Disease-associated mutations in the Ca ²⁺ sensor STIM1 alter its conformation and disrupt Ca ²⁺ ion binding.

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 (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 Ca²⁺ down-stream activation of autophagic processes.

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.

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.