-linking antibody (not shown).

A, whole-cell OAG-activated currents (means ±s.e.m.) taken at −100 and +100 mV from stable TRPC7 cells transfected with siGLO (n= 3), STIM1 siRNA (n= 4) or Orai1 siRNA (n= 3) 72 h prior to the experiments. The external HBSS contained 2 mm Ca²⁺, and OAG (50 μm) was externally applied as indicated by the line above the graph. B, current–voltage (I–V) relationships taken from stable TRPC7 cells before (leak; broken black trace) and after (siGLO, STIM1 siRNA, Orai1 siRNA) focal OAG application. In all conditions, the outwardly rectifying I–V relationships were nearly identical. C, mean Ba²⁺ entries (fura-2 AM) in response to OAG (50 μm) application in stable TRPC7 expressing HEK293 cells 72 h after transfection with siGLO (Control), STIM1 or Orai1 siRNA. D, summary of data in C, showing no significant difference (ANOVA) of OAG-mediated Ba²⁺ influx in stable TRPC7 expressing cells expressing siGLO (n= 7 coverslips), STIM1 siRNA (n= 8 coverslips) or Orai1 siRNA (n= 4 coverslips). E, summary of OAG-mediated whole-cell currents (−100 mV and +100 mV) in stable TRPC7 cells transfected with eYFP (control) (n= 10), Orai1 (n= 4), STIM1 (n= 6), STIM1 D76N/D78N (n= 4) or STIM2 (n= 4). Nominally Ca²⁺-free HBSS was used as the external bathing solution in these studies. F, I–V relationship showing the currents recorded under the same conditions as E.

A, whole-cell currents in HEK293 cells stably expressing TRPC7. Focally applied thapsigargin (n= 4, grey trace) increases outwardly rectifying currents in these cells, while DMSO does not (n= 5, black trace). Internal Ca²⁺ was clamped to around 100 nm free Ca²⁺, and external solutions were added as indicated by the arrows. Voltage ramps were applied from −100 mV to +100 mV (250 ms), every 2 s to record the currents which developed over time. B, representative current–voltage relationships showing the peak currents recorded after break-in (in the presence of 2 mm Ca²⁺, Control, dashed black trace), after switching to nominally Ca²⁺-free external solution (NF, grey dashed trace), after 1.5 min of focally applied thapsigargin (+TG, grey continuous trace), and after the application of 50 μm OAG (+OAG, black trace). C, experiments similar to A; however, using the ionophore ionomycin (n= 7, black trace) or the SERCA pump antagonist CPA (n= 6, grey trace), instead of thapsigargin. D, fura-2 AM imaging experiment showing the effects of ionomycin application (grey trace) compared to leak control (black trace). Ionomycin did not increase the rate of Ba²⁺ entry compared to the control. Shown are the means of single coverslips which are representative of three similar experiments for each condition. E, experiments (n= 7) similar to those inC (100 nm clamped Ca²⁺), in which ionomycin was used to deplete internal stores. However, in these cells, eYFP-STIM1 was transiently expressed and divalent cation-free (DVF) external solution was focally applied in order to demonstrate that ionomycin did indeed deplete the stores critical for I_CRAC development. The inward currents seen after the application of DVF solution represent I_CRAC. Note there is no depotentiation of these currents because the starting solution was devoid of Ca²⁺ (NF). F, same as in E; however, in conjunction with STIM1, Orai1 was also transiently expressed to further amplify the Na⁺-I_CRAC currents recorded (n= 4). All whole-cell currents shown in this figure are represented as means ±s.e.m.

A, a representative whole-cell current (−100 mV and +100 mV) recorded from an A10 cell before, during and after the focal application of 500 nm arginine–vasopressin (AVP). External solution contained 2 mm Ca²⁺ and the patch pipette contained 100 nm clamped Ca²⁺. AVP was applied as indicated by the line above the graph. Nimodipine (5 μm) was present throughout. B, bar graph depicting the effects of extracellular Ca²⁺ (HBSS + 2 mm Ca²⁺ or HBSS + 50 μm Ca²⁺) on the non-selective cation currents recorded in A10 cells after the application of AVP. C, single channel events recorded in the cell-attached mode from A10 cells bathed in 500 nm AVP application at the indicated holding potentials. The single channel events were not detected in experiments in which AVP was not applied (not shown). D, all points histogram showing the amplitude (pA) of the single channel events recorded after AVP treatment at a holding potential of +60 mV. E, current–voltage relationship of the single channel events and calculated slope conductance of the AVP-activated current.

A, time-controlled experiments showing the changes in near-plasma membrane fluorescence intensities measured by TIRF microscopy in HEK293 cells expressing eYFP-STIM1 and treated with 2 μm thapsigargin to deplete internal Ca²⁺ stores. Each trace represents an individual cell taken from a single coverslip. B, same as in A, however, in cells pre-treated with 10 mm MβCD. Lines above graphs indicate where thapsigargin was applied.

Ca²⁺ imaging experiments (fura-5f) were carried out on HEK293 cells expressing TRPC1 (A), TRPC3 (C), TRPC6 (E) and TRPC5 (G) with (grey traces) and without (black traces) the co-expression of STIM1. Carbachol (200 μm) was applied under nominally Ca²⁺-free (NF) external conditions and 1.8 mm Ca²⁺ was re-administered after 10 min in order to assess TRPC activities. Gd³⁺ was present as indicated in order to block endogenous SOCE. Bar graphs show no significant increase (unpaired t test) in TRPC-evoked Ca²⁺ entries when STIM1 is co-expressed with TRPC1 (B), TRPC3 (D), TRPC6 (F) or TRPC5 (H). Except for TRPC5, bar graphs also summarize experiments carried out following transfection with two different TRPC cDNA plasmid concentrations (Low: 0.32 μg per well or High: 1.0 μg per well using a 6-well plate) on TRPC-mediated Ca²⁺ influx. The data represent means ±s.e.m. from three to four coverslips with at least 25 cells for each coverslip.

A, Gd³⁺ (5 μm) was present throughout the TRPC7 experiments to block endogenous store-operated Ca²⁺ entry. Shown are mean traces (fura-2 AM) from stable TRPC7 HEK293 cells confirming increased TRPC7-dependent (Gd³⁺-insensitive) Ba²⁺ entry in response to 2 μm thapsigargin (leak control: dashed black trace vs. siGLO control: continuous black trace), and showing no significant affect of knocking down STIM1 (STIM1 siRNA: continuous grey trace) or Orai1 (Orai1 siRNA: dashed grey trace). Ba²⁺ was used as a surrogate for Ca²⁺ to avoid Ca²⁺ buffering problems, as previously described for TRPC7 cells (Lievremont et al. 2004). B, bar graph depicting no significant difference (ANOVA) between the rates of Ba²⁺ entry (under the presence of Gd³⁺) in TRPC7 cells transfected with Orai1 (n= 5 coverslips) or STIM1 (n= 7 coverslips) siRNA when compared to control cells (n= 7 coverslips) (siGLO). C, imaging experiments demonstrating that stable TRPC7 cells transfected with the constitutively active mutant of STIM1 (D76N/D78N; grey trace) show a significant increase in basal Ca²⁺ levels when compared to eYFP control cells (black trace), but do not show any increase in the basal activity of TRPC7 as seen by the influx of Ba²⁺ in the presence of 5 μm Gd³⁺. Nominally Ca²⁺-free (NF) and 2 mm external Ca²⁺ conditions, as well as the addition of Ba²⁺ and Gd³⁺ are indicated by the lines above the graph. D, bar graph depicting a significant increase in basal Ca²⁺ concentrations (unpaired t test, P= 0.00013) in TRPC7 cells transiently expressing STIM1 D76N/D78N; however, there was no difference between the rates of Ba²⁺ entry (under the presence of Gd³⁺; unpaired t test, P= 0.42387) in stable TRPC7 cells expressing either eYFP alone (n= 4 coverslips) or in conjunction with STIM1 D76N/D78N (n= 6 coverslips). E, ratiometric measurements showing stable TRPC7 cells transfected with STIM2 (grey trace) have no change in the rate of Ba²⁺ entry compared to eYFP control cells (black trace). F, bar graph depicting no significant difference (ANOVA) between the rates of Ba²⁺ entry in TRPC7 cells expressing either eYFP (n= 5 coverslips), or in conjunction with STIM1 (n= 5 coverslips), STIM2 (n= 5 coverslips) or Orai1 (n= 5 coverslips). Leak entry rates between conditions were also not significantly different (not shown). Each coverslip (each n) was taken as the mean of at least 25 individual cells.

A, representative whole-cell CRAC current in a HEK293 cell expressing STIM1. A divalent-free (DVF) solution exchange protocol was used, as previously described (DeHaven et al. 2007), in order to amplify the extremely small endogenous CRAC current in these cells. STIM1 was expressed to further amplify the small CRAC currents in HEK293 cells. Current was recorded at −100 and +100 mV and stores were depleted with 20 μm IP₃ and 20 mm BAPTA in the pipette. Lines above trace indicate where different extracellular solutions were focally applied. Gd³⁺ (10 μm) was used to block the Na⁺I_CRAC, revealing a linear increase in leak currents under these external conditions in which all Ca²⁺ and Mg²⁺ is removed and 10 μm Gd³⁺ is the only cation present. B, same as A, except in a HEK293 cell stably expressing TRPC7. Note that there is constitutive TRPC7 activity at break in, but this activity diminishes over time. In stable TRPC7-expressing HEK293 cells, normally functioning inwardly rectifying Na⁺-CRAC currents are detectable with similar current densities to control cells (A). C, current–voltage (I–V) relationships taken at specific time points indicated as i, ii and iii for current trace shown in B. D, I–V for the Gd³⁺-sensitive current shown in B and C.

A, whole-cell I_CRAC measurements taken at −120 mV from A10 smooth muscle cells with (n= 6; grey trace) or without (n= 7; black trace) the presence of 3 μm Gd³⁺. Stores were actively depleted with IP₃ and BAPTA in the patch pipette, and 5 μm nimodipine was present in the external HBSS containing 10 mm Ca²⁺. B, I_CRAC experiment showing in smooth muscle cells the Na⁺ currents are half-maximally blocked by 20 μm external Ca²⁺ at −120 mV. Ramps were applied from positive to negative potentials (see Methods) to avoid voltage-gated channel contamination of the CRAC currents. C, Ca²⁺ imaging experiment (fura-2 AM) showing the effects of RNAi targeted against STIM1 on thapsigargin-evoked SOCE in A10 cells. External solutions were exchanged as indicated by the lines above the graph. D, bar graph showing STIM1 siRNA-treated cells (-S1, n= 4 coverslips, grey bar) have significantly less (unpaired t test; P= 0.00152) SOCE compared to siControl-treated control (C, n= 5 coverslips, black bar) cells. E, representative whole-cell currents obtained as in Fig. 7, in which AVP-activated currents were reduced in cells treated with siRNA against TRPC6 (n= 9; broken black trace), but not in cells treated with STIM1 siRNA (n= 5; continuous grey trace), compared to siControl-treated cells (n= 7; continuous black trace). F, bar graph showing the summary of the data collected in A10 cells for the AVP-activated non-selective cation currents in which STIM1 siRNA had little effect, but TRPC6 siRNA reduced the overall current densities.

A, whole-cell voltage clamp experiments (−120 to +120 mV voltage ramp applied every 2 s) taken from HEK293 cells stably expressing TRPC3 and pre-treated with vehicle (0, black continuous trace), 1 mm (1, grey dashed trace), 5 mm (5, black dashed trace) or 10 mm (10, grey continuous trace) MβCD for 30 min. Whole-cell TRPC3 currents were activated by focal application of 30 μm OAG as indicated by the line above the graph. B, current–voltage relationships taken from the recordings shown in A after OAG application. Currents were not leak subtracted. C, bar graph showing significant inhibition of TRPC3 currents in cells treated with millimolar concentrations of MβCD (Control: n= 7; 5 mm MβCD: n= 8; 10 mm MβCD: n= 6). D, Na⁺-I_CRAC measurements taken from HEK293 cells expressing eYFP-STIM1 (+100 to −120 mV every 2 s) with (grey trace) or without (black trace) 10 mm MβCD treatment prior to break-in. Stores were depleted using 25 μm IP₃ in the patch pipette and all external divalent cations were removed in order to amplify CRAC currents. External solution exchanges were focally applied as indicated by the lines above the graph. E, leak subtracted I–V plots taken from the peak Na⁺ CRAC currents shown in D. F, bar graph showing in eYFP-STIM1-expressing cells that sequestration of cholesterol from the lipid bilayer by pre-treating the cells with MβCD has no effect on I_CRAC (Control: n= 7; 5 mm MβCD: n= 10; 10 mm MβCD: n= 5). Plotted are peak Na⁺ CRAC currents. Error bars represent means ±s.e.m. and data analyses were carried out using ANOVA followed by Tukey's test for pairwise comparisons.