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.

STIM1 and STIM2 are widely expressed endoplasmic reticulum (ER) Ca2+ sensor proteins able to translocate within the ER membrane to physically couple with and gate plasma membrane Orai Ca2+ channels. Although structurally similar, we reveal critical differences in the function of the short STIM-Orai activating regions (SOAR) from STIM1 and STIM2. We narrowed 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 headgroup, 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.

STIM proteins are sensors of endoplasmic reticulum (ER) luminalCa 2؉ changes and rapidly translocate into near plasma membrane (PM) junctions to activate Ca 2؉ entry through the Orai family of highly Ca 2؉ -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 Ca 2؉ release-activated Ca 2؉ (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-APB's action, the locked STIMct-Orai complex provides a potentially useful probe to structurally examine coupling.calcium signaling ͉ DT40 cells ͉ CRAC channel A dynamic interplay between 2 membrane proteins, STIM and Orai, underlies an intricate coupling between Ca 2ϩ release from endoplasmic reticulum (ER) stores and Ca 2ϩ entry across the plasma membrane (PM) (1-3). The single spanning transmembrane proteins STIM1 and STIM2 function as sensors of ER luminal Ca 2ϩ changes (4-7). Depletion of luminal Ca 2ϩ within ER stores triggers STIM proteins to aggregate and undergo rapid translocation into closely juxtaposed ER-PM junctions to activate Ca 2ϩ entry through one or more of the 3-member family of PM tetra-spanning channel proteins, Orai1, Orai2, and Orai3 (8-10). The Orai proteins function as highly Ca 2ϩ -selective ''storeoperated'' channels (SOCs) (11,12). The activation of SOCs is crucial in mediating longer-term cytosolic Ca 2ϩ signals, replenishing intracellular stores, and homeostatic control of both cytosolic and ER luminal Ca 2ϩ levels (3,(11)(12)(13)(14). Orai1 coexpressed with STIM1 reconstitutes high levels of the inwardly-rectifying Ca 2ϩ release-activated Ca 2ϩ (CRAC) current (10, 15-17), the hallmark of SOCs (11,12).Despite close interactions (5, 18), the coupling mechanism between STIM and Orai proteins to activate Ca 2ϩ entry remains to be elucidated. Our approach was to examine the STIM-Orai interactions by using the reactive borate 2-aminoethoxydiphenyl borate (2-APB), a powerful modifier of SOC ...

Aldosterone in some tissues increases expression of the mRNA encoding the small monomeric G protein Ki-RasA. Renal A6 epithelial cells were used to determine whether induction of Ki-ras leads to concomitant increases in the total as well as active levels of Ki-RasA and whether this then leads to subsequent activation of its effector mitogen-activated protein kinase (MAPK/extracellular signal-regulated kinase) cascade. The molecular basis and cellular consequences of this action were specifically investigated. We identified the intron 1-exon 1 region (rasI/E1) of the mouse Ki-ras gene as sufficient to reconstitute aldosterone responsiveness to a heterologous promotor. Aldosterone increased reporter gene activity containing rasI/E1 threefold. Aldosterone increased the absolute and GTP-bound levels of Ki-RasA by a similar extent, suggesting that activation resulted from mass action and not effects on GTP binding/hydrolysis rates. Aldosterone significantly increased Ki-RasA and MAPK activity as early as 15 min with activation peaking by 2 h and waning after 4 h. Inhibitors of transcription, translation, and a glucocorticoid receptor antagonist attenuated MAPK signaling. Similarly, rasI/E1-driven luciferase expression was sensitive to glucocorticoid receptor blockade. Overexpression of dominant-negative RasN17, addition of antisense Ki-rasA and inhibition of mitogen-activated protein kinase kinase also attenuated steroid-dependent increases in MAPK signaling. Thus, activation of MAPK by aldosterone is dependent, in part, on a genomic mechanism involving induction of Ki-ras transcription and subsequent activation of its downstream effectors. This genomic mechanism has a distinct time course from activation by traditional mitogens, such as serum, which affect the GTP-binding state and not absolute levels of Ras. The result of such a genomic mechanism is that peak activation of the MAPK cascade by adrenal corticosteroids is delayed but prolonged.