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The activation of the Ca 2+ -channel Orai1 via the physiological activator stromal interaction molecule 1 (STIM1) requires structural rearrangements within the entire channel complex involving a series of gating checkpoints. Focusing on the gating mechanism operating along the peripheral transmembrane domain (TM) 3/TM4-interface, we report here that some charged substitutions close to the center of TM3 or TM4 lead to constitutively active Orai1 variants triggering nuclear factor of activated T-cell (NFAT) translocation into the nucleus. Molecular dynamics simulations unveil that this gain-of-function correlates with enhanced hydration at peripheral TM-interfaces, leading to increased local structural flexibility of the channel periphery and global conformational changes permitting pore opening. Our findings indicate that efficient dehydration of the peripheral TM-interfaces driven by the hydrophobic effect is critical for maintaining the closed state of Orai1. We conclude that a charge close to the center of TM3 or TM4 facilitates concomitant hydration and widening of peripheral TM interfaces to trigger constitutive Orai1 pore opening to a level comparable to or exceeding that of native activated Orai1., Competing Interests: Competing interests The authors declare no competing interests. Consent to publish All authors have read and approved its submission to this journal., (© 2024. The Author(s).)

The single-pass transmembrane protein Stromal Interaction Molecule 1 (STIM1), located in the endoplasmic reticulum (ER) membrane, possesses two main functions: It senses the ER-Ca 2+ concentration and directly binds to the store-operated Ca 2+ channel Orai1 for its activation when Ca 2+ recedes. At high resting ER-Ca 2+ concentration, the ER-luminal STIM1 domain is kept monomeric but undergoes di/multimerization once stores are depleted. Luminal STIM1 multimerization is essential to unleash the STIM C-terminal binding site for Orai1 channels. However, structural basis of the luminal association sites has so far been elusive. Here, we employed molecular dynamics (MD) simulations and identified two essential di/multimerization segments, the α7 and the adjacent region near the α9-helix in the sterile alpha motif (SAM) domain. Based on MD results, we targeted the two STIM1 SAM domains by engineering point mutations. These mutations interfered with higher-order multimerization of ER-luminal fragments in biochemical assays and puncta formation in live-cell experiments upon Ca 2+ store depletion. The STIM1 multimerization impeded mutants significantly reduced Ca 2+ entry via Orai1, decreasing the Ca 2+ oscillation frequency as well as store-operated Ca 2+ entry. Combination of the ER-luminal STIM1 multimerization mutations with gain of function mutations and coexpression of Orai1 partially ameliorated functional defects. Our data point to a hydrophobicity-driven binding within the ER-luminal STIM1 multimer that needs to switch between resting monomeric and activated multimeric state. Altogether, these data reveal that interactions between SAM domains of STIM1 monomers are critical for multimerization and activation of the protein., Competing Interests: Competing interests statement:The authors declare no competing interest.

The family of stromal interaction molecules (STIM) includes two widely expressed single-pass endoplasmic reticulum (ER) transmembrane proteins and additional splice variants that act as precise ER-luminal Ca 2+ sensors. STIM proteins mainly function as one of the two essential components of the so-called Ca 2+ release-activated Ca 2+ (CRAC) channel. The second CRAC channel component is constituted by pore-forming Orai proteins in the plasma membrane. STIM and Orai physically interact with each other to enable CRAC channel opening, which is a critical prerequisite for various downstream signalling pathways such as gene transcription or proliferation. Their activation commonly requires the emptying of the intracellular ER Ca 2+ store. Using their Ca 2+ sensing capabilities, STIM proteins confer this Ca 2+ content-dependent signal to Orai, thereby linking Ca 2+ store depletion to CRAC channel opening. Here we review the conformational dynamics occurring along the entire STIM protein upon store depletion, involving the transition from the quiescent, compactly folded structure into an active, extended state, modulation by a variety of accessory components in the cell as well as the impairment of individual steps of the STIM activation cascade associated with disease., (© 2023 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

The store-operated calcium channels Orai1-3 form extraordinary long and funnel like pores, in stark contrast to a classical pore loop architecture. A hydrophobic segment centrally located in the Orai pore controls gating. Here, we comment on a recent work that describes decisive binding between three residues that controls the open and closed conformation of Orai channels., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The initial activation step in the gating of ubiquitously expressed Orai1 calcium (Ca 2+ ) ion channels represents the activation of the Ca 2+ -sensor protein STIM1 upon Ca 2+ store depletion of the endoplasmic reticulum. Previous studies using constitutively active Orai1 mutants gave rise to, but did not directly test, the hypothesis that STIM1-mediated Orai1 pore opening is accompanied by a global conformational change of all Orai transmembrane domain (TM) helices within the channel complex. We prove that a local conformational change spreads omnidirectionally within the Orai1 complex. Our results demonstrate that these locally induced global, opening-permissive TM motions are indispensable for pore opening and require clearance of a series of Orai1 gating checkpoints. We discovered these gating checkpoints in the middle and cytosolic extended TM domain regions. Our findings are based on a library of double point mutants that contain each one loss-of-function with one gain-of-function point mutation in a series of possible combinations. We demonstrated that an array of loss-of-function mutations are dominant over most gain-of-function mutations within the same as well as of an adjacent Orai subunit. We further identified inter- and intramolecular salt-bridge interactions of Orai subunits as a core element of an opening-permissive Orai channel architecture. Collectively, clearance and synergistic action of all these gating checkpoints are required to allow STIM1 coupling and Orai1 pore opening. Our results unravel novel insights in the preconditions of the unique fingerprint of CRAC channel activation, provide a valuable source for future structural resolutions, and help to understand the molecular basis of disease-causing mutations., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The Ca 2+ sensor STIM1 and the Ca 2+ channel Orai1 that form the store-operated Ca 2+ (SOC) channel complex are key targets for drug development. Selective SOC inhibitors are currently undergoing clinical evaluation for the treatment of auto-immune and inflammatory responses and are also deemed promising anti-neoplastic agents since SOC channels are linked with enhanced cancer cell progression. Here, we describe an investigation of the site of binding of the selective inhibitor Synta66 to the SOC channel Orai1 using docking and molecular dynamics simulations, and live cell recordings. Synta66 binding was localized to the extracellular site close to the transmembrane (TM)1 and TM3 helices and the extracellular loop segments, which, importantly, are adjacent to the Orai1-selectivity filter. Synta66-sensitivity of the Orai1 pore was, in fact, diminished by both Orai1 mutations affecting Ca 2+ selectivity and permeation of Na + in the absence of Ca 2+ . Synta66 also efficiently blocked SOC in three glioblastoma cell lines but failed to interfere with cell viability, division and migration. These experiments provide new structural and functional insights into selective drug inhibition of the Orai1 Ca 2+ channel by a high-affinity pore blocker.

Stromal interaction molecule 1 (STIM1) is a ubiquitously expressed Ca 2+ sensor protein that induces permeation of Orai Ca 2+ channels upon endoplasmic reticulum Ca 2+ -store depletion. A drop in luminal Ca 2+ causes partial unfolding of the N-terminal STIM1 domains and thus initial STIM1 activation. We compared the STIM1 structure upon Ca 2+ depletion from our molecular dynamics (MD) simulations with a recent 2D NMR structure. Simulation- and structure-based results showed unfolding of two α-helices in the canonical and in the non-canonical EF-hand. Further, we structurally and functionally evaluated mutations in the non-canonical EF-hand that have been shown to cause tubular aggregate myopathy. We found these mutations to cause full constitutive activation of Ca 2+ -release-activated Ca 2+ currents (I CRAC ) and to promote autophagic processes. Specifically, heterologously expressed STIM1 mutations in the non-canonical EF-hand promoted translocation of the autophagy transcription factors microphthalmia-associated transcription factor (MITF) and transcription factor EB (TFEB) into the nucleus. These STIM1 mutations additionally stimulated an enhanced production of autophagosomes. In summary, mutations in STIM1 that cause structural unfolding promoted Ca 2+ down-stream activation of autophagic processes.

The stromal interaction molecule 1 (STIM1) has two important functions, Ca 2+ sensing within the endoplasmic reticulum and activation of the store-operated Ca 2+ channel Orai1, enabling plasma-membrane Ca 2+ influx. We combined molecular dynamics (MD) simulations with live-cell recordings and determined the sequential Ca 2+ -dependent conformations of the luminal STIM1 domain upon activation. Furthermore, we identified the residues within the canonical and noncanonical EF-hand domains that can bind to multiple Ca 2+ ions. In MD simulations, a single Ca 2+ ion was sufficient to stabilize the luminal STIM1 complex. Ca 2+ store depletion destabilized the two EF hands, triggering disassembly of the hydrophobic cleft that they form together with the stable SAM domain. Point mutations associated with tubular aggregate myopathy or cancer that targeted the canonical EF hand, and the hydrophobic cleft yielded constitutively clustered STIM1, which was associated with activation of Ca 2+ entry through Orai1 channels. On the basis of our results, we present a model of STIM1 Ca 2+ binding and refine the currently known initial steps of STIM1 activation on a molecular level., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Highly Ca 2+ selective channels trigger a large variety of cellular signaling processes in both excitable and non-excitable cells. Among these channels, the Orai channel is unique in its activation mechanism and its structure. It mediates Ca 2+ influx into the cytosol with an extremely small unitary conductance over longer time-scales, ranging from minutes up to several hours. Its activation is regulated by the Ca 2+ content of the endoplasmic reticulum (ER). Depletion of luminal [Ca 2+ ] ER is sensed by the STIM1 single transmembrane protein that directly binds and gates the Orai1 channel. Orai mediated Ca 2+ influx increases cytosolic Ca 2+ from 100 nM up to low micromolar range close to the pore and thereby forms Ca 2+ microdomains. Hence, these features of the Orai channel can trigger long-term signaling processes without affecting the overall Ca 2+ content of a single living cell. Here we focus on the architecture and dynamic conformational changes within the Orai channel. This review summarizes current achievements of molecular dynamics simulations in combination with live cell recordings to address gating and permeation of the Orai channel with molecular precision., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The low bioavailability of most therapeutic compounds is often counterbalanced by association with molecular vectors capable of crossing cell membranes. Previous studies demonstrated that for vectors bearing titratable chemical groups, the translocation process might be accompanied by a change in the protonation state. For simple compounds e.g. a lysine analog, free energy calculations, using a single collective variable, namely the insertion depth, suggest that such a transition could only take place if the amino acid diffuses deep enough into the hydrophobic core of the membrane, a situation thermodynamically unfavorable. Here, we determined the 2D potential of mean force associated with the translocation of lysine across a model membrane using as reaction coordinates not only its location in the bilayer but also its hydration. Our results cogently demonstrate that the change in protonation can result from a small fluctuation in the latter, even at low insertion depth.

The channel Orai1 requires Ca 2+ store depletion in the endoplasmic reticulum and an interaction with the Ca 2+ sensor STIM1 to mediate Ca 2+ signaling. Alterations in Orai1-mediated Ca 2+ influx have been linked to several pathological conditions including immunodeficiency, tubular myopathy, and cancer. We screened large-scale cancer genomics data sets for dysfunctional Orai1 mutants. Five of the identified Orai1 mutations resulted in constitutively active gating and transcriptional activation. Our analysis showed that certain Orai1 mutations were clustered in the transmembrane 2 helix surrounding the pore, which is a trigger site for Orai1 channel gating. Analysis of the constitutively open Orai1 mutant channels revealed two fundamental gates that enabled Ca 2+ influx: Arginine side chains were displaced so they no longer blocked the pore, and a chain of water molecules formed in the hydrophobic pore region. Together, these results enabled us to identify a cluster of Orai1 mutations that trigger Ca 2+ permeation associated with gene transcription and provide a gating mechanism for Orai1., (Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Two approaches for modeling of the transmembrane potential, as present in all eukaryotic cells, are examined in detail and compared with each other. One approach uses an externally applied electric field, whereas the other maintains an imbalance of ions on the two sides of a membrane. We demonstrate that both methods provide converged results concerning structural parameters of the membrane which are practically indistinguishable from each other, at least for monovalent ions. Effects of the electric field on the detailed molecular structure of the phospholipid bilayer are also presented and discussed. In addition, we achieve a considerable speed-up of the underlying molecular dynamics simulations by implementing the virtual interaction sites method for the Slipids force field.

We report a novel nontoxic, high-yield, gene delivery system based on the synergistic use of nanosecond electric pulses (NPs) and nanomolar doses of the recently introduced CM18-Tat11 chimeric peptide (sequence of KWKLFKKIGAVLKVLTTGYGRKKRRQRRR, residues 1-7 of cecropin-A, 2-12 of melittin, and 47-57 of HIV-1 Tat protein). This combined use makes it possible to drastically reduce the required CM18-Tat11 concentration and confines stable nanopore formation to vesicle membranes followed by DNA release, while no detectable perturbation of the plasma membrane is observed. Two different experimental assays are exploited to quantitatively evaluate the details of NPs and CM18-Tat11 cooperation: (i) cytofluorimetric analysis of the integrity of synthetic 1,2-dioleoyl-sn-glycero-3-phosphocholine giant unilamellar vesicles exposed to CM18-Tat11 and NPs and (ii) the in vitro transfection efficiency of a green fluorescent protein-encoding plasmid conjugated to CM18-Tat11 in the presence of NPs. Data support a model in which NPs induce membrane perturbation in the form of transient pores on all cellular membranes, while the peptide stabilizes membrane defects selectively within endosomes. Interestingly, atomistic molecular dynamics simulations show that the latter activity can be specifically attributed to the CM18 module, while Tat11 remains essential for cargo binding and vector subcellular localization. We argue that this result represents a paradigmatic example that can open the way to other targeted delivery protocols.

The transfer of a lysine amino acid analogue across phospholipid membrane models was investigated using molecular-dynamics simulations. The evolution of the protonation state of this small peptide as a function of its position inside the membrane was studied by determining the local pKa by means of free-energy calculations. Permeability and mean-first-passage time were evaluated and showed that the transfer occurs on the submillisecond time scale. Comparative studies were conducted to evaluate changes in the pKa arising from differences in the phospholipid chemical structure. We compared, hence, the effect of an ether vs an ester linkage of the lipid headgroup as well as linear vs branched lipid tails. The study reveals that protonated lysine residues can be buried further inside an ether lipid membrane than an ester lipid membrane, while branched lipids are found to stabilize less the charged form compared to their unbranched lipid chain counterparts.

Electroporation relates to the cascade of events that follows the application of high electric fields and that leads to cell membrane permeabilization. Despite a wide range of applications, little is known about the electroporation threshold, which varies with membrane lipid composition. Here, using molecular dynamics simulations, we studied the response of dipalmitoyl-phosphatidylcholine, diphytanoyl-phosphocholine-ester and diphytanoyl-phosphocholine-ether lipid bilayers to an applied electric field. Comparing between lipids with acyl chains and methyl branched chains and between lipids with ether and ester linkages, which change drastically the membrane dipole potential, we found that in both cases the electroporation threshold differed substantially. We show, for the first time, that the electroporation threshold of a lipid bilayer depends not only on the "electrical" properties of the membrane, i.e., its dipole potential, but also on the properties of its component hydrophobic tails.

A hybrid nanoporous membrane made of a solid-state polymeric thin film in which an ion channel is confined is realized. The primary and extremely encouraging results obtained by confocal fluorescence spectroscopy and ion diffusion measurement demonstrate respectively that (i) the considered ion channel, that is, Gramicidin-A, can be confined selectively inside the nanopores and (ii) the ionic permeability of the membrane is enhanced. Atomistic molecular simulations are also reported and fruitfully compared to the experimental findings.