A Cytosolic Homomerization and a Modulatory Domain within STIM1 C Terminus Determine Coupling to ORAI1 Channels

Abstract

In immune cells, generation of sustained Ca(2+) levels is mediated by the Ca(2+) release-activated Ca(2+) (CRAC) current. Molecular key players in this process comprise the stromal interaction molecule 1 (STIM1) that functions as a Ca(2+) sensor in the endoplasmic reticulum and ORAI1 located in the plasma membrane. Depletion of endoplasmic reticulum Ca(2+) stores leads to STIM1 multimerization into discrete puncta, which co-cluster with ORAI1 to couple to and activate ORAI1 channels. The cytosolic C terminus of STIM1 is sufficient to activate ORAI1 currents independent of store depletion. Here we identified an ORAI1-activating small fragment (OASF, amino acids 233-450/474) within STIM1 C terminus comprising the two coiled-coil domains and additional 50-74 amino acids that exhibited enhanced interaction with ORAI1, resulting in 3-fold increased Ca(2+) currents. This OASF, similar to the complete STIM1 C terminus, displayed the ability to homomerize by a novel assembly domain that occurred subsequent to the coiled-coil domains. A smaller fragment (amino acids 233-420) generated by a further deletion of 30 amino acids substantially reduced the ability to homomerize concomitant to a loss of coupling to as well as activation of ORAI1. Extending OASF by 35 amino acids (233-485) did not alter homomerization but substantially decreased efficiency in coupling to and activation of ORAI1. Expressing OASF in rat basophilic leukemia (RBL) mast cells demonstrated its enhanced plasma membrane targeting associated with 2.5-fold larger CRAC currents in comparison with the complete STIM1 C terminus. In aggregate, we have identified two cytosolic key regions within STIM1 C terminus that control ORAI1/CRAC activation: a homomerization domain indispensable for coupling to ORAI1 and a modulatory domain that controls the extent of coupling to ORAI1.

FIGURE 1.

a, time course of constitutive whole-cell inward currents at –74 mV of HEK293 cells expressing the following C-terminal STIM1 fragments: 233–420, 233–450, 233–474, 233–485, and 233–685 (STIM1 C terminus wild-type) with ORAI1. b, respective I/V curves of a from representative cells taken at t = 0 s. c, block diagram summarizing constitutive STIM1 C terminus-mediated current densities at t = 0 s from a. d, block diagram displaying FRET of STIM1 fragments with ORAI1. e, localization, overlay, and calculated FRET life cell image series of YFP-STIM1 fragments and CFP-ORAI1. Additional intensity plots represent localization of STIM1 fragments across the cell as indicated by the dashed line. f, a model depicting STIM1 fragments in correlation with their function regarding stimulation of as well as coupling to ORAI1. a.u., arbitrary units.

FIGURE 2.

a, localization, overlay, and calculated FRET life cell image series of YFP- and CFP-STIM1 fragments 233–420, 233–474, and 233–485. b, a block diagram summarizing homomerization FRET of STIM1 fragments: 233–420, 233–450, 233–474, 233–485, and 233–685 (complete STIM1 C terminus). c, PFO plot depicting monomers and possible dimers for the indicated STIM1 fragments. d, localization, overlay, and calculated FRET life cell image series of YFP- and CFP-STIM1 fragments 233–420, 233–474, and 233–485 in the presence of co-expressed ORAI1. e, block diagram summarizing homomerization FRET of STIM1 fragments as in b but now in the presence of co-expressed ORAI1. f, model depicting STIM1 fragments in correlation with their ability to homomultimerize.

FIGURE 3.

a, time course of constitutive whole-cell inward currents at –74 mV of RBL cells expressing the following C-terminal STIM1 fragments: 233–420, 233–450, 233–474, 233–485, and 233–685 (STIM1 C terminus wild-type). b, respective I/V curves from a from representative cells taken at t = 0 s density activation. c, confocal fluorescence microscopy images of RBL cells expressing YFP-labeled STIM1 fragments: 233–420, 233–450, 233–474, 233–485, and 233–685 (complete STIM1 C terminus) and intensity plots representing localization of STIM1 fragments in regions close to the cell membrane as indicated by the dashed line. a.u., arbitrary units.