A basic sequence in STIM1 promotes Ca2+ influx by interacting with the C-terminal acidic coiled coil of Orai1

Abstract

Store-operated Ca(2+) entry (SOCE) is a ubiquitous signaling process in eukaryotic cells in which the endoplasmic reticulum (ER)-localized Ca(2+) sensor, STIM1, activates the plasma membrane-localized Ca(2+) release-activated Ca(2+) (CRAC) channel, Orai1, in response to emptying of ER Ca(2+) stores. In efforts to understand this activation mechanism, we recently identified an acidic coiled-coil region in the C-terminus of Orai1 that contributes to physical association between these two proteins, as measured by fluorescence resonance energy transfer, and is necessary for Ca(2+) influx, as measured by an intracellular Ca(2+) indicator. Here, we present evidence that a positively charged sequence of STIM1 in its CRAC channel activating domain, human residues 384-386, is necessary for activation of SOCE, most likely because this sequence interacts directly with the acidic coiled coil of Orai1 to gate Ca(2+) influx. We find that mutation to remove positive charges in these residues in STIM1 prevents its stimulated association with wild-type Orai1. However, association does occur between this mutant STIM1 and Orai1 that is mutated to remove negative charges in its C-terminal coiled coil, indicating that other structural features are sufficient for this interaction. Despite this physical association, we find that thapsigargin fails to activate SOCE following coexpression of mutant STIM1 with either wild type or mutant Orai1, implicating STIM1 residues 384-386 in transmission of the Ca(2+) gating signal to Orai1 following store depletion.

Figure 1

Thapsigargin-mediated FRET. Thapsigargin (150 nM)-stimulated FRET between STIM1-mRFP and AcGFP-Orai1 constructs in RBL mast cells: wtSTIM1-mRFP and AcGFP-wtOrai1 (black line), wtSTIM1-mRFP and AcGFP-Orai1ΔDE (mut-Orai1; grey line), STIM1 K(384-6)Q-mRFP (mut-STIM1) and AcGFP-Orai1ΔDE (○), and STIM1 K(384-6)Q-mRFP and AcGFP-wtOrai1 (●). Error bars show SEM for 12 to 18 cells from 4-5 different experiments, and every fifth error bar is shown for clarity.

Figure 2

Cellular distributions of Orai1 and STIM1. Ventral surface confocal microscopy images of transfected RBL cells showing distributions of (A-B) AcGFP-Orai1ΔDE (green) and wtSTIM1-mRFP (red), (C-D) AcGFP-wtOrai1 (green) and STIM1 K(384-6)Q-mRFP (red), or (E-F) AcGFP-Orai1ΔDE and STIM1 K(384-6)Q-mRFP in the absence (A,C,E) or presence (B,D,F) of 150 nM thapsigargin for 30 min at 37°C. Scale bars represent 10 m.

Figure 3

Stimulated Ca²⁺ responses. A) Ca²⁺ responses to 150 nM thapsigargin in COS7 cells expressing wtSTIM1-mRFP and wtOrai1 (), STIM1 K(384-6)Q-mRFP and wtOrai1 (), wtSTIM1-mRFP and Orai1ΔDE (), STIM1 K(384-6)Q-mRFP and Orai1ΔDE (), and untransfected cells (). Fluo-4 fluorescence was normalized relative to the average prestimulation basal Ca²⁺ level in untransfected cells in the same field. Each time course represents the average of 10-18 cells, and error bars show SEM. Cells with basal Ca²⁺ levels >3x larger than the average for untransfected cells were excluded. B) Mean Ca²⁺ levels before and 10 min after addition of 150 nM thapsigargin for averaged individual COS7 cells expressing a combination of wtSTIM1-mRFP or mutant STIM1 K(384-6)Q-mRFP with wtOrai1 or mutant Orai1ΔDE. Each bar represents the mean for 20-60 cells from 3 or more different experiments, and error bars show SEM.

Figure 4

Cartoon depicting basic features of proposed model for wild type and mutant STIM1 and Orai1 interactions. A) In unstimulated cells, the negatively charged acidic amino acids on wtOrai1 and the positively charged lysine sequence (K384-386) in the STIM1 CAD domain (dark grey) defined by our mutations are shown associated with solvated counter ions. Upon stimulation, these ions are displaced from the binding cleft between the two proteins, allowing these charged regions to interact with each other and causing the Ca²⁺-selective channel to open in an allosteric manner. B) When K384-386 in STIM1 is mutated to glutamines, STIM1 no longer binds and activates wtOrai1 due to the loss of electrostatic attraction and resulting repulsion at this interaction subsite. C) When these charged sequences on Orai1 and STIM1 are both mutated, STIM1 CAD regains the capacity to bind Orai1 via interactions at other sites because the electrostatic repulsion has been eliminated. However, this association does not cause allosteric activation of the Ca²⁺ gate. The oligomerization state of these proteins is omitted for clarity: Orai1 is oligomerized in the active wtSTIM-wtOrai1 complex and in all cases involving Orai1ΔDE.