Figure 3. TRPC7 previously reported to function in a store-dependent manner is not regulated by STIM or Orai.

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.