A minimal regulatory domain in the C terminus of STIM1 binds to and activates ORAI1 CRAC channels

Abstract

Store-operated Ca(2+) entry (SOCE) is a universal mechanism to increase intracellular Ca(2+) concentrations in non-excitable cells. It is initiated by the depletion of ER Ca(2+) stores, activation of stromal interaction molecule (STIM) 1 and gating of the Ca(2+) release activated Ca(2+) (CRAC) channel ORAI1 in the plasma membrane. We identified a minimal activation domain in the cytoplasmic region of STIM1 (CCb9) which activated Ca(2+) influx and CRAC currents (I(CRAC)) in the absence of store depletion similar to but more potently than the entire C terminus of STIM1. A STIM1 fragment (CCb7) that is longer by 39 [corrected] amino acids than CCb9 at its C terminal end showed reduced ability to constitutively activate I(CRAC) consistent with our observation that CCb9 but not CCb7 efficiently colocalized with and bound to ORAI1. Intracellular application of a 31 amino acid peptide contained in CCb7 but not CCb9 inhibited constitutive and store-dependent CRAC channel activation. In summary, these findings suggest that CCb9 represents a minimal ORAI1 activation domain within STIM1 that is masked by an adjacent 31 amino acid peptide preventing efficient CRAC channel activation in cells with replete Ca(2+) stores.

Figure 1. Identification of CRAC channel activating regions in STIM1

(A) Schematic representation of STIM1 protein domains and C-terminal fragments generated in this study. CC, coiled-coil; CT, STIM1 C-terminus; EF, EF hand; ERM, ezrin/radixin/moesin; SAM, sterile alpha motif; TM, transmembrane; S/P, serine-proline; K, lysine. (B) Western blot of STIM1 C-terminal fragments. HEK293 cells were transfected with FLAG-Cherry-tagged STIM1 fragments, cell lysates separated by SDS-PAGE and STIM1 fragments detected with anti-FLAG antibody. (C) Store-independent and store-dependent Ca²⁺ influx in cells expressing STIM1 fragments. HEK293 cells were transfected with Cherry-tagged STIM1 fragments and [Ca²⁺]_i was measured by single cell time-lapse imaging in 2 mM Ca²⁺_o before and after addition of 1 μM thapsigargin (TG). Traces represent mean 340/380 Fura-2 emission ratios from one representative experiment; error bars represent s.e.m. (D) Summary of experiments similar to those shown in (C). Bar graphs represent average peak [Ca²⁺]_i in 2 mM Ca²⁺_o before (dark gray) and after thapsigargin (light gray) addition from 3-6 independent experiments; error bars represent standard deviation.

Figure 2. Constitutive Ca²⁺ influx and CRAC channel activation by STIM1 fragments CCb7 and CCb9

(A) CCb7 and CCb9 fragments induce store-independent, constitutive Ca²⁺ influx when co-expressed with ORAI1. HEK293 cells were transfected with ORAI1-IRES-GFP and Cherry-tagged STIM1 fragments and [Ca²⁺]_i was measured as described in Fig 1. Traces are representative of > 3 similar experiments; error bars represent s.e.m. (B) Summary of experiments similar to those shown in (A). Bar graphs represent peak [Ca²⁺]_i before (dark gray) and after (light gray) addition of thapsigargin (TG) in the presence of 2 mM Ca²⁺_o. (C) Constitutive CRAC channel activation by CCb7 and CCb9. HEK293 cells were transfected as described above. Shown are the time courses of currents obtained after establishing whole-cell configuration. To prevent passive store depletion during I_CRAC recordings, [Ca²⁺]_I was clamped to ∼ 150 nM by addition of 10 mM BAPTA and 4 mM Ca²⁺ to the internal pipette solution. (D) Representative current-voltage relationships (I-V) extracted from currents shown in (C). (E) Averages of current amplitudes at -80 mV obtained in the first 100 seconds after break-in from experiments shown in (C,D). STIM1-CT (n=10), CCb7 (n=21), CCb9 (n=21), CCb10 (n=6), full-length STIM1 + IP₃ (n=8); error bars represent s.e.m.

Figure 3. STIM1_445-475 peptide interferes with store-dependent and independent CRAC channel activation

(A-C) Synthetic STIM1_445-475 peptide inhibits constitutive I_CRAC HEK293 cells were transfected with ORAI1 and CCb9 and I_CRAC was recorded in the presence of 20 μM STIM1_445-475 peptide or control myelin oligodendrocyte glycoprotein (MOG) peptide in the internal pipette solution. (A) Averages of time-dependent current development (STIM1_445-475, n=17; MOG, n=18). (B) Representative I-Vs from the same experiments shown in A extracted at 200 sec. (C) Averages of current values recorded during the time indicated by the black bar in (A). Error bars represent s.e.m. (D-F) Store-dependent activation of native CRAC channel currents in T cells is inhibited by STIM1_445-475 peptide. Endogenous I_CRAC was activated in Jurkat T cells by passive store depletion with 10 mM BAPTA in the pipette solution in the presence of 20 μM STIM1_445-475 or MOG control peptide. (D) Averages of time-dependent current development extracted at -80 mV. (E) Representative I-Vs from the same experiments shown in (D) at 275 sec. (F) Averages of I_CRAC values recorded from 280-300 sec (black bar) in experiments shown in (D). STIM1_445-475, n=13; MOG, n=13. Error bars represent s.e.m.

Figure 4. STIM1 fragment CCb9 but not CCb7 efficiently colocalizes with and binds to ORAI1

(A) CCb9 colocalizes with ORAI1 at the plasma membrane. HEK293 cells were transfected with Cherry-STIM1 fragments CCb7, CCb9 and CCb10 alone or together with GFP-ORAI1. The subcellular distribution of CCb fragments was analyzed by confocal microscopy. Representative cells from 2 independent experiments are shown. (B) Quantification of subcellular distribution of CCb fragments from the experiment shown in (A). ≥ 200 cells per condition were analyzed as described in Materials and Methods. (C) STIM1 fragment CCb9 binds to ORAI1. For pulldown experiments, HEK293 cells were transfected with FLAG-tagged wild-type ORAI1 or mutant ORAI1-L273S. Cell lysates were incubated with purified GST-tagged STIM1 fragments or GST alone followed by pull-down of protein complexes with glutathione sepharose resin. Proteins were detected by SDS-PAGE, immunoblotting with anti-FLAG antibody (top) and Poinceau Red staining (bottom). (D) Model of ORAI1 activation by STIM1. In the resting state with replete Ca²⁺ stores, the CCb9 ORAI1-binding domain in STIM1 is masked by the STIM1_445-475 peptide. Upon store depletion, the CCb9 domain is released, binds to and activates the ORAI1 CRAC channel. For abbreviations see Fig. 1.