Rebecca R. Boyles

Large store-operated calcium selective currents due to co-expression of Orai1 or Orai2 with the intracellular calcium sensor, Stim1.

The molecular nature of store-operated Ca2+-selective channels has remained an enigma, due largely to the continued inability to convincingly demonstrate Ca2+-selective store-operated currents resulting from exogenous expression of known genes. Recent findings have implicated two proteins, Stim1 and Orai1, as having essential roles in store-operated Ca2+ entry across the plasma membrane. However, transient overexpression of these proteins on their own results in little or no increase in store-operated entry. Here we demonstrate dramatic synergism between these two mediators; co-transfection of HEK293 cells with Stim1 and Orai1 results in an approximate 20-fold increase in store-operated Ca2+ entry and Ca2+-selective current. This demonstrates that these two proteins are limiting for both the signaling and permeation mechanisms for Ca2+-selective store-operated Ca2+ entry. There are three mammalian homologs of Orai1, and in expression experiments they all produced or augmented store-operated Ca2+ entry with efficacies in the order Orai1 > Orai2 > Orai3. Stim1 apparently initiates the signaling process by acting as a Ca2+ sensor in the endoplasmic reticulum. This results in rearrangement of Stim1 within the cell and migration toward the plasma membrane to regulate in some manner Orai1 located in the plasma membrane. However, we demonstrate that Stim1 does not incorporate in the surface membrane, and thus likely regulates or interacts with Orai1 at sites of close apposition between the plasma membrane and an intracellular Stim1-containing organelle.

Calcium Inhibition and Calcium Potentiation of Orai1, Orai2, and Orai3 Calcium Release-activated Calcium Channels

The recent discoveries of Stim1 and Orai proteins have shed light on the molecular makeup of both the endoplasmic reticulum Ca2+ sensor and the calcium release-activated calcium (CRAC) channel, respectively. In this study, we investigated the regulation of CRAC channel function by extracellular Ca2+ for channels composed primarily of Orai1, Orai2, and Orai3, by co-expressing these proteins together with Stim1, as well as the endogenous channels in HEK293 cells. As reported previously, Orai1 or Orai2 resulted in a substantial increase in CRAC current (Icrac), but Orai3 failed to produce any detectable Ca2+-selective currents. However, sodium currents measured in the Orai3-expressing HEK293 cells were significantly larger in current density than Stim1-expressing cells. Moreover, upon switching to divalent free external solutions, Orai3 currents were considerably more stable than Orai1 or Orai2, indicating that Orai3 channels undergo a lesser degree of depotentiation. Additionally, the difference between depotentiation from Ca2+ and Ba2+ or Mg2+ solutions was significantly less for Orai3 than for Orai1 or -2. Nonetheless, the Na+ currents through Orai1, Orai2, and Orai3, as well as the endogenous store-operated Na+ currents in HEK293 cells, were all inhibited by extracellular Ca2+ with a half-maximal concentration of ∼20 μm. We conclude that Orai1, -2, and -3 channels are similarly inhibited by extracellular Ca2+, indicating similar affinities for Ca2+ within the selectivity filter. Orai3 channels appeared to differ from Orai1 and -2 in being somewhat resistant to the process of Ca2+ depotentiation.

Complex Actions of 2-Aminoethyldiphenyl Borate on Store-operated Calcium Entry

Store-operated Ca2+ entry (SOCE) is likely the most common mode of regulated influx of Ca2+ into cells. However, only a limited number of pharmacological agents have been shown to modulate this process. 2-Aminoethyldiphenyl borate (2-APB) is a widely used experimental tool that activates and then inhibits SOCE and the underlying calcium release-activated Ca2+ current (ICRAC). The mechanism by which depleted stores activates SOCE involves complex cellular movements of an endoplasmic reticulum Ca2+ sensor, STIM1, which redistributes to puncta near the plasma membrane and, in some manner, activates plasma membrane channels comprising Orai1, -2, and -3 subunits. We show here that 2-APB blocks puncta formation of fluorescently tagged STIM1 in HEK293 cells. Accordingly, 2-APB also inhibited SOCE and ICRAC-like currents in cells co-expressing STIM1 with the CRAC channel subunit, Orai1, with similar potency. However, 2-APB inhibited STIM1 puncta formation less well in cells co-expressing Orai1, indicating that the inhibitory effects of 2-APB are not solely dependent upon STIM1 reversal. Further, 2-APB only partially inhibited SOCE and current in cells co-expressing STIM1 and Orai2 and activated sustained currents in HEK293 cells expressing Orai3 and STIM1. Interestingly, the Orai3-dependent currents activated by 2-APB showed large outward currents at potentials greater than +50 mV. Finally, Orai3, and to a lesser extent Orai1, could be directly activated by 2-APB, independently of internal Ca2+ stores and STIM1. These data reveal novel and complex actions of 2-APB effects on SOCE that can be attributed to effects on both STIM1 as well as Orai channel subunits.

Phosphorylation of STIM1 underlies suppression of store-operated calcium entry during mitosis

Store-operated Ca2+ entry (SOCE) and Ca2+ release-activated Ca2+ currents (Icrac) are strongly suppressed during cell division, the only known physiological situation in which Ca2+ store depletion is uncoupled from the activation of Ca2+ influx. We found that the endoplasmic reticulum (ER) Ca2+ sensor STIM1 failed to rearrange into near-plasma membrane puncta in mitotic cells, a critical step in the SOCE-activation pathway. We also found that STIM1 from mitotic cells is recognized by the phospho-specific MPM-2 antibody, suggesting that STIM1 is phosphorylated during mitosis. Removal of ten MPM-2 recognition sites by truncation at amino acid 482 abolished MPM-2 recognition of mitotic STIM1, and significantly rescued STIM1 rearrangement and SOCE response in mitosis. We identified Ser 486 and Ser 668 as mitosis-specific phosphorylation sites, and STIM1 containing mutations of these sites to alanine also significantly rescued mitotic SOCE. Therefore, phosphorylation of STIM1 at Ser 486 and Ser 668, and possibly other sites, underlies suppression of SOCE during mitosis.

STIM1 Is a Calcium Sensor Specialized for Digital Signaling

When cells are activated by calcium-mobilizing agonists at low, physiological concentrations, the resulting calcium signals generally take the form of repetitive regenerative discharges of stored calcium, termed calcium oscillations [1]. These intracellular calcium oscillations have long fascinated biologists as a mode of digitized intracellular signaling. Recent work has highlighted the role of calcium influx as an essential component of calcium oscillations [2]. This influx occurs through a process known as store-operated calcium entry, which is initiated by calcium sensor proteins, STIM1 and STIM2, in the endoplasmic reticulum [3]. STIM2 is activated by changes in endoplasmic reticulum calcium near the resting level, whereas a threshold of calcium depletion is required for STIM1 activation [4]. Here we show that, surprisingly, it is STIM1 and not STIM2 that is exclusively involved in calcium entry during calcium oscillations. The implication is that each oscillation produces a transient drop in endoplasmic reticulum calcium and that this drop is sufficient to transiently activate STIM1. This transient activation of STIM1 can be observed in some cells by total internal reflection fluorescence microscopy. This arrangement nicely provides a clearly defined and unambiguous signaling system, translating a digital calcium release signal into calcium influx that can signal to downstream effectors.

Role of the store-operated calcium entry proteins Stim1 and Orai1 in muscarinic cholinergic receptor-stimulated calcium oscillations in human embryonic kidney cells

We have investigated the nature of the Ca2+ entry supporting [Ca2+]i oscillations in human embryonic kidney (HEK293) cells by examining the roles of recently described store‐operated Ca2+ entry proteins, Stim1 and Orai1. Knockdown of Stim1 by RNA interference (RNAi) reduced the frequency of [Ca2+]i oscillations in response to a low concentration of methacholine to the level seen in the absence of external Ca2+. However, knockdown of Stim1 did not block oscillations in canomical transient receptor potential 3 channel (TRPC3)‐expressing cells and did not affect Ca2+ entry in response to arachidonic acid. The effects of knockdown of Stim1 could be reversed by inhibiting Ca2+ extrusion with a high concentration of Gd3+, or by rescuing the knockdown by overexpression of Stim1. Similarly, knockdown of Orai1 abrogated [Ca2+]i oscillations, and this was reversed by use of high concentrations of Gd3+; however, knockdown of Orai1 did not affect arachidonic acid‐activated entry. RNAi targeting 34 members of the transient receptor potential (TRP) channel superfamily did not reveal a role for any of these channel proteins in store‐operated Ca2+ entry in HEK293 cells. These findings indicate that the Ca2+ entry supporting [Ca2+]i oscillations in HEK293 cells depends upon the Ca2+ sensor, Stim1, and calcium release‐activated Ca2+ channel protein, Orai1, and provide further support for our conclusion that it is the store‐operated mechanism that plays the major role in this pathway.

Calcium Influx Mechanisms Underlying Calcium Oscillations in Rat Hepatocytes

The process of capacitative or store‐operated Ca2+ entry has been extensively investigated, and recently two major molecular players in this process have been described. Stromal interacting molecule (STIM) 1 acts as a sensor for the level of Ca2+ stored in the endoplasmic reticulum, and Orai proteins constitute pore‐forming subunits of the store‐operated channels. Store‐operated Ca2+ entry is readily demonstrated with protocols that provide extensive Ca2+ store depletion; however, the role of store‐operated entry with modest and more physiological cell stimuli is less certain. Recent studies have addressed this question in cell lines; however, the role of store‐operated entry during physiological activation of primary cells has not been extensively investigated, and there is little or no information on the roles of STIM and Orai proteins in primary cells. Also, the nature of the Ca2+ influx mechanism with hormone activation of hepatocytes is controversial. Hepatocytes respond to physiological levels of glycogenolytic hormones with well‐characterized intracellular Ca2+ oscillations. In the current study, we have used both pharmacological tools and RNA interference (RNAi)‐based techniques to investigate the role of store‐operated channels in the maintenance of hormone‐induced Ca2+ oscillations in rat hepatocytes. Pharmacological inhibitors of store‐operated channels blocked thapsigargin‐induced Ca2+ entry but only partially reduced the frequency of Ca2+ oscillations. Similarly, RNAi knockdown of STIM1 or Orai1 substantially reduced thapsigargin‐induced calcium entry, and more modestly diminished the frequency of vasopressin‐induced oscillations. Conclusion: Our findings establish that store‐operated Ca2+ entry plays a role in the maintenance of agonist‐induced oscillations in primary rat hepatocytes but indicate that other agonist‐induced entry mechanisms must be involved to a significant extent. (HEPATOLOGY 2008.)

Laying a Community-Based Foundation for Data-Driven Semantic Standards in Environmental Health Sciences.

Background: Despite increasing availability of environmental health science (EHS) data, development, and implementation of relevant semantic standards, such as ontologies or hierarchical vocabularies, has lagged. Consequently, integration and analysis of information needed to better model environmental influences on human health remains a significant challenge. Objectives: We aimed to identify a committed community and mechanisms needed to develop EHS semantic standards that will advance understanding about the impacts of environmental exposures on human disease. Methods: The National Institute of Environmental Health Sciences sponsored the “Workshop for the Development of a Framework for Environmental Health Science Language” hosted at North Carolina State University on 15–16 September 2014. Through the assembly of data generators, users, publishers, and funders, we aimed to develop a foundation for enabling the development of community-based and data-driven standards that will ultimately improve standardization, sharing, and interoperability of EHS information. Discussion: Creating and maintaining an EHS common language is a continuous and iterative process, requiring community building around research interests and needs, enabling integration and reuse of existing data, and providing a low barrier of access for researchers needing to use or extend such a resource. Conclusions: Recommendations included developing a community-supported web-based toolkit that would enable a) collaborative development of EHS research questions and use cases, b) construction of user-friendly tools for searching and extending existing semantic resources, c) education and guidance about standards and their implementation, and d) creation of a plan for governance and sustainability. Citation: Mattingly CJ, Boyles R, Lawler CP, Haugen AC, Dearry A, Haendel M. 2016. Laying a community-based foundation for data-driven semantic standards in environmental health sciences. Environ Health Perspect 124:1136–1140; http://dx.doi.org/10.1289/ehp.1510438

Workshop report: Identifying opportunities for global integration of toxicogenomics databases, 26–27 June 2013, Research Triangle Park, NC, USA

A joint US-EU workshop on enhancing data sharing and exchange in toxicogenomics was held at the National Institute for Environmental Health Sciences. Currently, efficient reuse of data is hampered by problems related to public data availability, data quality, database interoperability (the ability to exchange information), standardization and sustainability. At the workshop, experts from universities and research institutes presented databases, studies, organizations and tools that attempt to deal with these problems. Furthermore, a case study showing that combining toxicogenomics data from multiple resources leads to more accurate predictions in risk assessment was presented. All participants agreed that there is a need for a web portal describing the diverse, heterogeneous data resources relevant for toxicogenomics research. Furthermore, there was agreement that linking more data resources would improve toxicogenomics data analysis. To outline a roadmap to enhance interoperability between data resources, the participants recommend collecting user stories from the toxicogenomics research community on barriers in data sharing and exchange currently hampering answering to certain research questions. These user stories may guide the prioritization of steps to be taken for enhancing integration of toxicogenomics databases.

Metadata Capital: Automating Metadata Workflows in the NIEHS Viral Vector Core Laboratory

This paper presents research examining metadata capital in the context of the Viral Vector Core Laboratory at the National Institute of Environmental Health Sciences (NIEHS). Methods include collaborative workflow modeling and a metadata analysis. Models of the laboratory’s workflow and metadata activity are generated to identify potential opportunities for defining microservices that may be supported by iRODS rules. Generic iRODS rules are also shared along with images of the iRODS prototype. The discussion includes an exploration of a modified capital sigma equation to understand metadata as an asset. The work aims to raise awareness of metadata as an asset and to incentivize investment in metadata R&D.