Figure 6. Thapsigargin-evoked increases in TRPC7 current.

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.