Essential role of N-terminal SAM regions in STIM1 multimerization and function.

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-Ca2+ concentration and directly binds to the store-operated Ca2+ channel Orai1 for its activation when Ca2+ recedes. At high resting ER-Ca2+ 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 Ca2+ store depletion. The STIM1 multimerization impeded mutants significantly reduced Ca2+ entry via Orai1, decreasing the Ca2+ oscillation frequency as well as store-operated Ca2+ 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.

Mechanistic insights into the Orai channel by molecular dynamics simulations.

Highly Ca2+ 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 Ca2+ 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 Ca2+ content of the endoplasmic reticulum (ER). Depletion of luminal [Ca2+]ER is sensed by the STIM1 single transmembrane protein that directly binds and gates the Orai1 channel. Orai mediated Ca2+ influx increases cytosolic Ca2+ from 100 nM up to low micromolar range close to the pore and thereby forms Ca2+ microdomains. Hence, these features of the Orai channel can trigger long-term signaling processes without affecting the overall Ca2+ 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.

Luminal STIM1 Mutants that Cause Tubular Aggregate Myopathy Promote Autophagic Processes.

Stromal interaction molecule 1 (STIM1) is a ubiquitously expressed Ca2+ sensor protein that induces permeation of Orai Ca2+ channels upon endoplasmic reticulum Ca2+-store depletion. A drop in luminal Ca2+ causes partial unfolding of the N-terminal STIM1 domains and thus initial STIM1 activation. We compared the STIM1 structure upon Ca2+ 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 Ca2+-release-activated Ca2+ currents (ICRAC) 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 Ca2+ down-stream activation of autophagic processes.

Postmortem MR diffusion-weighted imaging of the liver: time-behavior of the hepatic apparent diffusion coefficient in the early death interval.

OBJECTIVE: The objective of this study is to assess postmortem changes of the hepatic apparent diffusion coefficient (ADC) at multiple time points in the time interval of 16 hours postmortem in comparison to in vivo controls and to literature data. MATERIAL AND METHODS: Hepatic diffusion-weighted imaging (DWI) was repeatedly performed at 1.5 Tesla (b values 50, 400, and 800 s/mm2) in 2-hourly steps within 16 hours postmortem in 19 cases (male to female 13:6, mean age 68.5 ± 12.2 years) and 5 in vivo controls. The core body temperature was measured rectally prior to every scan. Mean ADC values were calculated from regions of interest (ROIs) and compared to in vivo healthy controls and to literature data of normal liver parenchyma. Spearman rank correlation and Levenberg-Marquardt algorithm were used to assess a relationship between postmortem core body temperature and ADC values. RESULTS: Mean hepatic ADC values were significantly lower in postmortem cases than in in vivo controls (52.0 ± 15.0 · 10-5 mm2/s vs. 111.0 ± 15.7 · 10-5 mm2/s, p < 0.0001). The ex vivo liver ADC correlated inversely to calculated liver temperature (-3.5 ± 0.8) · 10-5 mm2/s/°C, r = -0.44, p < 0.0001. At low calculated liver temperature (< 30 °C), the ADC described an average increase of (22 ± 10) · 10-5 mm2/s/°C. CONCLUSION: Hepatic ADC values show a characteristic change in the immediate 16 hours postmortem, which is influenced by the postmortem liver temperature change. With the knowledge of characteristic postmortem liver changes, diffusion-weighted imaging could be added to conventional postmortem MRI for virtual autopsy.

Photo-Chemical Stimulation of Neurons with Organic Semiconductors.

Recent advances in light-responsive materials enabled the development of devices that can wirelessly activate tissue with light. Here it is shown that solution-processed organic heterojunctions can stimulate the activity of primary neurons at low intensities of light via photochemical reactions. The p-type semiconducting polymer PDCBT and the n-type semiconducting small molecule ITIC (a non-fullerene acceptor) are coated on glass supports, forming a p-n junction with high photosensitivity. Patch clamp measurements show that low-intensity white light is converted into a cue that triggers action potentials in primary cortical neurons. The study shows that neat organic semiconducting p-n bilayers can exchange photogenerated charges with oxygen and other chemical compounds in cell culture conditions. Through several controlled experimental conditions, photo-capacitive, photo-thermal, and direct hydrogen peroxide effects on neural function are excluded, with photochemical delivery being the possible mechanism. The profound advantages of low-intensity photo-chemical intervention with neuron electrophysiology pave the way for developing wireless light-based therapy based on emerging organic semiconductors.

Light Stimulation of Neurons on Organic Photocapacitors Induces Action Potentials with Millisecond Precision.

Nongenetic optical control of neurons is a powerful technique to study and manipulate the function of the nervous system. This research has benchmarked the performance of organic electrolytic photocapacitor (OEPC) optoelectronic stimulators at the level of single mammalian cells: human embryonic kidney (HEK) cells with heterologously expressed voltage-gated K+ channels and hippocampal primary neurons. OEPCs act as extracellular stimulation electrodes driven by deep red light. The electrophysiological recordings show that millisecond light stimulation of OEPC shifts conductance-voltage plots of voltage-gated K+ channels by ≈30 mV. Models are described both for understanding the experimental findings at the level of K+ channel kinetics in HEK cells, as well as elucidating interpretation of membrane electrophysiology obtained during stimulation with an electrically floating extracellular photoelectrode. A time-dependent increase in voltage-gated channel conductivity in response to OEPC stimulation is demonstrated. These findings are then carried on to cultured primary hippocampal neurons. It is found that millisecond time-scale optical stimuli trigger repetitive action potentials in these neurons. The findings demonstrate that OEPC devices enable the manipulation of neuronal signaling activities with millisecond precision. OEPCs can therefore be integrated into novel in vitro electrophysiology protocols, and the findings can inspire in vivo applications.

Immobilization of Recombinant Fluorescent Biosensors Permits Imaging of Extracellular Ion Signals.

Given the importance of ion gradients and fluxes in biology, monitoring ions locally at the exterior of the plasma membrane of intact cells in a noninvasive manner is highly desirable but challenging. Classical targeting of genetically encoded biosensors at the exterior of cell surfaces would be a suitable approach; however, it often leads to intracellular accumulation of the tools in vesicular structures and adverse modifications, possibly impairing sensor functionality. To tackle these issues, we generated recombinant fluorescent ion biosensors fused to traptavidin (TAv) specifically coupled to a biotinylated AviTag expressed on the outer cell surface of cells. We show that purified chimeras of TAv and pH-Lemon or GEPII 1.0, Förster resonance energy transfer-based pH and K+ biosensors, can be immobilized directly and specifically on biotinylated surfaces including glass platelets and intact cells, thereby remaining fully functional for imaging of ion dynamics. The immobilization of recombinant TAv-GEPII 1.0 on the extracellular cell surface of primary cortical rat neurons allowed imaging of excitotoxic glutamate-induced K+ efflux in vitro. We also performed micropatterning of purified TAv biosensors using a microperfusion system to generate spatially separated TAv-pH-Lemon and TAv-GEPII 1.0 spots for simultaneous pH and K+ measurements on cell surfaces. Our results suggest that the approach can be greatly expanded by immobilizing various biosensors on extracellular surfaces to quantitatively visualize microenvironmental transport and signaling processes in different cell culture models and other experimental settings.

Potassium ions promote hexokinase-II dependent glycolysis.

High expression levels of mitochondria-associated hexokinase-II (HKII) represent a hallmark of metabolically highly active cells such as fast proliferating cancer cells. Typically, the enzyme provides a crucial metabolic switch towards aerobic glycolysis. By imaging metabolic activities on the single-cell level with genetically encoded fluorescent biosensors, we here demonstrate that HKII activity requires intracellular K+. The K+ dependency of glycolysis in cells expressing HKII was confirmed in cell populations using extracellular flux analysis and nuclear magnetic resonance-based metabolomics. Reductions of intracellular K+ by gramicidin acutely disrupted HKII-dependent glycolysis and triggered energy stress pathways, while K+ re-addition promptly restored glycolysis-dependent adenosine-5'-triphosphate generation. Moreover, expression and activation of KV1.3, a voltage-gated K+ channel, lowered cellular K+ content and the glycolytic activity of HEK293 cells. Our findings unveil K+ as an essential cofactor of HKII and provide a mechanistic link between activities of distinct K+ channels and cell metabolism.

Erratum: Potassium ions promote hexokinase-II dependent glycolysis.

[This corrects the article DOI: 10.1016/j.isci.2021.102346.].

Targeted Chemotherapy of Glioblastoma Spheroids with an Iontronic Pump.

Successful treatment of glioblastoma multiforme (GBM), the most lethal tumor of the brain, is presently hampered by (i) the limits of safe surgical resection and (ii) "shielding" of residual tumor cells from promising chemotherapeutic drugs such as Gemcitabine (Gem) by the blood brain barrier (BBB). Here, the vastly greater GBM cell-killing potency of Gem compared to the gold standard temozolomide is confirmed, moreover, it shows neuronal cells to be at least 104-fold less sensitive to Gem than GBM cells. The study also demonstrates the potential of an electronically-driven organic ion pump ("GemIP") to achieve controlled, targeted Gem delivery to GBM cells. Thus, GemIP-mediated Gem delivery is confirmed to be temporally and electrically controllable with pmol min-1 precision and electric addressing is linked to the efficient killing of GBM cell monolayers. Most strikingly, GemIP-mediated GEM delivery leads to the overt disintegration of targeted GBM tumor spheroids. Electrically-driven chemotherapy, here exemplified, has the potential to radically improve the efficacy of GBM adjuvant chemotherapy by enabling exquisitely-targeted and controllable delivery of drugs irrespective of whether these can cross the BBB.

Modeling External Stimulation of Excitable Cells Using a Novel Light-Activated Organic Semiconductor Technology.

Optoelectronic neurostimulation is a promising, minimally invasive treatment modality for neuronal damage, in particular for patients with traumatic brain injury. In this work, a newly developed optoelectronic device, a so-called photocap, based on light-activated organic semiconductor structures with high spatial and temporal resolution is investigated. To prove and verify the feasibility of this new technology, a mathematical model was developed, simulating the electrical response of excitable cells to photocap stimulation. In the first step, a comprehensive technical review of the device concept was performed, building the basis for setting up the simulation model. The simulations demonstrate that photocaps may serve as a stimulation device, triggering action potentials in neural or cardiac cells. Our first results show that the model serves as a perfect tool for evaluating and further developing this new technology, showing high potential for introducing new and innovative therapy methods in the field of optoelectronic cell stimulation.

Blockage of Store-Operated Ca2+ Influx by Synta66 is Mediated by Direct Inhibition of the Ca2+ Selective Orai1 Pore.

The Ca2+ sensor STIM1 and the Ca2+ channel Orai1 that form the store-operated Ca2+ (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 Ca2+ selectivity and permeation of Na+ in the absence of Ca2+. 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 Ca2+ channel by a high-affinity pore blocker.

STIM1 and Orai1 regulate Ca2+ microdomains for activation of transcription.

Since calcium (Ca2+) regulates a large variety of cellular signaling processes in a cell's life, precise control of Ca2+ concentrations within the cell is essential. This enables the transduction of information via Ca2+ changes in a time-dependent and spatially defined manner. Here, we review molecular and functional aspects of how the store-operated Ca2+ channel Orai1 creates spatiotemporal Ca2+ microdomains. The architecture of this channel is unique, with a long helical pore and a six-fold symmetry. Energetic barriers within the Ca2+ channel pathway limit permeation to allow an extensive local Ca2+ increase in close proximity to the channel. The precise timing of the Orai1 channel function is controlled by direct binding to STIM proteins upon Ca2+ depletion in the endoplasmic reticulum. These induced Ca2+ microdomains are tailored to, and sufficient for, triggering long-term activation processes, such as transcription factor activation and subsequent gene regulation. We describe the principles of spatiotemporal activation of the transcription factor NFAT and compare its signaling characteristics to those of the autophagy regulating transcription factors, MITF and TFEB.

Optoelectronic control of single cells using organic photocapacitors.

Optical control of the electrophysiology of single cells can be a powerful tool for biomedical research and technology. Here, we report organic electrolytic photocapacitors (OEPCs), devices that function as extracellular capacitive electrodes for stimulating cells. OEPCs consist of transparent conductor layers covered with a donor-acceptor bilayer of organic photoconductors. This device produces an open-circuit voltage in a physiological solution of 330 mV upon illumination using light in a tissue transparency window of 630 to 660 nm. We have performed electrophysiological recordings on Xenopus laevis oocytes, finding rapid (time constants, 50 µs to 5 ms) photoinduced transient changes in the range of 20 to 110 mV. We measure photoinduced opening of potassium channels, conclusively proving that the OEPC effectively depolarizes the cell membrane. Our results demonstrate that the OEPC can be a versatile nongenetic technique for optical manipulation of electrophysiology and currently represents one of the simplest and most stable and efficient optical stimulation solutions.

Sequential activation of STIM1 links Ca2+ with luminal domain unfolding.

The stromal interaction molecule 1 (STIM1) has two important functions, Ca2+ sensing within the endoplasmic reticulum and activation of the store-operated Ca2+ channel Orai1, enabling plasma-membrane Ca2+ influx. We combined molecular dynamics (MD) simulations with live-cell recordings and determined the sequential Ca2+-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 Ca2+ ions. In MD simulations, a single Ca2+ ion was sufficient to stabilize the luminal STIM1 complex. Ca2+ 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 Ca2+ entry through Orai1 channels. On the basis of our results, we present a model of STIM1 Ca2+ binding and refine the currently known initial steps of STIM1 activation on a molecular level.

Postmortem 31P magnetic resonance spectroscopy of the skeletal muscle: α-ATP/Pi ratio as a forensic tool?

PURPOSE: Phosphor magnetic resonance spectroscopy ((31)P MRS) is an established method for metabolic examinations of resting and exercising skeletal muscle. So far, there are few MRS investigations of human corpses. The aim of this study was to investigate the temporal postmortem pattern of phosphor metabolites in the adductor magnus muscle and to check the value of MRS as a forensic tool, especially for the determination of the time of death. MATERIAL AND METHODS: Eight corpses, died of natural cause, were examined (5 males, 3 females; age: 73±7 y, weight 65.8±15.9 kg). A control group of 3 subjects (2 males, 1 female, mean age: 51±24 y, range: 24-69 y, mean body weight: 84.0±16.5 kg) was examined at a single time point as well. (31)P MRS was performed on a 1.5 T MRI (TR 700 ms, TE 0.35 ms, averages 256, flip angle 90°). A standard (31)P/(1)H heart/liver coil was employed (receiver coil diameter 12 cm). The (31)P MRS scans were repeated in intervals of 1 h over a period from 4.5 to 24 h postmortem (p.m.). The core temperature was rectally measured throughout the MRI examination. RESULTS: The mean core temperature decreased from 36.0°C to 25.7°C. In vivo and ex vivo spectra showed characteristic differences, especially the PCr metabolite was no longer detectable after 10 h p.m. The α-ATP/Pi ratio decreased with time from 0.445 to 0.032 over 24 h p.m. CONCLUSION: There is a characteristic postmortem time pattern of the phosphor metabolites. Especially the acquired α-ATP/Pi ratio could be described by a significant exponential time course (r(2)=0.92, p<0.001). (31)P MRS might be added to the postmortem imaging methods.

DWI of the brain: postmortal DWI of the brain in comparison with in vivo data.

PURPOSE: Changes in water diffusion can be quantified by diffusion-weighted MR imaging. However, there are only few reports about changes in post mortem brain. The aim of this study was to investigate the temporal pattern of the apparent diffusion coefficient (ADC) in the brain after death, to compare the values to in vivo brain and to assess the value of ex vivo DWI as a forensic tool. MATERIAL AND METHODS: The study was approved by the local Ethics Committee, and informed consent was obtained from all relatives and the control subjects. Twenty-one corpses, died of natural cause, were examined (13 males, 8 females; age: 70.5±8.7 y, weight 74±18 kg). Diffusion-Weighted Imaging (DWI) was performed with b-values of 0 and 1000 s/mm(2) at 1.5 T. Scans were repeated in intervals of 1 h. ADC-maps were calculated in thalamus, cerebrum and cerebellum. The obtained values were statistically compared to healthy volunteers (n=3) and to literature data. RESULTS: The ADC in the three regions decreased characteristically during the examination time. In the cerebrum there was a significant difference between ex vivo and in vivo ADC (p<0.001) as well as in the other regions (thalamus: p<0.001, cerebellum: p=0.045). CONCLUSION: DWI of the postmortal brain can be added to the MRI methods for a post mortem imaging.