Orai channel C-terminal peptides are key modulators of STIM-Orai coupling and calcium signal generation.

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.

A time course for markers of protein synthesis and degradation with hindlimb unloading and the accompanying anabolic resistance to refeeding.

Skeletal muscle atrophy is associated with disease, aging, and disuse. Hindlimb unloading (HU) in animals provides an experimental model to study muscle atrophy. A comprehensive time course for how HU affects biomarkers of protein synthesis and degradation acutely and chronically and the associated resistance to an anabolic stimulus following disuse remain undocumented. Sixteen-week-old C57BL/6 mice underwent 0, 1, 12, 24, 72, 168, or 336 h of HU. Following 336 h of HU, mice were reloaded for 1, 24, or 72 h. Another group of mice underwent 120 h of HU, were fasted or refed, and were then compared with similar condition control animals (CTL). Protein content and phosphorylation of biomarkers of protein synthesis, degradation, and autophagy were assessed in the soleus muscle. Gastrocnemius, soleus, and plantaris muscles atrophied within 120 h of HU. Protein synthesis trended toward decrease following 24 h of HU. p70S6K phosphorylation and protein synthesis increased with reloading. Following HU, changes in MAFbx and DEPTOR expression and DEPTOR phosphorylation were consistent with development of a catabolic state. DEPTOR expression recovered following reloading. Animals that underwent 120 h of HU exhibited attenuation of refeeding-induced p70S6K phosphorylation compared with CTL counterparts. Following 120 h of HU, protein synthesis, eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) phosphorylation, and DEPTOR, MAFbx, and Sestrin1 expression indicated a catabolic state. Following 120 h of HU, autophagy markers, including p62 expression, REDD1 expression, LC3 ratio, and Unc-51-like autophagy-activating kinase 1 (ULK1) phosphorylation, indicated impaired autophagy. HU promotes a deleterious balance between protein synthesis and degradation. The time course herein provides scientists information about when the associated biomarkers become affected.NEW & NOTEWORTHY Hindlimb unloading causes significant skeletal muscle atrophy by adversely affecting the balance between protein synthesis and breakdown. This study demonstrates a more complete time course for changes in biomarkers associated with protein synthesis and breakdown and investigates the associated anabolic resistance to an anabolic stimulus following hindlimb unloading. These data in concert with information from other studies provide a basis for designing future experiments to optimally interrogate a desired cellular biomarker or pathway.