Youjun Wang

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.

S-glutathionylation activates STIM1 and alters mitochondrial homeostasis

Oxidant stress induces constitutive calcium entry by tacking glutathiones onto the Orai CRAC channel activator STIM1.

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.

STIM, ORAI AND TRPC CHANNELS IN THE CONTROL OF CALCIUM ENTRY SIGNALS IN SMOOTH MUSCLE

1 Ca2+ entry signals are crucial in the control of smooth muscle contraction. Smooth muscle cells are unusual in containing plasma membrane (PM) Ca2+ entry channels that respond to voltage changes, receptor activation and Ca2+ store depletion. 2 Activation of these channel subtypes is highly coordinated. The TRPC6 channel, widely expressed in most smooth muscle cell types, is largely non‐selective to cations and is activated by diacylglycerol arising from receptor‐induced phosholipase C activation. 3 Receptor activation results largely in Na+ ion movement through TRPC6 channels, depolarization and subsequent activation of voltage‐dependent L‐type Ca2+ channels. The TRPC6 channels also appear to be activated by mechanical stretch, resulting again in depolarization and L‐type Ca2+ channel activation. Such a coupling may be crucial in mediating the myogenic tone response in vascular smooth muscle. 4 The emptying of stores mediated by inositol 1,4,5‐trisphosphate receptors triggers the endoplasmic reticulum (ER) Ca2+ sensing protein stromal‐interacting molecule (STIM) 1 to translocate into defined ER–PM junctional areas in which coupling occurs to Orai proteins, which serve as highly Ca2+‐selective low‐conductance Ca2+ entry channels. 5 These ER‐PM junctional domains may serve as crucial sites of interaction and integration between the function of store‐operated, receptor‐operated and voltage‐operated Ca2+ channels. The STIM, Orai and TRPC channels represent highly promising new pharmacological targets through which such control may be induced.

STIM and Orai – dynamic intermembrane coupling to control cellular calcium signals

Ca2+ signals controlling a vast array of cell functions involve both Ca2+ store release and external Ca2+ entry. These two events are coordinated through a dynamic intermembrane coupling between two distinct membrane proteins, STIM and Orai. STIM proteins are endoplasmic reticulum (ER) luminal Ca2+ sensors that undergo a profound redistribution into discrete junctional ER domains closely juxtaposed with the plasma membrane (PM). Orai proteins are PM Ca2+ channels that migrate and become tethered by STIM within the ER-PM junctions, where they mediate exceedingly selective Ca2+ entry. We describe a new understanding of the nature of the proteins and how they function to mediate this remarkable intermembrane signaling process controlling Ca2+ signals.

The Short N-terminal Domains of STIM1 and STIM2 Control the Activation Kinetics of Orai1 Channels

STIM1 and STIM2 are dynamic transmembrane endoplasmic reticulum Ca2+ sensors, coupling directly to activate plasma membrane Orai Ca2+ entry channels. Despite extensive sequence homology, the STIM proteins are functionally distinct. We reveal that the short variable N-terminal random coil sequences of STIM1 and STIM2 confer profoundly different activation properties. Using Orai1-expressing HEK293 cells, chimeric replacement of the 43-amino-acid STIM1 N terminus with that of STIM2 attenuates Orai1-mediated Ca2+ entry and drastically slows store-induced Orai1 channel activation. Conversely, the 55-amino-acid STIM2 terminus substituted within STIM1 strikingly enhances both Orai1-mediated Ca2+ entry and constitutive coupling to activate Orai1 channels. Hence, STIM N termini are powerful coupling modifiers, functioning in STIM2 to “brake” the otherwise constitutive activation of Orai1 channels afforded by its high sensitivity to luminal Ca2+.

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.

Proteomic mapping of ER-PM junctions identifies STIMATE as a regulator of Ca2+ influx

Specialized junctional sites that connect the plasma membrane (PM) and endoplasmic reticulum (ER) play critical roles in controlling lipid metabolism and Ca2+ signalling. Store-operated Ca2+ entry mediated by dynamic STIM1–ORAI1 coupling represents a classical molecular event occurring at ER–PM junctions, but the protein composition and how previously unrecognized protein regulators facilitate this process remain ill-defined. Using a combination of spatially restricted biotin labelling in situ coupled with mass spectrometry and a secondary screen based on bimolecular fluorescence complementation, we mapped the proteome of intact ER–PM junctions in living cells without disrupting their architectural integrity. Our approaches led to the discovery of an ER-resident multi-transmembrane protein that we call STIMATE (STIM-activating enhancer, encoded by TMEM110) as a positive regulator of Ca2+ influx in vertebrates. STIMATE physically interacts with STIM1 to promote STIM1 conformational switch. Genetic depletion of STIMATE substantially reduces STIM1 puncta formation at ER–PM junctions and suppresses the Ca2+–NFAT signalling. Our findings enable further genetic studies to elucidate the function of STIMATE in normal physiology and disease, and set the stage to uncover more uncharted functions of hitherto underexplored ER–PM junctions.

Inside-out Ca2+ signalling prompted by STIM1 conformational switch

Store-operated Ca2+ entry mediated by STIM1 and ORAI1 constitutes one of the major Ca2+ entry routes in mammalian cells. The molecular choreography of STIM1–ORAI1 coupling is initiated by endoplasmic reticulum (ER) Ca2+ store depletion with subsequent oligomerization of the STIM1 ER-luminal domain, followed by its redistribution towards the plasma membrane to gate ORAI1 channels. The mechanistic underpinnings of this inside-out Ca2+ signalling were largely undefined. By taking advantage of a unique gain-of-function mutation within the STIM1 transmembrane domain (STIM1-TM), here we show that local rearrangement, rather than alteration in the oligomeric state of STIM1-TM, prompts conformational changes in the cytosolic juxtamembrane coiled-coil region. Importantly, we further identify critical residues within the cytoplasmic domain of STIM1 (STIM1-CT) that entail autoinhibition. On the basis of these findings, we propose a model in which STIM1-TM reorganization switches STIM1-CT into an extended conformation, thereby projecting the ORAI-activating domain to gate ORAI1 channels.

Calcium Signaling by STIM and Orai: Intimate Coupling Details Revealed

Intramolecular interactions within STIM1 control activation of Orai1 channels. STIM (stromal interaction molecule) and Orai, two recently identified protein families, mediate cellular Ca2+ signals through a remarkably dynamic interaction. STIM proteins are sensors of Ca2+ stored within the endoplasmic reticulum (ER). Orai proteins are highly selective plasma membrane (PM) channels that allow only Ca2+ ions to flow into cells. Although present in separate membranes, the two proteins undergo profound reorganization culminating in an exquisite pas de deux within small junctional regions between the ER and PM. Before these proteins can embrace, STIM undergoes an activation process triggered by depletion of Ca2+ stores. During its union with Orai, STIM induces the channel pore within Orai to open, allowing Ca2+ ions to flow through the PM and provide crucial intracellular signals. Recent studies on the activation of STIM and its coupling to Orai provide valuable new insights into the nature of the liaison between these two proteins and the intricate mechanism through which activation of Ca2+ signals occurs.