A plasma membrane-targeted cytosolic domain of STIM1 selectively activates ARC channels, an arachidonate-regulated store-independent Orai channel

Figures & data

Figure 1. ARC and CRAC channel currents in HEK293 cells expressing the STIM1-CT construct. (A) Mean (± SE) I/V curves for the endogenous AA-activated ARC channels (black) and store-operated CRAC channels (gray) in HEK293 cells. (B) Representative western blot showing STIM1 levels in untransfected cells (C), in the same cells following treatment with a STIM1 siRNA (siR) and in the siRNA-treated cells following expression of the siRNA-resistant STIM1-CT construct (+ST). (C) Mean (± SE) ARC currents and CRAC currents measured at -80 mV in untransfected cells (white columns), in STIM1 siRNA-treated cells (gray columns) and in siRNA-treated cells following expression of the siRNA-resistant STIM1-CT construct (black columns). (D) Representative live confocal image of cells expressing the eGFP-tagged STIM1-CT construct.

Figure 2. Expression of the Lck-STIM1-C construct in HEK293 cells. (A) Representative live confocal image of cells expressing the Lck-STIM1-CT construct bearing a C-terminal eGFP-tag. (B) Mean (± SE) AA-activated ARC channel currents (black) and store-operated CRAC channel currents (gray) measured at -80 mV in siRNA-treated cells (siRNA), and in the same cells following expression of an siRNA-resistant wild-type STIM1 (+wt STIM1) or the siRNA-resistant Lck-STIM1-C construct (+Lck-STIM1-C). (C) Mean (± SE) I/V curves for the ARC channels (black) and CRAC channels (gray) in the siRNA-treated cells expressing the wild-type STIM1, and (D) the corresponding I/V curves for the ARC channels and CRAC channels in the siRNA-treated cells expressing the Lck-STIM1-C construct.

Figure 3. Effect of the Lck-STIM1-C construct on CRAC channel activity in RBL cells. (A) Mean (± SE) store-operated CRAC channel currents measured at -80 mV in untransfected cells (untran), in cells expressing a control siRNA (cont siRNA), a STIM1 siRNA (siRNA) and in the same STIM1 siRNA-treated cells expressing an siRNA-resistant wild-type STIM1 (siRNA⁺WT) or an siRNA-resistant Lck-STIM1-C construct (siRNA⁺Lck). (B) Mean (± SE) I/V curves for CRAC channels in untransfected RBL cells (black), in STIM1 siRNA-treated cells (open circles) and in the same cells expressing the siRNA-resistant Lck-STIM1-C construct (gray circles). (C) Representative live confocal image of RBL cells expressing the Lck-STIM1-CT construct bearing a C-terminal eGFP-tag.

Figure 4. Expression of the S to A mutant Lck-STIM1-C construct in HEK293 cells. (A) Representative live confocal image of cells expressing the mutant Lck-STIM1-CT construct bearing a C-terminal eGFP-tag. (B) Mean (± SE) AA-activated ARC channel currents measured at -80 mV in siRNA-treated cells expressing the siRNA-resistant Lck-STIM1-C construct (black), or the same siRNA-treated cells expressing the S to A mutant Lck-STIM1-C (gray). (C) The corresponding mean (± SE) I/V curves for the Lck-STIM1-C-expressing cells (black) and the mutant Lck-STIM1-C-expressing cells (gray). (D) Representative western blot showing relative expression of eGFP-tagged wild-type Lck-STIM1-C and the S to A mutant Lck-STIM1-C levels transfected into cells, as assessed by an eGFP Ab.

Figure 5. Carbachol-activated currents in HEK cells expressing the Lck-STIM1-C construct. (A) Mean (± SE) carbachol-activated Ca²⁺ currents measured at -80 mV in HEK cells expressing the m3 muscarinic receptor. Shown are the total agonist-activated currents (gray), and the same currents recorded in cells after depletion of ER Ca²⁺ stores by intracellular adenophostin A (black) in untransfected control cells (control) and in STIM1 siRNA-treated cells expressing the Lck-STIM1-C construct. (B) Mean (± SE) I/V curves for the total agonist-activated currents (gray), and adenophostin-insensitive currents (black) in control cells and (C) the same for the Lck-STIM1-C expressing cells (see text for details). (D) Mean (± SE) curves comparing the activation kinetics of carbachol-activated currents in m3-HEK cells expressing the Lck-STIM1-C construct compared with those in wild-type m3-HEK cells. Total currents were normalized to their final maximal values to allow direct comparison of their respective rates of activation following addition of carbachol (10 µM) at time zero.