STIM1 gates the store-operated calcium channel ORAI1 in vitro

Abstract

Store-operated Ca(2+) entry through the plasma membrane Ca(2+) release-activated Ca(2+) (CRAC) channel in mammalian T cells and mast cells depends on the sensor protein stromal interaction molecule 1 (STIM1) and the channel subunit ORAI1. To study STIM1-ORAI1 signaling in vitro, we have expressed human ORAI1 in a sec6-4 strain of the yeast Saccharomyces cerevisiae and isolated sealed membrane vesicles carrying ORAI1 from the Golgi compartment to the plasma membrane. We show by in vitro Ca(2+) flux assays that bacterially expressed recombinant STIM1 opens wild-type ORAI1 channels but not channels assembled from the ORAI1 pore mutant E106Q or the ORAI1 severe combined immunodeficiency (SCID) mutant R91W. These experiments show that the STIM1-ORAI1 interaction is sufficient to gate recombinant human ORAI1 channels in the absence of other proteins of the human ORAI1 channel complex, and they set the stage for further biochemical and biophysical dissection of ORAI1 channel gating.

Figure 1

Recombinant ORAI1 and STIM1 proteins used in the present study. (a) Above, ORAI1 (red shading) is incorporated into the plasma membrane of sec6-4 yeast at the permissive temperature, 25 °C, but accumulates in vesicles within the cell at the nonpermissive temperature, 37 °C. Cytoplasmic portions of ORAI1 in isolated vesicles face the external solution. Below, immunocytochemical localization of Myc-ORAI1 in sec6-4 cells grown at 25 °C and at 37 °C, respectively. (b) Western blot (WB) for Myc-ORAI1 in vesicles isolated from control yeast or from yeast expressing wild-type ORAI1, ORAI1_R91W or ORAI1_E106Q. (c) Above, sequence conservation in the STIM C-terminal region. Each horizontal black bar represents the human STIM1 sequence, with gaps introduced to maintain alignment with fish or insect orthologs. Vertical magenta lines indicate identity between human STIM1 and at least four of five fish orthologs; vertical blue lines indicate identity with at least two of three insect Stim proteins. Below, STIM1 cytoplasmic fragments with predicted coiled-coil (CC), Ser-Pro-rich (SP) and polybasic (K) regions indicated. (d) SEC-MALLS analysis of STIM1^CT. Recombinant STIM1^CT (red) migrated as a single symmetrical peak, with no evidence of aggregated protein in the void volume at ~5–8 ml. Plotted molecular mass estimates refer to the axis at right. STIM1^CT experimental molecular weight (MW), 110.5 kDa; theoretical monomer MW, 54.7 kDa. Standards were BSA monomer and dimer (black) and glutathione S-transferase dimer (GST, gray). (e) SEC-MALLS profiles of STIM1_233–498 (experimental MW, 70.1 kDa; theoretical monomer MW, 34.8 kDa) and STIM1_233–463 (experimental MW, 87.5 and 119.6 kDa; theoretical monomer MW, 30.9 kDa). Standards (omitted from the plot for clarity) were the same as in d.

Figure 2

STIM1 cytoplasmic fragments interact with ORAI1 assembled in yeast membranes and with two cytoplasmic fragments of ORAI1. (a) Above, the part of ORAI1 expressed as a membrane protein in P. pastoris is highlighted in red. Below, P. pastoris membranes containing Flag-ORAI1_65–301 E106Q or control membranes, with or without His₆-STIM1 protein, were loaded at the bottom of a discontinuous sucrose density gradient and subjected to centrifugation. Flag-ORAI1 and His₆-STIM1 in individual gradient fractions were detected by western blotting (WB). The fraction of unbound STIM1 remaining at the bottom of the gradient is probably due to the presence of a moderate excess of STIM1 over ORAI1 in the assay. (b) Above, the C-terminal cytoplasmic tail of ORAI1 expressed as a GST fusion protein is highlighted in red. Below, the indicated STIM1 fragments were incubated with immobilized GST-ORAI1_259–301 or with GST. Bound proteins were analyzed by SDS-PAGE and staining with Coomassie Brilliant Blue R-250. Samples on the input gel correspond to 20% of protein in the binding assay. (c) Above, the segment ORAI1_65–87 is highlighted in red. Below, STIM1 fragments were incubated with immobilized GST-ORAI1_65–87 or with GST and analyzed as in Figure 2b. Samples on the input gel correspond to 5% of protein in the binding assay.

Figure 3

STIM1 triggers ORAI1-dependent Ca²⁺ efflux from membrane vesicles of S. cerevisiae. (a) Above, principle of the Ca²⁺ flux assay using Fura-2. Initially, the external Ca²⁺ concentration is low and the peak of the free Fura-2 excitation spectrum is ~365 nm. When STIM1 gates the ORAI1 channel, Ca²⁺ is released from the vesicles, Fura-2 binds Ca²⁺ and the excitation peak is shifted to ~340 nm. Middle, Fura-2 excitation spectra of control and ORAI1-containing vesicles, with no addition or after the addition of ionomycin (20 μM) or STIM1 cytoplasmic fragments (2 μM). Below, graph of the fluorescence intensity ratio of Fura-2 (F_340nm/F_380nm) in each condition. Error bars indicate the range of duplicate measurements. (b) Above, principle of the Ca²⁺ flux assay using the FRET-based Ca²⁺ sensor cameleon D4cpV. In unstimulated vesicles, internal Ca²⁺ concentration is sufficient for binding to a fraction of the sensor, and cyan fluorescent protein-yellow fluorescent protein (CFP-YFP) FRET is evident in the peak at ~528 nm. When STIM1 gates the ORAI1 channel, Ca²⁺ is released from the vesicles, Ca²⁺ dissociates from the sensor, and there is a decline in the YFP peak at ~528 nm accompanied by an increase in the CFP peak at ~475 nm. Middle, D4cpV fluorescence emission spectra of control and ORAI1-containing vesicles, with no addition or after the addition of ionomycin (20 μM) or STIM1 cytoplasmic fragments (2 μM). Below, graph of the fluorescence intensity ratio of D4cpV (F_528nm/F_475nm) in each condition. Error bars indicate the range of duplicate measurements.

Figure 4

STIM1 activates ORAI1 channels in membrane vesicles from S. cerevisiae. The experiment was carried out as in Figure 3b except for the use of D3cpV sensor. Fluorescence emission spectra were obtained from vesicles containing wild-type ORAI1, ORAI1 R91W or ORAI1 E106Q, either with no addition or following addition of STIM1^CT (2.6 μM). The bar graph plots mean fluorescence intensity ratio F_528nm/F_475nm ± s.e.m. for each condition. STIM1^CT changes the ratio significantly for wild-type ORAI1 (P < 0.001, two-tailed Welch’s t-test), but not for ORAI1 R91W and ORAI1 E106Q.