Eunan Hendron

The Calcium Store Sensor, STIM1, Reciprocally Controls Orai and CaV1.2 Channels

Channel STIMulation The STIM1 protein functions as a calcium sensor and regulates entry of calcium into cells across the plasma membrane. When cell surface receptors are stimulated and cause release of calcium from internal stores in the endoplasmic reticulum (ER), STIM proteins in the ER membrane interact with the Orai channel pore protein in the plasma membrane to allow calcium entry from the outside of the cell (see the Perspective by Cahalan). Park et al. (p. 101) and Wang et al. (p. 105) now show that STIM also acts to suppress conductance by another calcium channel—the voltage-operated CaV1.2 channel. STIM1 appeared to interact directly with CaV1.2 channels in multiple cell types, including vascular smooth muscle cells, neurons, and cultured cells derived from T lymphocytes. The interaction inhibited opening of the CaV1.2 channels and caused depletion of the channel from the cell surface. The sensor protein that monitors depletion of intracellular calcium regulates two classes of calcium entry channels. Calcium signals, pivotal in controlling cell function, can be generated by calcium entry channels activated by plasma membrane depolarization or depletion of internal calcium stores. We reveal a regulatory link between these two channel subtypes mediated by the ubiquitous calcium-sensing STIM proteins. STIM1 activation by store depletion or mutational modification strongly suppresses voltage-operated calcium (CaV1.2) channels while activating store-operated Orai channels. Both actions are mediated by the short STIM-Orai activating region (SOAR) of STIM1. STIM1 interacts with CaV1.2 channels and localizes within discrete endoplasmic reticulum/plasma membrane junctions containing both CaV1.2 and Orai1 channels. Hence, STIM1 interacts with and reciprocally controls two major calcium channels hitherto thought to operate independently. Such coordinated control of the widely expressed CaV1.2 and Orai channels has major implications for Ca2+ signal generation in excitable and nonexcitable cells.

STIM protein coupling in the activation of Orai channels

STIM proteins are sensors of endoplasmic reticulum (ER) luminal Ca2+ changes and rapidly translocate into near plasma membrane (PM) junctions to activate Ca2+ entry through the Orai family of highly Ca2+-selective “store-operated” channels (SOCs). Dissecting the STIM–Orai coupling process is restricted by the abstruse nature of the ER–PM junctional domain. To overcome this problem, we studied coupling by using STIM chimera and cytoplasmic C-terminal domains of STIM1 and STIM2 (S1ct and S2ct) and identifying a fundamental action of the powerful SOC modifier, 2-aminoethoxydiphenyl borate (2-APB), the mechanism of which has eluded recent scrutiny. We reveal that 2-APB induces profound, rapid, and direct interactions between S1ct or S2ct and Orai1, effecting full Ca2+ release-activated Ca2+ (CRAC) current activation. The short 235-505 S1ct coiled-coil region was sufficient for functional Orai1 coupling. YFP-tagged S1ct or S2ct fragments cleared from the cytosol seconds after 2-APB addition, binding avidly to Orai1-CFP with a rapid increase in FRET and transiently increasing CRAC current 200-fold above basal levels. Functional S1ct–Orai1 coupling occurred in STIM1/STIM2−/− DT40 chicken B cells, indicating ct fragments operate independently of native STIM proteins. The 2-APB-induced S1ct–Orai1 and S2-ct–Orai1 complexes undergo rapid reorganization into discrete colocalized PM clusters, which remain stable for >100 s, well beyond CRAC activation and subsequent deactivation. In addition to defining 2-APBs action, the locked STIMct–Orai complex provides a potentially useful probe to structurally examine coupling.

Distinct Orai-coupling domains in STIM1 and STIM2 define the Orai-activating site

STIM1 and STIM2 are widely expressed endoplasmic reticulum (ER) Ca(2+) sensor proteins able to translocate within the ER membrane to physically couple with and gate plasma membrane Orai Ca(2+) channels. Although they are structurally similar, we reveal critical differences in the function of the short STIM-Orai-activating regions (SOAR) of STIM1 and STIM2. We narrow these differences in Orai1 gating to a strategically exposed phenylalanine residue (Phe-394) in SOAR1, which in SOAR2 is substituted by a leucine residue. Remarkably, in full-length STIM1, replacement of Phe-394 with the dimensionally similar but polar histidine head group prevents both Orai1 binding and gating, creating an Orai1 non-agonist. Thus, this residue is critical in tuning the efficacy of Orai activation. While STIM1 is a full Orai1-agonist, leucine-replacement of this crucial residue in STIM2 endows it with partial agonist properties, which may be critical for limiting Orai1 activation stemming from its enhanced sensitivity to store-depletion.

Initial steps of inactivation at the K+ channel selectivity filter

Significance C-type inactivation represents a key process that governs cellular K+ channel activity. Although C-type inactivation seems to be inextricably linked with dissociation of K+ from the channel’s pore, the structural connection between K+ dissociation and initiation of C-type inactivation has been unclear. Here, we combine electrophysiology and molecular simulation of MthK, a prototypical K+ channel of known structure, to determine relations between K+ dissociation and entry into the inactivated state. We find that Ca2+ can bind to a site in the pore favored by outward movement of K+. K+ subsequently dissociates, favoring a conformational change to the inactivated state. This study, thus, establishes a direct link between K+ dissociation and initiation of C-type inactivation. K+ efflux through K+ channels can be controlled by C-type inactivation, which is thought to arise from a conformational change near the channel’s selectivity filter. Inactivation is modulated by ion binding near the selectivity filter; however, the molecular forces that initiate inactivation remain unclear. We probe these driving forces by electrophysiology and molecular simulation of MthK, a prototypical K+ channel. Either Mg2+ or Ca2+ can reduce K+ efflux through MthK channels. However, Ca2+, but not Mg2+, can enhance entry to the inactivated state. Molecular simulations illustrate that, in the MthK pore, Ca2+ ions can partially dehydrate, enabling selective accessibility of Ca2+ to a site at the entry to the selectivity filter. Ca2+ binding at the site interacts with K+ ions in the selectivity filter, facilitating a conformational change within the filter and subsequent inactivation. These results support an ionic mechanism that precedes changes in channel conformation to initiate inactivation.

Distinct Orai-Coupling Domains in Stim1 and Stim2 Define the Orai-Activating Site

The ER membrane-spanning STIM1 protein is a finely-tuned sensor of ER luminal Ca2+. Small changes in ER Ca2+ induce STIM1 to undergo an intricate self-triggering process, causing it to translocate into ER-PM junctions where it couples with and activates the highly Ca2+-selective family of Orai channels in the PM. The entering Ca2+ sustains Ca2+ oscillations, maintains Ca2+ homeostasis, and provides crucial long-term Ca2+ signals in many cell types which control gene expression and cellular growth. Similar in structure and also widely expressed among cells, the little-studied STIM2 protein is reported to differ subtly from STIM1 in its N-terminal domain, affecting luminal Ca2+-sensitivity and the rate of unfolding and self-activation. The STIM1 cytoplasmic C-terminus contains the STIM-Orai activating region (SOAR) which has been structurally resolved. While the corresponding SOAR sequence in STIM2 is highly conserved, we reveal it has a profoundly diminished interaction with and ability to gate Orai1 channels. We narrowed this distinction in Orai1 activation to a small sequence in SOAR, within which substitution of a single phenylalanine in STIM1 with leucine in STIM2 confers a severe decrease in Orai1 channel-gating efficacy. This residue is strategically positioned at the structural apex of the SOAR domain. Modification of this single residue within the intact STIM1 protein reveals its pivotal role in both interaction with and gating of the Orai1 channel. The results not only pinpoint a crucial locus of STIM-Orai coupling but also reveal a physiologically profound distinction between STIM1 and STIM2.