Photoswitching alters fluorescence readout of jGCaMP8 Ca2+ indicators tethered to Orai1 channels.

We used electrophysiology and Ca2+ channel tethering to evaluate the performance of jGCaMP8 genetically encoded Ca2+ indicators (GECIs). Orai1 Ca2+ channel-jGCaMP8 fusions were transfected into HEK 293A cells and jGCaMP8 fluorescence responses recorded by simultaneous total internal reflection fluorescence microscopy and whole-cell patch clamp electrophysiology. Noninactivating currents from the Orai1 Y80E mutant provided a steady flux of Ca2+ controlled on a millisecond time scale by step changes in membrane potential. Test pulses to -100 mV produced Orai1 Y80E-jGCaMP8f fluorescence traces that unexpectedly declined by ~50% over 100 ms before reaching a stable plateau. Testing of Orai1-jGCaMP8f using unroofed cells further demonstrated that rapid and partial fluorescence inactivation is a property of the indicator itself, rather than channel function. Photoinactivation spontaneously recovered over 5 min in the dark, and recovery was accelerated in the absence of Ca2+. Mutational analysis of residues near the tripeptide fluorophore of jGCaMP8f pointed to a mechanism: Q69M/C70V greatly increased (~90%) photoinactivation, reminiscent of fluorescent protein fluorophore cis-trans photoswitching. Indeed, 405-nm illumination of jGCaMP8f or 8m/8s/6f led to immediate photorecovery, and simultaneous illumination with 405 and 488-nm light blocked photoinactivation. Subsequent mutagenesis produced a variant, V203Y, that lacks photoinactivation but largely preserves the desirable properties of jGCaMP8f. Our results point to caution in interpreting rapidly changing Ca2+ signals using jGCaMP8 and earlier series GECIs, suggest strategies to avoid photoswitching, and serve as a starting point to produce more photostable, and thus more accurate, GECI derivatives.

Regulatory T cells suppress Th17 cell Ca2+ signaling in the spinal cord during murine autoimmune neuroinflammation.

T lymphocyte motility and interaction dynamics with other immune cells are vital determinants of immune responses. Regulatory T (Treg) cells prevent autoimmune disorders by suppressing excessive lymphocyte activity, but how interstitial motility patterns of Treg cells limit neuroinflammation is not well understood. We used two-photon microscopy to elucidate the spatial organization, motility characteristics, and interactions of endogenous Treg and Th17 cells together with antigen-presenting cells (APCs) within the spinal cord leptomeninges in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Th17 cells arrive before the onset of clinical symptoms, distribute uniformly during the peak, and decline in numbers during later stages of EAE. In contrast, Treg cells arrive after Th17 cells and persist during the chronic phase. Th17 cells meander widely, interact with APCs, and exhibit cytosolic Ca2+ transients and elevated basal Ca2+ levels before the arrival of Treg cells. In contrast, Treg cells adopt a confined, repetitive-scanning motility while contacting APCs. These locally confined but highly motile Treg cells limit Th17 cells from accessing APCs and suppress Th17 cell Ca2+ signaling by a mechanism that is upstream of store-operated Ca2+ entry. Finally, Treg cell depletion increases APC numbers in the spinal cord and exaggerates ongoing neuroinflammation. Our results point to fundamental differences in motility characteristics between Th17 and Treg cells in the inflamed spinal cord and reveal three potential cellular mechanisms by which Treg cells regulate Th17 cell effector functions: reduction of APC density, limiting access of Th17 cells to APCs, and suppression of Th17 Ca2+ signaling.

Nanoscale patterning of STIM1 and Orai1 during store-operated Ca2+ entry.

Stromal interacting molecule (STIM) and Orai proteins constitute the core machinery of store-operated calcium entry. We used transmission and freeze-fracture electron microscopy to visualize STIM1 and Orai1 at endoplasmic reticulum (ER)-plasma membrane (PM) junctions in HEK 293 cells. Compared with control cells, thin sections of STIM1-transfected cells possessed far more ER elements, which took the form of complex stackable cisternae and labyrinthine structures adjoining the PM at junctional couplings (JCs). JC formation required STIM1 expression but not store depletion, induced here by thapsigargin (TG). Extended molecules, indicative of STIM1, decorated the cytoplasmic surface of ER, bridged a 12-nm ER-PM gap, and showed clear rearrangement into small clusters following TG treatment. Freeze-fracture replicas of the PM of Orai1-transfected cells showed extensive domains packed with characteristic "particles"; TG treatment led to aggregation of these particles into sharply delimited "puncta" positioned upon raised membrane subdomains. The size and spacing of Orai1 channels were consistent with the Orai crystal structure, and stoichiometry was unchanged by store depletion, coexpression with STIM1, or an Orai1 mutation (L273D) affecting STIM1 association. Although the arrangement of Orai1 channels in puncta was substantially unstructured, a portion of channels were spaced at ∼15 nm. Monte Carlo analysis supported a nonrandom distribution for a portion of channels spaced at ∼15 nm. These images offer dramatic, direct views of STIM1 aggregation and Orai1 clustering in store-depleted cells and provide evidence for the interaction of a single Orai1 channel with small clusters of STIM1 molecules.

Molecular biophysics of Orai store-operated Ca2+ channels.

Upon endoplasmic reticulum Ca(2+) store depletion, Orai channels in the plasma membrane are activated directly by endoplasmic reticulum-resident STIM proteins to generate the Ca(2+)-selective, Ca(2+) release-activated Ca(2+) (CRAC) current. After the molecular identification of Orai, a plethora of functional and biochemical studies sought to compare Orai homologs, determine their stoichiometry, identify structural domains responsible for the biophysical fingerprint of the CRAC current, identify the physiological functions, and investigate Orai homologs as potential therapeutic targets. Subsequently, the solved crystal structure of Drosophila Orai (dOrai) substantiated many findings from structure-function studies, but also revealed an unexpected hexameric structure. In this review, we explore Orai channels as elucidated by functional and biochemical studies, analyze the dOrai crystal structure and its implications for Orai channel function, and present newly available information from molecular dynamics simulations that shed light on Orai channel gating and permeation.

The CRAC channel consists of a tetramer formed by Stim-induced dimerization of Orai dimers.

Ca(2+)-release-activated Ca(2+) (CRAC) channels underlie sustained Ca(2+) signalling in lymphocytes and numerous other cells after Ca(2+) liberation from the endoplasmic reticulum (ER). RNA interference screening approaches identified two proteins, Stim and Orai, that together form the molecular basis for CRAC channel activity. Stim senses depletion of the ER Ca(2+) store and physically relays this information by translocating from the ER to junctions adjacent to the plasma membrane, and Orai embodies the pore of the plasma membrane calcium channel. A close interaction between Stim and Orai, identified by co-immunoprecipitation and by Förster resonance energy transfer, is involved in the opening of the Ca(2+) channel formed by Orai subunits. Most ion channels are multimers of pore-forming subunits surrounding a central channel, which are preassembled in the ER and transported in their final stoichiometry to the plasma membrane. Here we show, by biochemical analysis after cross-linking in cell lysates and intact cells and by using non-denaturing gel electrophoresis without cross-linking, that Orai is predominantly a dimer in the plasma membrane under resting conditions. Moreover, single-molecule imaging of green fluorescent protein (GFP)-tagged Orai expressed in Xenopus oocytes showed predominantly two-step photobleaching, again consistent with a dimeric basal state. In contrast, co-expression of GFP-tagged Orai with the carboxy terminus of Stim as a cytosolic protein to activate the Orai channel without inducing Ca(2+) store depletion or clustering of Orai into punctae yielded mostly four-step photobleaching, consistent with a tetrameric stoichiometry of the active Orai channel. Interaction with the C terminus of Stim thus induces Orai dimers to dimerize, forming tetramers that constitute the Ca(2+)-selective pore. This represents a new mechanism in which assembly and activation of the functional ion channel are mediated by the same triggering molecule.

Mutations in Orai1 transmembrane segment 1 cause STIM1-independent activation of Orai1 channels at glycine 98 and channel closure at arginine 91.

Stim and Orai proteins comprise the molecular machinery of Ca(2+) release-activated Ca(2+) (CRAC) channels. As an approach toward understanding the gating of Orai1 channels, we investigated effects of selected mutations at two conserved sites in the first transmembrane segment (TM1): arginine 91 located near the cytosolic end of TM1 and glycine 98 near the middle of TM1. Orai1 R91C, when coexpressed with STIM1, was activated normally by Ca(2+)-store depletion. Treatment with diamide, a thiol-oxidizing agent, induced formation of disulfide bonds between R91C residues in adjacent Orai1 subunits and rapidly blocked STIM1-operated Ca(2+) current. Diamide-induced blocking was reversed by disulfide bond-reducing agents. These results indicate that R91 forms a very narrow part of the conducting pore at the cytosolic side. Alanine replacement at G98 prevented STIM1-induced channel activity. Interestingly, mutation to aspartate (G98D) or proline (G98P) caused constitutive channel activation in a STIM1-independent manner. Both Orai1 G98 mutants formed a nonselective Ca(2+)-permeable conductance that was relatively resistant to block by Gd(3+). The double mutant R91W/G98D was also constitutively active, overcoming the normal inhibition of channel activity by tryptophan at the 91 position found in some patients with severe combined immunodeficiency (SCID), and the double mutant R91C/G98D was resistant to diamide block. These data suggest that the channel pore is widened and ion selectivity is altered by mutations at the G98 site that may perturb α-helical structure. We propose distinct functional roles for G98 as a gating hinge and R91 as part of the physical gate at the narrow inner mouth of the channel.

Subunit stoichiometry of human Orai1 and Orai3 channels in closed and open states.

We applied single-molecule photobleaching to investigate the stoichiometry of human Orai1 and Orai3 channels tagged with eGFP and expressed in mammalian cells. Orai1 was detected predominantly as dimers under resting conditions and as tetramers when coexpressed with C-STIM1 to activate Ca(2+) influx. Orai1 was also found to be tetrameric when coexpressed with STIM1 and evaluated following fixation. We show that fixation rapidly causes release of Ca(2+), redistribution of STIM1 to the plasma membrane, and STIM1/Orai1 puncta formation, and may cause the channel to be in the activated state. Consistent with this possibility, Orai1 was found predominantly as a dimer when coexpressed with STIM1 in living cells under resting conditions. We further show that Orai3, like Orai1, is dimeric under resting conditions and is predominantly tetrameric when activated by C-STIM1. Interestingly, a dimeric Orai3 stoichiometry was found both before and during application of 2-aminoethyldiphenyl borate (2-APB) to activate a nonselective cation conductance in its STIM1-independent mode. We conclude that the human Orai1 and Orai3 channels undergo a dimer-to-tetramer transition to form a Ca(2+)-selective pore during store-operated activation and that Orai3 forms a dimeric nonselective cation pore upon activation by 2-APB.

Molecular identification of the CRAC channel by altered ion selectivity in a mutant of Orai.

Recent RNA interference screens have identified several proteins that are essential for store-operated Ca2+ influx and Ca2+ release-activated Ca2+ (CRAC) channel activity in Drosophila and in mammals, including the transmembrane proteins Stim (stromal interaction molecule) and Orai. Stim probably functions as a sensor of luminal Ca2+ content and triggers activation of CRAC channels in the surface membrane after Ca2+ store depletion. Among three human homologues of Orai (also known as olf186-F), ORAI1 on chromosome 12 was found to be mutated in patients with severe combined immunodeficiency disease, and expression of wild-type Orai1 restored Ca2+ influx and CRAC channel activity in patient T cells. The overexpression of Stim and Orai together markedly increases CRAC current. However, it is not yet clear whether Stim or Orai actually forms the CRAC channel, or whether their expression simply limits CRAC channel activity mediated by a different channel-forming subunit. Here we show that interaction between wild-type Stim and Orai, assessed by co-immunoprecipitation, is greatly enhanced after treatment with thapsigargin to induce Ca2+ store depletion. By site-directed mutagenesis, we show that a point mutation from glutamate to aspartate at position 180 in the conserved S1-S2 loop of Orai transforms the ion selectivity properties of CRAC current from being Ca2+-selective with inward rectification to being selective for monovalent cations and outwardly rectifying. A charge-neutralizing mutation at the same position (glutamate to alanine) acts as a dominant-negative non-conducting subunit. Other charge-neutralizing mutants in the same loop express large inwardly rectifying CRAC current, and two of these exhibit reduced sensitivity to the channel blocker Gd3+. These results indicate that Orai itself forms the Ca2+-selectivity filter of the CRAC channel.

Piezo1 channels restrain regulatory T cells but are dispensable for effector CD4+ T cell responses.

T lymphocytes encounter complex mechanical cues during an immune response. The mechanosensitive ion channel, Piezo1, drives inflammatory responses to bacterial infections, wound healing, and cancer; however, its role in helper T cell function remains unclear. In an animal model for multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), we found that mice with genetic deletion of Piezo1 in T cells showed diminished disease severity. Unexpectedly, Piezo1 was not essential for lymph node homing, interstitial motility, Ca2+ signaling, T cell proliferation, or differentiation into proinflammatory T helper 1 (TH1) and TH17 subsets. However, Piezo1 deletion in T cells resulted in enhanced transforming growth factor-ß (TGFß) signaling and an expanded pool of regulatory T (Treg) cells. Moreover, mice with deletion of Piezo1 specifically in Treg cells showed significant attenuation of EAE. Our results indicate that Piezo1 selectively restrains Treg cells, without influencing activation events or effector T cell functions.

STIM1, an essential and conserved component of store-operated Ca2+ channel function.

Store-operated Ca2+ (SOC) channels regulate many cellular processes, but the underlying molecular components are not well defined. Using an RNA interference (RNAi)-based screen to identify genes that alter thapsigargin (TG)-dependent Ca2+ entry, we discovered a required and conserved role of Stim in SOC influx. RNAi-mediated knockdown of Stim in Drosophila S2 cells significantly reduced TG-dependent Ca2+ entry. Patch-clamp recording revealed nearly complete suppression of the Drosophila Ca2+ release-activated Ca2+ (CRAC) current that has biophysical characteristics similar to CRAC current in human T cells. Similarly, knockdown of the human homologue STIM1 significantly reduced CRAC channel activity in Jurkat T cells. RNAi-mediated knockdown of STIM1 inhibited TG- or agonist-dependent Ca2+ entry in HEK293 or SH-SY5Y cells. Conversely, overexpression of STIM1 in HEK293 cells modestly enhanced TG-induced Ca2+ entry. We propose that STIM1, a ubiquitously expressed protein that is conserved from Drosophila to mammalian cells, plays an essential role in SOC influx and may be a common component of SOC and CRAC channels.

Cell-wide mapping of Orai1 channel activity reveals functional heterogeneity in STIM1-Orai1 puncta.

Upon Ca2+ store depletion, Orai1 channels cluster and open at endoplasmic reticulum-plasma membrane (ER-PM) junctions in signaling complexes called puncta. Little is known about whether and how Orai1 channel activity may vary between individual puncta. Previously, we developed and validated optical recording of Orai channel activity, using genetically encoded Ca2+ indicators fused to Orai1 or Orai3 N or C termini. We have now combined total internal reflection fluorescence microscopy with whole-cell recording to map functional properties of channels at individual puncta. After Ca2+ store depletion in HEK cells cotransfected with mCherry-STIM1 and Orai1-GCaMP6f, Orai1-GCaMP6f fluorescence increased progressively with increasingly negative test potentials and robust responses could be recorded from individual puncta. Cell-wide fluorescence half-rise and -fall times during steps to -100 mV test potential indicated probe response times of <50 ms. The in situ Orai1-GCaMP6f affinity for Ca2+ was 620 nM, assessed by monitoring fluorescence using buffered Ca2+ solutions in "unroofed" cells. Channel activity and temporal activation profile were tracked in individual puncta using image maps and automated puncta identification and recording. Simultaneous measurement of mCherry-STIM1 fluorescence uncovered an unexpected gradient in STIM1/Orai1 ratio that extends across the cell surface. Orai1-GCaMP6f channel activity was found to vary across the cell, with inactive channels occurring in the corners of cells where the STIM1/Orai1 ratio was lowest; low-activity channels typically at edges displayed a slow activation phase lasting hundreds of milliseconds. Puncta with high STIM1/Orai1 ratios exhibited a range of channel activity that appeared unrelated to the stoichiometric requirements for gating. These findings demonstrate functional heterogeneity of Orai1 channel activity between individual puncta and establish a new experimental platform that facilitates systematic comparisons between puncta composition and activity.

Molecular basis of the CRAC channel.

Ca(2+) release-activated Ca(2+) (CRAC) channels, located in the plasma membrane, are opened upon release of Ca(2+) from intracellular stores, permitting Ca(2+) entry and sustained [Ca(2+)](i) signaling that replenishes the store in numerous cell types. This mechanism is particularly important in T lymphocytes of the immune system, providing the missing link in the signal transduction cascade that is initiated by T cell receptor engagement and leads to altered expression of genes that results ultimately in the production of cytokines and cell proliferation. In the past three years, RNA interference screens together with over-expression and site-directed mutagenesis have identified the triggering molecule (Stim) that links store depletion to CRAC channel-mediated Ca(2+) influx and the pore subunit (Orai) of the CRAC channel that allows highly selective entry of Ca(2+) ions into cells.

iPSC-Derived Human Microglia-like Cells to Study Neurological Diseases.

Microglia play critical roles in brain development, homeostasis, and neurological disorders. Here, we report that human microglial-like cells (iMGLs) can be differentiated from iPSCs to study their function in neurological diseases, like Alzheimer's disease (AD). We find that iMGLs develop in vitro similarly to microglia in vivo, and whole-transcriptome analysis demonstrates that they are highly similar to cultured adult and fetal human microglia. Functional assessment of iMGLs reveals that they secrete cytokines in response to inflammatory stimuli, migrate and undergo calcium transients, and robustly phagocytose CNS substrates. iMGLs were used to examine the effects of Aß fibrils and brain-derived tau oligomers on AD-related gene expression and to interrogate mechanisms involved in synaptic pruning. Furthermore, iMGLs transplanted into transgenic mice and human brain organoids resemble microglia in vivo. Together, these findings demonstrate that iMGLs can be used to study microglial function, providing important new insight into human neurological disease.

A store-operated calcium channel in Drosophila S2 cells.

Using whole-cell recording in Drosophila S2 cells, we characterized a Ca(2+)-selective current that is activated by depletion of intracellular Ca2+ stores. Passive store depletion with a Ca(2+)-free pipette solution containing 12 mM BAPTA activated an inwardly rectifying Ca2+ current with a reversal potential >60 mV. Inward currents developed with a delay and reached a maximum of 20-50 pA at -110 mV. This current doubled in amplitude upon increasing external Ca2+ from 2 to 20 mM and was not affected by substitution of choline for Na+. A pipette solution containing approximately 300 nM free Ca2+ and 10 mM EGTA prevented spontaneous activation, but Ca2+ current activated promptly upon application of ionomycin or thapsigargin, or during dialysis with IP3. Isotonic substitution of 20 mM Ca2+ by test divalent cations revealed a selectivity sequence of Ba2+ > Sr2+ > Ca2+ >> Mg2+. Ba2+ and Sr2+ currents inactivated within seconds of exposure to zero-Ca2+ solution at a holding potential of 10 mV. Inactivation of Ba2+ and Sr2+ currents showed recovery during strong hyperpolarizing pulses. Noise analysis provided an estimate of unitary conductance values in 20 mM Ca2+ and Ba2+ of 36 and 420 fS, respectively. Upon removal of all external divalent ions, a transient monovalent current exhibited strong selectivity for Na+ over Cs+. The Ca2+ current was completely and reversibly blocked by Gd3+, with an IC50 value of approximately 50 nM, and was also blocked by 20 microM SKF 96365 and by 20 microM 2-APB. At concentrations between 5 and 14 microM, application of 2-APB increased the magnitude of Ca2+ currents. We conclude that S2 cells express store-operated Ca2+ channels with many of the same biophysical characteristics as CRAC channels in mammalian cells.

Store-dependent and -independent modes regulating Ca2+ release-activated Ca2+ channel activity of human Orai1 and Orai3.

We evaluated currents induced by expression of human homologs of Orai together with STIM1 in human embryonic kidney cells. When co-expressed with STIM1, Orai1 induced a large inwardly rectifying Ca(2+)-selective current with Ca(2+)-induced slow inactivation. A point mutation of Orai1 (E106D) altered the ion selectivity of the induced Ca(2+) release-activated Ca(2+) (CRAC)-like current while retaining an inwardly rectifying I-V characteristic. Expression of the C-terminal portion of STIM1 with Orai1 was sufficient to generate CRAC current without store depletion. 2-APB activated a large relatively nonselective current in STIM1 and Orai3 co-expressing cells. 2-APB also induced Ca(2+) influx in Orai3-expressing cells without store depletion or co-expression of STIM1. The Orai3 current induced by 2-APB exhibited outward rectification and an inward component representing a mixed calcium and monovalent current. A pore mutant of Orai3 inhibited store-operated Ca(2+) entry and did not carry significant current in response to either store depletion or addition of 2-APB. Analysis of a series of Orai1-3 chimeras revealed the structural determinant responsible for 2-APB-induced current within the sequence from the second to third transmembrane segment of Orai3. The Orai3 current induced by 2-APB may reflect a store-independent mode of CRAC channel activation that opens a relatively nonselective cation pore.

Genome-wide RNAi screen of Ca(2+) influx identifies genes that regulate Ca(2+) release-activated Ca(2+) channel activity.

Recent studies by our group and others demonstrated a required and conserved role of Stim in store-operated Ca(2+) influx and Ca(2+) release-activated Ca(2+) (CRAC) channel activity. By using an unbiased genome-wide RNA interference screen in Drosophila S2 cells, we now identify 75 hits that strongly inhibited Ca(2+) influx upon store emptying by thapsigargin. Among these hits are 11 predicted transmembrane proteins, including Stim, and one, olf186-F, that upon RNA interference-mediated knockdown exhibited a profound reduction of thapsigargin-evoked Ca(2+) entry and CRAC current, and upon overexpression a 3-fold augmentation of CRAC current. CRAC currents were further increased to 8-fold higher than control and developed more rapidly when olf186-F was cotransfected with Stim. olf186-F is a member of a highly conserved family of four-transmembrane spanning proteins with homologs from Caenorhabditis elegans to human. The endoplasmic reticulum (ER) Ca(2+) pump sarco-/ER calcium ATPase (SERCA) and the single transmembrane-soluble N-ethylmaleimide-sensitive (NSF) attachment receptor (SNARE) protein Syntaxin5 also were required for CRAC channel activity, consistent with a signaling pathway in which Stim senses Ca(2+) depletion within the ER, translocates to the plasma membrane, and interacts with olf186-F to trigger CRAC channel activity.