Irene Frischauf

Dynamic Coupling of the Putative Coiled-coil Domain of ORAI1 with STIM1 Mediates ORAI1 Channel Activation

STIM1 and ORAI1 (also termed CRACM1) are essential components of the classical calcium release-activated calcium current; however, the mechanism of the transmission of information of STIM1 to the calcium release-activated calcium/ORAI1 channel is as yet unknown. Here we demonstrate by Förster resonance energy transfer microscopy a dynamic coupling of STIM1 and ORAI1 that culminates in the activation of Ca2+ entry. Förster resonance energy transfer imaging of living cells provided insight into the time dependence of crucial events of this signaling pathway comprising Ca2+ store depletion, STIM1 multimerization, and STIM1-ORAI1 interaction. Accelerated store depletion allowed resolving a significant time lag between STIM1-STIM1 and STIM1-ORAI1 interactions. Store refilling reversed both STIM1 multimerization and STIM1-ORAI1 interaction. The cytosolic STIM1 C terminus itself was able, in vitro as well as in vivo, to associate with ORAI1 and to stimulate channel function, yet without ORAI1-STIM1 cluster formation. The dynamic interaction occurred via the C terminus of ORAI1 that includes a putative coiled-coil domain structure. An ORAI1 C terminus deletion mutant as well as a mutant (L273S) with impeded coiled-coil domain formation lacked both interaction as well as functional communication with STIM1 and failed to generate Ca2+ inward currents. An N-terminal deletion mutant of ORAI1 as well as the ORAI1 R91W mutant linked to severe combined immune deficiency syndrome was similarly impaired in terms of current activation despite being able to interact with STIM1. Hence, the C-terminal coiled-coil motif of ORAI1 represents a key domain for dynamic coupling to STIM1.

A Cytosolic Homomerization and a Modulatory Domain within STIM1 C Terminus Determine Coupling to ORAI1 Channels.

In immune cells, generation of sustained Ca2+ levels is mediated by the Ca2+ release-activated Ca2+ (CRAC) current. Molecular key players in this process comprise the stromal interaction molecule 1 (STIM1) that functions as a Ca2+ sensor in the endoplasmic reticulum and ORAI1 located in the plasma membrane. Depletion of endoplasmic reticulum Ca2+ stores leads to STIM1 multimerization into discrete puncta, which co-cluster with ORAI1 to couple to and activate ORAI1 channels. The cytosolic C terminus of STIM1 is sufficient to activate ORAI1 currents independent of store depletion. Here we identified an ORAI1-activating small fragment (OASF, amino acids 233–450/474) within STIM1 C terminus comprising the two coiled-coil domains and additional 50–74 amino acids that exhibited enhanced interaction with ORAI1, resulting in 3-fold increased Ca2+ currents. This OASF, similar to the complete STIM1 C terminus, displayed the ability to homomerize by a novel assembly domain that occurred subsequent to the coiled-coil domains. A smaller fragment (amino acids 233–420) generated by a further deletion of 30 amino acids substantially reduced the ability to homomerize concomitant to a loss of coupling to as well as activation of ORAI1. Extending OASF by 35 amino acids (233–485) did not alter homomerization but substantially decreased efficiency in coupling to and activation of ORAI1. Expressing OASF in rat basophilic leukemia (RBL) mast cells demonstrated its enhanced plasma membrane targeting associated with 2.5-fold larger CRAC currents in comparison with the complete STIM1 C terminus. In aggregate, we have identified two cytosolic key regions within STIM1 C terminus that control ORAI1/CRAC activation: a homomerization domain indispensable for coupling to ORAI1 and a modulatory domain that controls the extent of coupling to ORAI1.

Proliferation of aligned mammalian cells on laser-nanostructured polystyrene.

Biomaterial surface chemistry and nanoscale topography are important for many potential applications in medicine and biotechnology as they strongly influence cell function, adhesion and proliferation. In this work, we present periodic surface structures generated by linearly polarized KrF laser light (248 nm) on polystyrene (PS) foils. These structures have a periodicity of 200-430 nm and a depth of 30-100 nm, depending on the angle of incidence of the laser beam. The changes in surface topography and chemistry were analysed by atomic force microscopy (AFM), advancing water contact-angle measurements, Fourier-transform infrared spectroscopy using an attenuated total reflection device (ATR-FTIR) and X-ray photoelectron spectroscopy (XPS). We show that the surface laser modification results in a significantly enhanced adhesion and proliferation of human embryonic kidney cells (HEK-293) compared to the unmodified polymer foil. Furthermore, we report on the alignment of HEK-293 cells, Chinese hamster ovary (CHO-K1) cells and skeletal myoblasts along the direction of the structures. The results indicate that the presence of nanostructures on the substrates can guide cell alignment along definite directions, and more importantly, in our opinion, that this alignment is only observed when the periodicity is above a critical periodicity value that is cell-type specific.

STIM1 couples to ORAI1 via an intramolecular transition into an extended conformation.

Stromal interaction molecule (STIM1) and ORAI1 are key components of the Ca2+ release‐activated Ca2+ (CRAC) current having an important role in T‐cell activation and mast cell degranulation. CRAC channel activation occurs via physical interaction of ORAI1 with STIM1 when endoplasmic reticulum Ca2+ stores are depleted. Here we show, utilizing a novel STIM1‐derived Förster resonance energy transfer sensor, that the ORAI1 activating small fragment (OASF) undergoes a C‐terminal, intramolecular transition into an extended conformation when activating ORAI1. The C‐terminal rearrangement of STIM1 does not require a functional CRAC channel, suggesting interaction with ORAI1 as sufficient for this conformational switch. Extended conformations were also engineered by mutations within the first and third coiled‐coil domains in the cytosolic portion of STIM1 revealing the involvement of hydrophobic residues in the intramolecular transition. Corresponding full‐length STIM1 mutants exhibited enhanced interaction with ORAI1 inducing constitutive CRAC currents, even in the absence of store depletion. We suggest that these mutant STIM1 proteins imitate a physiological activated state, which mimics the intramolecular transition that occurs in native STIM1 upon store depletion.

Molecular Determinants of the Coupling between STIM1 and Orai Channels DIFFERENTIAL ACTIVATION OF Orai1–3 CHANNELS BY A STIM1 COILED-COIL MUTANT

STIM1 and Orai1 have been reported to interact upon store depletion culminating in Ca2+ release-activated Ca2+ current activation. Recently, the essential region has been identified within the STIM1 C terminus that includes the second coiled-coil domain C-terminally extended by ∼50 amino acids and exhibits a strong binding to the Orai1 C terminus. Based on the homology within the Orai family, an analogous scenario might be assumed for Orai2 as well as Orai3 channels as both are activated in a similar STIM1-dependent manner. A combined approach of electrophysiology and Foerster resonance energy transfer microscopy uncovered a general mechanism in the communication of STIM1 with Orai proteins that involved the conserved putative coiled-coil domains in the respective Orai C terminus and the second coiled-coil motif in the STIM1 C terminus. A coiled-coil single mutation in the Orai1 C terminus abrogated communication with the STIM1 C terminus, whereas an analogous mutation in Orai2 and Orai3 still allowed for their moderate activation. However, increasing coiled-coil probability by a gain of function deletion in Orai1 or by generating an Orai1-Orai3 chimera containing the Orai3 C terminus recovered stimulation to a similar extent as with Orai2/3. At the level of STIM1, decreasing probability of the second coiled-coil domain by a single mutation within the STIM1 C terminus abolished activation of Orai1 but still enabled partial stimulation of Orai2/3 channels. A double mutation within the second coiled-coil motif of the STIM1 C terminus fully disrupted communication with all three Orai channels. In aggregate, the impairment in the overall communication between STIM1 and Orai channels upon decreasing probabilities of either one of the putative coiled-coil domains in the C termini might be compatible with the concept of their functional, heteromeric interaction.

2-Aminoethoxydiphenyl Borate Alters Selectivity of Orai3 Channels by Increasing Their Pore Size

Stim1 in the endoplasmic reticulum and the three Orai (also termed CRACM) channels in the plasma-membrane are main components of native Ca2+ release-activated Ca2+ channels. A pharmacological hallmark of these channels is their distinct sensitivity to 2-aminoethoxydiphenyl borate (2-APB). Here we report that Orai3 currents can be robustly stimulated by 75 μm 2-APB independent of Stim1, whereas 2-APB at similar concentrations inhibited store-operated Orai1 currents. 2-APB did not only promote currents through Orai3 channels but also dramatically altered ion selectivity of Orai3 channels. This allowed for permeation of monovalent cations both in the inward as well as outward direction, which is in sharp contrast to the high Ca2+ selectivity of store-operated Orai3 currents. An Orai3-R66W mutant, which lacked in analogy to the severe combined immune deficiency mutant Orai1-R91W store-operated activation, was also found to be resistant to 2-APB stimulation. The change in selectivity by 2-APB was associated with an increase in Orai3 minimum pore size from about 3.8Å to more than 5.34Å. In line with a potential interaction of 2-APB with the Orai3 pore, among three pore mutants tested, the Orai3 E165Q mutant particularly resembled in its permeation properties those of 2-APB stimulated Orai3 and additionally exhibited a reduced response to 2-APB. In aggregate, stimulation of Orai3 currents by 2-APB occurred along with an alteration of the permeation pathway that represents a unique mechanism for regulating ion channel selectivity by chemical compounds.

The STIM/Orai coupling machinery

Calcium (Ca2+) entry into non-excitable cells is mainly carried by store-operated channels (SOCs), which serve essential functions ranging from regulation of transcription to cell growth. The best-characterised store-operated current, ICRAC, is the calcium release-activated calcium (CRAC) current initially discovered in T-lymphocytes and mast cells. The search for the molecular components of the CRAC channel lasted over 20 years. Recently STIM1 has been identified as the Ca2+ sensor in the endoplasmic reticulum (ER) that accumulates into punctae close to the plasma-membrane following store-depletion. The identification of STIM1 has been closely followed by the discovery of Orai1 as the CRAC channel pore in human T-cells. Upon punctae formation STIM1 activates Ca2+ influx via Orai1 channels. This review covers functional details concerning the activation cascade of the STIM1 / Orai1 complex from ER Ca2+ sensing to Ca2+ influx through Orai1. Furthermore, functional domains within STIM1 and Orai1 in comparison to their structural homologs STIM2 as well as Orai2 and Orai3, respectively, are displayed together with recent findings on the pore architecture and selectivity filter of Orai channels. A broad tissue expression of STIM and Orai proteins together with substantial effects in STIM1 / Orai1 knock-out mice suggests an essential physiological role in store-operated Ca2+ signaling in human health and disease.

Mechanistic view on domains mediating STIM1–Orai coupling

Summary:  Calcium (Ca2+) entry into non‐excitable cells is mainly carried by store‐operated channels, which serve essential functions ranging from regulation of transcription to cell growth. The best‐characterized store‐operated current, initially discovered in T lymphocytes and mast cells, is the Ca2+ release‐activated Ca2+ (CRAC) current. The search for the molecular components of the CRAC channel has recently identified stromal interaction molecule 1 (STIM1) as the Ca2+ sensor in the endoplasmic reticulum (ER) and Orai1 as the CRAC channel pore. ER store depletion results in formation of STIM1 puncta that trigger Ca2+ influx via Orai1 channels. This review covers the role of domains within STIM1 and Orai and enlightens their function in the STIM1/Orai coupling process. Moreover, a molecular interpretation focuses on interactions between cytosolic portions of STIM1 and Orai together with a mechanistic view on the loss of function of the SCID (severe combined immunodeficiency)‐linked Orai1 R91W mutant channel. The architecture of the selectivity filter of Orai channels is finally elucidated based on permeation properties of Orai pore mutants.

Resting-State Orai1 Diffuses as Homotetramer in the Plasma Membrane of Live Mammalian Cells

Store-operated calcium entry is essential for many signaling processes in nonexcitable cells. The best studied store-operated calcium current is the calcium release-activated calcium (CRAC) current in T-cells and mast cells, with Orai1 representing the essential pore forming subunit. Although it is known that functional CRAC channels in store-depleted cells are composed of four Orai1 subunits, the stoichiometric composition in quiescent cells is still discussed controversially: both a tetrameric and a dimeric stoichiometry of resting state Orai1 have been reported. We obtained here robust and similar FRET values on labeled tandem repeat constructs of Orai1 before and after store depletion, suggesting an unchanged tetrameric stoichiometry. Moreover, we directly visualized the stoichiometry of mobile Orai1 channels in live cells using a new single molecule recording modality that combines single molecule tracking and brightness analysis. By alternating imaging and photobleaching pulses, we recorded trajectories of single, fluorescently labeled Orai1 channels, with each trajectory consisting of bright and dim segments, corresponding to higher and lower numbers of colocalized active GFP label. The according brightness values were used for global fitting and statistical analysis, yielding a tetrameric subunit composition of mobile Orai1 channels in resting cells.

Increased Hydrophobicity at the N Terminus/Membrane Interface Impairs Gating of the Severe Combined Immunodeficiency-related ORAI1 Mutant

Patients with severe combined immune deficiency (SCID) suffer from defective T-cell Ca2+ signaling. A loss of Ca2+ entry has been linked at the molecular level to single missense mutation R91W in the store-operated Ca2+ channel ORAI1. However, the mechanistic impact of this mutation on ORAI1 function remains unclear. Confocal Förster resonance energy transfer microscopy revealed that dynamic store-operated coupling of STIM1 to ORAI1 R91W was largely sustained similar to wild-type ORAI1. Characterization of various point mutants at position 91 by whole cell patch clamp recordings displayed that neutral or even negatively charged amino acids did not abolish ORAI1 function. However, substitution by hydrophobic leucine, valine, or phenylalanine resulted in non-functional ORAI1 channels, despite preserved STIM1 coupling. Besides conformational constraints at the N terminus/membrane interface predicted for the hydrophobic mutants, additional key factor(s) were suggested to determine ORAI1 functionality. Calculation of the probability for the 1st transmembrane domain and its hydrophobicity revealed a substantial increase for all hydrophobic substitutions that lead to non-functional ORAI1 R91X mutants in contrast to those with hydrophilic residues. Hence, increased hydrophobicity might lead to disrupted permeation/gating, as an ORAI1 channel with increased pore size and R91W mutation failed to recover activity. In conclusion, the increase in hydrophobicity at the N terminus/membrane interface represents the major cause for yielding non-functional ORAI1 channels.