Phosphorylation-mediated structural changes within the SOAR domain of stromal interaction molecule 1 enable specific activation of distinct Orai channels

Abstract

Low-conductance, highly calcium-selective channels formed by the Orai proteins exist as store-operated CRAC channels and store-independent, arachidonic acid-activated ARC channels. Both are activated by stromal interaction molecule 1 (STIM1), but CRAC channels are activated by STIM1 located in the endoplasmic reticulum membrane, whereas ARC channels are activated by the minor plasma membrane-associated pool of STIM1. Critically, maximally activated CRAC channel and ARC channel currents are completely additive within the same cell, and their selective activation results in their ability to each induce distinct cellular responses. We have previously shown that specific ARC channel activation requires a PKA-mediated phosphorylation of a single threonine residue (Thr³⁸⁹) within the cytoplasmic region of STIM1. Here, examination of the molecular basis of this phosphorylation-dependent activation revealed that phosphorylation of the Thr³⁸⁹ residue induces a significant structural change in the STIM1-Orai-activating region (SOAR) that interacts with the Orai proteins, and it is this change that determines the selective activation of the store-independent ARC channels versus the store-operated CRAC channels. In conclusion, our findings reveal the structural changes underlying the selective activation of STIM1-induced CRAC or ARC channels that determine the specific stimulation of these two functionally distinct Ca²⁺ entry pathways.

Keywords: A-kinase anchoring protein (AKAP); Orai channels; calcium release-activated calcium channel protein 1 (ORAI1); calcium signaling; phosphorylation; stromal interaction molecule 1 (STIM1); structural biology.

Figure 1.

The effect of mutation of the Lys³⁸⁶ residue located in the BR of STIM1 on CRAC channel currents. The data shown are the whole-cell inward CRAC channel currents (mean ± S.D., n = 8) measured at −80 mV in WT cells and in the same cells bearing the indicated mutations of the Lys³⁸⁶ residue in STIM1 (n = 6–9). Error bars represent the mean ± S.D. channel currents induced by inclusion of adenophostin A (2 μm) in the patch pipette solution with individual measurements indicated by open circles.

Figure 2.

The effect of BR mutations (Lys³⁸² and Lys³⁸⁴–Lys³⁸⁶) on ARC channel currents either alone or in cells incorporating the T389E phosphomimetic mutation. Currents in cells expressing the ARC-specific T389E-STIM1 are shown for comparison. Channel currents were activated by exogenous addition of arachidonic acid (8 μm) in cells expressing a Lck-STIM1 construct, which specifically records ARC channel activity induced by plasma membrane–located STIM1 (8). A, error bars represent the mean ± S.D. current magnitudes measured at −80 mV (n = 5–7) with individual measurements indicated by open circles. B, representative current–voltage relationship for each of the three constructs.

Figure 3.

A, effects of the STIM1 L390Q mutation on AA-activated ARC channel currents either alone or when included with T389E mutation. B, effects of the STIM1 L390Q mutation on adenophostin-activated CRAC channel activity either alone or when included with T389L mutation. Whole-cell inward ARC and CRAC channel currents (error bars represent the mean ± S.D.; n = 4–6) were measured at −80 mV using the standard protocols in WT cells and in the same cells bearing the STIM1 L390Q mutation and the same mutation along with either the T389E mutation (ARC channels; A) or the T389L mutation (CRAC channels; B). Individual measurements are indicated by open circles. ARC channel and CRAC channel currents were activated by standard procedures (see “Experimental procedures”).

Figure 4.

The effects of mutations of the Leu³⁹⁰ residue in STIM1 on adenophostin-activated CRAC channel currents and AA-activated ARC channel currents compared with the same in the wildtype channels. Data shown are individual measurements (circles) of inward currents (with mean ± SD, n = 6–9, indicated by error bars) measured at −80 mV in cells expressing the wildtype STIM1 or STIM1 bearing the indicated mutations of the Leu³⁹⁰ residue. ARC channel and CRAC channel currents were activated by standard procedures (see “Experimental procedures”).

Figure 5.

A, SEC of the STIM1 sequence 234–491 in wildtype STIM1 and the T389A-, T389E-, or L390Q-STIM1 mutant. Apparent molecular masses of WT, T389E, T389A, and L390Q were 2.4, 2.5, 2.4, and 2.3× the theoretical monomeric molecular mass of STIM1(234–491) (30.8 kDa), consistent with the well-established notion that the cytosolic domains of STIM1 recapitulate the dimeric nature of full-length STIM1 when expressed in live mammalian cells. Data are representative of at least two distinct protein preparations. B, secondary structure obtained by far-UV CD spectroscopy for the STIM1 sequence 234–491 in wildtype STIM1 and the T389A, T389E, or L390Q mutant. Far-UV CD spectra of STIM1(234–491) were acquired at 0.3–0.5 mg ml⁻¹. Data are representative of at least two distinct protein preparations. C, thermal stability analyses by changes in far-UV CD spectroscopy at 222 nm for the STIM1 sequence 234–491 in wildtype STIM1 and the T389E-, T389A-, or L390Q-STIM1 mutants. Thermal stability was measured at protein concentrations of 0.3–0.5 mg ml⁻¹. The apparent T_m values were 48.0, 50.5, 48.0, and 47.0 °C for the wildtype, T389E, T389A, and L390Q proteins, respectively. Data are representative of at least two distinct protein preparations. A.U., absorbance units.

Figure 6.

Side-chain contact probability maps determined from MD simulations. The probability that a particular pair of side chains is in contact over the course of the simulation is indicated by the grayscale (lighter indicates the contact is more probable). The results are the average of results from the four replicas for each protein type. Note the increased contact probability in both the phos-STIM1 and the T389E-STIM1 data in the region spanning residues Phe³⁹⁴–Ser⁴⁰⁰ compared with the same region in wildtype STIM1.

Figure 7.

Contact probability “difference maps” obtained from MD simulations. These plots are the differences between the maps shown in Fig. 6, focusing on residues 386–399. Red indicates that a particular contact is more likely in the first system listed, whereas blue indicates that the contact becomes less populated. A represents the difference (diff) between the phosphorylated STIM1 sequence and the same sequence in wildtype STIM1, whereas B represents the difference between the same sequence in the phosphomimetic T389E mutant STIM1 and wildtype STIM1. The image in C shows the difference between the data for the phosphorylated STIM1 and the corresponding data for the T389E-STIM1.

Figure 8.

A, whole-cell current–voltage relationships for both CRAC channels and ARC channels in WT cells. B, the same currents in cells bearing the G392A-STIM1 mutation. C, the same currents obtained with G392A along with the “CRAC-specific” T389A mutation. The data shown are the whole-cell adenophostin-activated CRAC channel currents (open circles) and AA-activated ARC channel currents (filled circles). Error bars represent the mean ± S.E. (n = 6–7) in each case.

Figure 9.

Representative molecular dynamic snapshots of the STIM1 CC2 terminal helical region. The individual images demonstrate the STIM1 CC2 helical region in wildtype STIM1 and the elongation of this region upon phosphorylation of residue Thr³⁸⁹ (phos-STIM1) or introduction of the T389E phosphomimetic mutation (T389E-STIM1). For comparison, the three images have been aligned at the position of the Thr³⁸⁹ residue (blue dashed line).