Anna Amcheslavsky

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 Ca2+ release-activated Ca2+ (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 Ca2+-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 Ca2+ 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 Ca2+-permeable conductance that was relatively resistant to block by Gd3+. 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 Ca2+ influx. Orai1 was also found to be tetrameric when coexpressed with STIM1 and evaluated following fixation. We show that fixation rapidly causes release of Ca2+, 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 Ca2+-selective pore during store-operated activation and that Orai3 forms a dimeric nonselective cation pore upon activation by 2-APB.

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.

State-dependent block of Orai3 TM1 and TM3 cysteine mutants: Insights into 2-APB activation

Residue E165, in transmembrane helix 3, participates in formation of the dilated pore of the 2-APB–activated Orai3 channel but not that of the more selective store-operated Orai3 pore.

Genetically targeted single-channel optical recording reveals multiple Orai1 gating states and oscillations in calcium influx

Significance Calcium ions serve as intracellular signals controlling many aspects of cell behavior. Here, we create fusions of genetically encoded calcium indicators and cell surface Orai calcium channels that report calcium influx through the channel. These fluorescent fusion proteins will allow us to see in living cells Orai signals that underlie cell functions, especially those essential for mounting an adaptive immune response. Their output is bright enough to record the intermittent openings of a single Orai channel for the first time to our knowledge. Our recordings reveal several patterns of channel activity, including oscillations. These responses help us understand Orai channels as molecular machines, and our work enables Orai calcium signals to be identified, mapped, and related to the functional systems of the body. Orai1 comprises the pore-forming subunit of the Ca2+ release-activated Ca2+ (CRAC) channel. When bound and activated by stromal interacting molecule 1 (STIM1), an endoplasmic reticulum (ER)-resident calcium sensor, Orai1 channels possess high selectivity for calcium but extremely small conductance that has precluded direct recording of single-channel currents. We have developed an approach to visualize Orai1 activity by fusing Orai1 to fluorescent, genetically encoded calcium indicators (GECIs). The GECI–Orai1 probes reveal local Ca2+ influx at STIM1–Orai1 puncta. By whole cell recording, these fusions are fully functional as CRAC channels. When GECI–Orai1 and the CRAC-activating domain (CAD) of STIM1 were coexpressed at low levels and imaged using a total internal reflectance fluorescence microscope, cells exhibited sporadic fluorescence transients the size of diffraction-limited spots and the brightness of a few activated GECI proteins. Transients typically rose rapidly and fell into two classes according to duration: briefer “flickers” lasting only a few hundred milliseconds, and longer “pulses” lasting one to several seconds. The size, intensity, trace shape, frequency, distribution, physiological characteristics, and association with CAD binding together demonstrate that GECI–Orai1 fluorescence transients correspond to single-channel Orai1 responses. Single Orai1 channels gated by CAD, and small Orai1 puncta gated by STIM1, exhibit repetitive fluctuations in single-channel output. CAD binding supports a role in open state maintenance and reveals a second phase of CAD/STIM1 binding after channel opening. These first recordings of single-channel Orai1 currents reveal unexpected dynamics, and when paired with CAD association, support multiple single-channel states.

Orai3 TM3 point mutation G158C alters kinetics of 2-APB-induced gating by disulfide bridge formation with TM2 C101.

After endoplasmic reticulum (ER) Ca2+ store depletion, Orai channels in the plasma membrane (PM) are activated directly by ER-resident STIM proteins to form the Ca2+-selective Ca2+ release–activated Ca2+ (CRAC) channel. However, in the absence of Ca2+ store depletion and STIM interaction, the mammalian homologue Orai3 can be activated by 2-aminoethyl diphenylborinate (2-APB), resulting in a nonselective cation conductance characterized by biphasic inward and outward rectification. Here, we use site-directed mutagenesis and patch-clamp analysis to better understand the mechanism by which 2-APB activates Orai3. We find that point mutation of glycine 158 in the third transmembrane (TM) segment to cysteine, but not alanine, slows the kinetics of 2-APB activation and prevents complete channel closure upon 2-APB washout. The “slow” phenotype exhibited by Orai3 mutant G158C reveals distinct open states, characterized by variable reversal potentials. The slow phenotype can be reversed by application of the reducing reagent bis(2-mercaptoethylsulfone) (BMS), but in a state-dependent manner, only during 2-APB activation. Moreover, the double mutant C101G/G158C, in which an endogenous TM2 cysteine is changed to glycine, does not exhibit altered kinetics of 2-APB activation. We suggest that a disulfide bridge, formed between the introduced cysteine at TM3 position 158 and the endogenous cysteine at TM2 position 101, hinders transitions between Orai3 open and closed states. Our data provide functional confirmation of the proximity of these two residues and suggest a location within the Orai3 protein that is sensitive to the actions of 2-APB.

Store-Operated Calcium Channels

Store-operated calcium (SOC) channels mediate calcium (Ca 2+ ) influx from the extracellular environment into the cell cytosol upon depletion of endoplasmic reticulum (ER) Ca 2+ stores. The molecular components responsible for SOC entry (SOCE) remained a mystery until two recent discoveries. RNA-interference (RNAi) screening of candidate genes led first to the discovery of dStim/STIM1, an ER-resident transmembrane protein responsible for mediating the signal that activates SOCE. Genome-wide RNAi screening then led to identification of the ORAI proteins that form the Ca 2+ -selective pore of the SOC channel known as the calcium release-activated calcium channel. STIM dimers sense ER Ca 2+ store depletion through Ca 2+ unbinding from STIMs low-affinity, N-terminal EF-hand in the lumen of the ER. This induces STIM dimers to oligomerize and to translocate toward the plasma membrane (PM). STIM then forms dense clusters at ER–PM junctions, organizes ORAI dimers into tetramers in mirror-image PM clusters, and finally activates ORAI tetramers to conduct Ca 2+ selectively across the PM. STIM proteins also organize canonical transient receptor potential channels into clusters and open them to bring about SOCE through Ca 2+ -permeable but relatively nonselective pores. STIM proteins serve multiple signaling functions that have important biomedical implications, particularly in the immune system.

Subunit Stoichiometry of Human Orai1 and Orai3 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 HEK cells. At low expression, GFP-tagged subunits were detected in TIRF microscopy as single fluorescent spots that decayed in a step-wise manner as individual GFP molecules bleached. By counting the number of photobleaching steps, the number of subunits per channel complex could be deduced. In fixed cells, Orai1 was detected primarily as dimers when expressed alone and as tetramers when co-expressed with the cytosolic STIM1 fragment, C-STIM1, to activate Ca2+ influx, as previously found for Drosophila Orai with and without activation by C-Stim (Penna et al., 2008, Nature 456:116-120). When co-expressed with full-length STIM1, Orai1 was also found to be predominantly dimeric in living cells under resting conditions with Ca2+ stores filled. We also investigated Orai3 alone and when activated by either C-STIM1 to form a Ca2+-selective channel in its store-operated mode, or by addition of 2-APB to form a Ca2+-permeable but relatively nonselective cation channel in its STIM1-independent mode. Similar to our observations with Orai1, eGFP-Orai3 alone was detected mostly as dimers under basal conditions, but predominantly as tetramers when co-expressed with C-STIM1. On the other hand, cells expressing only eGFP-Orai3 that were exposed to 2APB before fixation showed a distribution of bleaching steps closely similar to that observed without 2APB, with a predominance of dimers. These results indicate a predominantly dimeric state for Orai3 at rest or when activated by 2-APB, and a tetrameric channel when activated by C-STIM1. We conclude that the human Orai1 and Orai3 channels undergo a dimer-to-tetramer transition to form a Ca2+-selective pore during store-operated activation, and that Orai3 forms a dimeric non-selective cation pore upon activation by 2-APB.