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Summary: This protocol outlines steps to visualize and detect Ca2+ puffs following photo-liberation of caged inositol-1,4,5-trisphosphate (IP3) from HEK-293 cells expressing only the native IP3R type 1 receptor using total internal reflection fluorescence (TIRF) microscopy. TIRF microscopy offers high axial resolution and allows imaging at high speed, with a higher signal-to-background ratio. Additionally, we shed light on commonly encountered pitfalls, which should be considered while recording Ca2+ puffs using TIRF microscopy.For complete details on the use and execution of this protocol, please refer to Emrich et al. (2021) and Lock et al. (2015a).

Summary: Junctional coupling between endoplasmic reticulum (ER) Ca2+-sensor STIM proteins and plasma membrane (PM) Orai channels mediates Ca2+ signals in most cells. We reveal that PM-tethered, fluorescently tagged C-terminal M4x (fourth transmembrane helix contains a cytoplasmic C-terminal extension) peptides from Orai channels undergo a Leu-specific signature of direct interaction with the STIM1 Orai-activating region (SOAR), exactly mimicking STIM1 binding to gate Orai channels. The 20-amino-acid Orai3-M4x peptide associates avidly with STIM1 within ER-PM junctions, functions to competitively block native Ca2+ signals, and mediates a key modification of STIM-Orai coupling induced by 2-aminoethoxydiphenyl borate. By blocking STIM-Orai coupling, the Orai3-M4x peptide reveals the critical role of Orai channels in driving Ca2+ oscillatory signals and transcriptional control through NFAT. The M4x peptides interact independently with SOAR dimers consistent with unimolecular coupling between Orai subunits and STIM1 dimers. We reveal the critical role of M4x helices in defining the coupling interface between STIM and Orai proteins to mediate store-operated Ca2+ signals.

The pro-inflammatory cytokine Interleukin-1α (IL-1α) has recently emerged as a susceptibility marker for a wide array of inflammatory diseases associated with oxidative stress including Alzheimer's, arthritis, atherosclerosis, diabetes and cancer. In the present study, we establish that expression and nuclear localization of IL-1α are redox-dependent. Shifts in steady-state H2O2 concentrations (SS-[H2O2]) resulting from enforced expression of manganese superoxide dismutase (SOD2) drive IL-1α mRNA and protein expression. The redox-dependent expression of IL-1α is accompanied by its increased nuclear localization. Both IL-1α expression and its nuclear residency are abrogated by catalase co-expression. Sub-lethal doses of H2O2 also cause IL-1α nuclear localization. Mutagenesis revealed IL-1α nuclear localization does not involve oxidation of cysteines within its N terminal domain. Inhibition of the processing enzyme calpain prevents IL-1α nuclear localization even in the presence of H2O2. H2O2 treatment caused extracellular Ca2+ influx suggesting oxidants may influence calpain activity indirectly through extracellular Ca2+ mobilization. Functionally, as a result of its nuclear activity, IL-1α overexpression promotes NF-kB activity, but also interacts with the histone acetyl transferase (HAT) p300. Together, these findings demonstrate a mechanism by which oxidants impact inflammation through IL-1α and suggest that antioxidant-based therapies may prove useful in limiting inflammatory disease progression.