L-type Ca2+ channel blockers promote vascular remodeling through activation of STIM proteins.

Voltage-gated L-type Ca2+ channel (Cav1.2) blockers (LCCBs) are major drugs for treating hypertension, the preeminent risk factor for heart failure. Vascular smooth muscle cell (VSMC) remodeling is a pathological hallmark of chronic hypertension. VSMC remodeling is characterized by molecular rewiring of the cellular Ca2+ signaling machinery, including down-regulation of Cav1.2 channels and up-regulation of the endoplasmic reticulum (ER) stromal-interacting molecule (STIM) Ca2+ sensor proteins and the plasma membrane ORAI Ca2+ channels. STIM/ORAI proteins mediate store-operated Ca2+ entry (SOCE) and drive fibro-proliferative gene programs during cardiovascular remodeling. SOCE is activated by agonists that induce depletion of ER Ca2+, causing STIM to activate ORAI. Here, we show that the three major classes of LCCBs activate STIM/ORAI-mediated Ca2+ entry in VSMCs. LCCBs act on the STIM N terminus to cause STIM relocalization to junctions and subsequent ORAI activation in a Cav1.2-independent and store depletion-independent manner. LCCB-induced promotion of VSMC remodeling requires STIM1, which is up-regulated in VSMCs from hypertensive rats. Epidemiology showed that LCCBs are more associated with heart failure than other antihypertensive drugs in patients. Our findings unravel a mechanism of LCCBs action on Ca2+ signaling and demonstrate that LCCBs promote vascular remodeling through STIM-mediated activation of ORAI. Our data indicate caution against the use of LCCBs in elderly patients or patients with advanced hypertension and/or onset of cardiovascular remodeling, where levels of STIM and ORAI are elevated.

Targeting the NFAT:AP-1 transcriptional complex on DNA with a small-molecule inhibitor.

The transcription factor nuclear factor of activated T cells (NFAT) has a key role in both T cell activation and tolerance and has emerged as an important target of immune modulation. NFAT directs the effector arm of the immune response in the presence of activator protein-1 (AP-1), and T cell anergy/exhaustion in the absence of AP-1. Envisioning a strategy for selective modulation of the immune response, we designed a FRET-based high-throughput screen to identify compounds that disrupt the NFAT:AP-1:DNA complex. We screened ∼202,000 small organic compounds and identified 337 candidate inhibitors. We focus here on one compound, N-(3-acetamidophenyl)-2-[5-(1H-benzimidazol-2-yl)pyridin-2-yl]sulfanylacetamide (Compound 10), which disrupts the NFAT:AP-1 interaction at the composite antigen-receptor response element-2 site without affecting the binding of NFAT or AP-1 alone to DNA. Compound 10 binds to DNA in a sequence-selective manner and inhibits the transcription of the Il2 gene and several other cyclosporin A-sensitive cytokine genes important for the effector immune response. This study provides proof-of-concept that small molecules can inhibit the assembly of specific DNA-protein complexes, and opens a potential new approach to treat human diseases where known transcription factors are deregulated.

STIM calcium sensing and conformational change.

The control of calcium influx at the plasma membrane by endoplasmic reticulum (ER) calcium stores, a process common to invertebrates and vertebrates, is central to physiological calcium signalling and cellular calcium balance. Stromal interaction molecule 1 (STIM1) is a calcium sensor and regulatory protein localized to the ER. ORAI1 is a calcium channel in the plasma membrane (PM). In outline, STIM1 senses an ER-luminal calcium decrease, relocalizes to ER-PM junctions, and recruits and gates ORAI1 channels. Recent work, reviewed here, has offered detailed insight into the process of sensing and communicating ER calcium-store depletion, and particularly into the STIM1 conformational change that is the basis for communication between the ER and the PM.

TMEM110 regulates the maintenance and remodeling of mammalian ER-plasma membrane junctions competent for STIM-ORAI signaling.

The stromal interaction molecule (STIM)-ORAI calcium release-activated calcium modulator (ORAI) pathway controls store-dependent calcium entry, a major mechanism of physiological calcium signaling in mammalian cells. The core elements of the pathway are the regulatory protein STIM1, located in the endoplasmic reticulum (ER) membrane, the calcium channel ORAI1 in the plasma membrane, and sites of close contact between the ER and the plasma membrane that permit the two proteins to interact. Research on calcium signaling has centered on STIM1, ORAI1, and a few proteins that directly modulate STIM-ORAI function. However, little is known about proteins that organize ER-plasma membrane junctions for STIM-ORAI-dependent calcium signaling. Here, we report that an ER-resident membrane protein identified in a previous genome-wide RNAi screen, transmembrane protein 110 (TMEM110), regulates the long-term maintenance of ER-plasma membrane junctions and the short-term physiological remodeling of the junctions during store-dependent calcium signaling.

The STIM-Orai Pathway: Orai, the Pore-Forming Subunit of the CRAC Channel.

This chapter focuses on the Orai proteins, Orai1-Orai3, with special emphasis on Orai1, in humans and other mammals, and on the definitive evidence that Orai is the pore subunit of the CRAC channel. It begins by reviewing briefly the defining characteristics of the CRAC channel, then discusses the studies that implicated Orai as part of the store-operated Ca2+ entry pathway and as the CRAC channel pore subunit, and finally examines ongoing work that is providing insights into CRAC channel structure and gating.

STIM-ORAI interactions that control the CRAC channel.

The Ca(2+) sensor STIM1 and the Ca(2+) channel ORAI1 are the fundamental working machinery of the CRAC channel, a classical pathway for store-operated Ca(2+) entry. This chapter focuses on the protein-protein interactions of STIM and ORAI proteins that control the channel.

Coiled-Coil Formation Conveys a STIM1 Signal from ER Lumen to Cytoplasm.

STIM1 and STIM2 are endoplasmic reticulum (ER) membrane proteins that sense decreases in ER-luminal free Ca2+ and, through a conformational change in the STIM cytoplasmic domain, control gating of the plasma membrane Ca2+ channel ORAI1. To determine how STIM1 conveys a signal from the ER lumen to the cytoplasm, we studied the Ca2+-dependent conformational change of engineered STIM1 proteins in isolated ER membranes and, in parallel, physiological activation of these proteins in cells. We find that conserved "sentinel" features of the CC1 region help to prevent activation while Ca2+ is bound to STIM ER-luminal domains. Reduced ER-luminal Ca2+ drives a concerted conformational change, in which STIM luminal domains rearrange and the STIM transmembrane helices and initial parts of the CC1 regions pair in an extended coiled coil. This intradimer rearrangement overcomes the relatively weak CC1-SOAR/CAD interactions that hold STIM in an inactive conformation, releasing the SOAR/CAD domain to activate ORAI channels.

Calcium sensing by the STIM1 ER-luminal domain.

Stromal interaction molecule 1 (STIM1) monitors ER-luminal Ca2+ levels to maintain cellular Ca2+ balance and to support Ca2+ signalling. The prevailing view has been that STIM1 senses reduced ER Ca2+ through dissociation of bound Ca2+ from a single EF-hand site, which triggers a dramatic loss of secondary structure and dimerization of the STIM1 luminal domain. Here we find that the STIM1 luminal domain has 5-6 Ca2+-binding sites, that binding at these sites is energetically coupled to binding at the EF-hand site, and that Ca2+ dissociation controls a switch to a second structured conformation of the luminal domain rather than protein unfolding. Importantly, the other luminal-domain Ca2+-binding sites interact with the EF-hand site to control physiological activation of STIM1 in cells. These findings fundamentally revise our understanding of physiological Ca2+ sensing by STIM1, and highlight molecular mechanisms that govern the Ca2+ threshold for activation and the steep Ca2+ concentration dependence.

A secretory pathway kinase regulates sarcoplasmic reticulum Ca2+ homeostasis and protects against heart failure.

Ca2+ signaling is important for many cellular and physiological processes, including cardiac function. Although sarcoplasmic reticulum (SR) proteins involved in Ca2+ signaling have been shown to be phosphorylated, the biochemical and physiological roles of protein phosphorylation within the lumen of the SR remain essentially uncharacterized. Our laboratory recently identified an atypical protein kinase, Fam20C, which is uniquely localized to the secretory pathway lumen. Here, we show that Fam20C phosphorylates several SR proteins involved in Ca2+ signaling, including calsequestrin2 and Stim1, whose biochemical activities are dramatically regulated by Fam20C mediated phosphorylation. Notably, phosphorylation of Stim1 by Fam20C enhances Stim1 activation and store-operated Ca2+ entry. Physiologically, mice with Fam20c ablated in cardiomyocytes develop heart failure following either aging or induced pressure overload. We extended these observations to show that non-muscle cells lacking Fam20C display altered ER Ca2+ signaling. Overall, we show that Fam20C plays an overarching role in ER/SR Ca2+ homeostasis and cardiac pathophysiology.

STIM1 triggers a gating rearrangement at the extracellular mouth of the ORAI1 channel.

The ER-resident regulatory protein STIM1 triggers store-operated Ca(2+) entry by direct interaction with the plasma membrane Ca(2+) channel ORAI1. The mechanism of channel gating remains undefined. Here we establish that STIM1 gates the purified recombinant ORAI1 channel in vitro, and use Tb(3+) luminescence and, separately, disulfide crosslinking to probe movements of the pore-lining helices. We show that interaction of STIM1 with the cytoplasmic face of the human ORAI1 channel elicits a conformational change near the external entrance to the pore, detectable at the pore Ca(2+)-binding residue E106 and the adjacent pore-lining residue V102. We demonstrate that a short nonpolar segment of the pore including V102 forms a barrier to ion flux in the closed channel, implicating the STIM1-dependent movement in channel gating. Our data explain the close coupling between ORAI1 channel gating and ion selectivity, and open a new avenue to dissect the gating, modulation and inactivation of ORAI-family channels.

Initial activation of STIM1, the regulator of store-operated calcium entry.

Physiological Ca(2+) signaling in T lymphocytes and other cells depends on the STIM-ORAI pathway of store-operated Ca(2+) entry. STIM1 and STIM2 are Ca(2+) sensors in the endoplasmic reticulum (ER) membrane, with ER-luminal domains that monitor cellular Ca(2+) stores and cytoplasmic domains that gate ORAI channels in the plasma membrane. The STIM ER-luminal domain dimerizes or oligomerizes upon dissociation of Ca(2+), but the mechanism transmitting activation to the STIM cytoplasmic domain was previously undefined. Using Tb(3+)-acceptor energy transfer, we show that dimerization of STIM1 ER-luminal domains causes an extensive conformational change in mouse STIM1 cytoplasmic domains. The conformational change, triggered by apposition of the predicted coiled-coil 1 (CC1) regions, releases the ORAI-activating domains from their interaction with the CC1 regions and allows physical extension of the STIM1 cytoplasmic domain across the gap between ER and plasma membrane and communication with ORAI channels.