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.

Adipose triglyceride lipase (ATGL) catalyzes the rate limiting step in triacylglycerol breakdown in adipocytes but is expressed in most tissues. The enzyme was shown to be lost in many human tumors, and its loss may play a role in early stages of cancer development. Here, we report that loss of ATGL supports a more-aggressive cancer phenotype in a model system in which ATGL was deleted in A549 lung cancer cells by CRISPR/Cas9. We observed that loss of ATGL led to triacylglycerol accumulation in lipid droplets and higher levels of cellular phospholipid and bioactive lipid species (lyso- and ether-phospholipids). Label-free quantitative proteomics revealed elevated expression of the pro-oncogene SRC kinase in ATGL depleted cells, which was also found on mRNA level and confirmed on protein level by Western blot. Consistently, higher expression of phosphorylated (active) SRC (Y416 phospho-SRC) was observed in ATGL-KO cells. Cells depleted of ATGL migrated faster, which was dependent on SRC kinase activity. We propose that loss of ATGL may thus increase cancer aggressiveness by activation of pro-oncogenic signaling via SRC kinase and increased levels of bioactive lipids.

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.

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.

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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.

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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.