The interaction between Ca²⁺ sensors STIM1 and STIM2 and Ca²⁺ channel-forming protein ORAI1 is a crucial element of store-operated calcium entry (SOCE) in non-excitable cells. However, the molecular mechanism of SOCE in neurons remains unclear. We addressed this issue by establishing the presence and function of STIM proteins. Real-time polymerase chain reaction from cortical neurons showed that these cells contain significant amounts of Stim1 and Stim2 mRNA. Thapsigargin (TG) treatment increased the amount of both endogenous STIM proteins in neuronal membrane fractions. The number of YFP-STIM1/ORAI1 and YFP-STIM2/ORAI1 complexes was also enhanced by such treatment. The differences observed in the number of STIM1 and STIM2 complexes under SOCE conditions and the differential sensitivity to SOCE inhibitors suggest their distinct roles. Endoplasmic reticulum (ER) store depletion by TG enhanced intracellular Ca²⁺ levels in loaded with Fura-2 neurons transfected with YFP-STIM1 and ORAI1, but not with YFP-STIM2 and ORAI1, which correlated well with the number of complexes formed. Moreover, the SOCE inhibitors ML-9 and 2-APB reduced Ca²⁺ influx in neurons expressing YFP-STIM1/ORAI1 but produced no effect in cells transfected with YFP-STIM2/ORAI1. Moreover, in neurons transfected with YFP-STIM2/ORAI1, the increase in constitutive calcium entry was greater than with YFP-STIM1/ORAI1. Our data indicate that both STIM proteins are involved in calcium homeostasis in neurons. STIM1 mainly activates SOCE, whereas STIM2 regulates resting Ca²⁺ levels in the ER and Ca²⁺ leakage with the additional involvement of STIM1.

CRNDE, recently described as the lncRNA-coding gene, is overexpressed at RNA level in human malignancies. Its role in gametogenesis, cellular differentiation and pluripotency has been suggested as well. Herein, we aimed to verify our hypothesis that the CRNDE gene may encode a protein product, CRNDEP. By using bioinformatics methods, we identified the 84-amino acid ORF encoded by one of two CRNDE transcripts, previously described by our research team. This ORF was cloned into two expression vectors, subsequently utilized in localization studies in HeLa cells. We also developed a polyclonal antibody against CRNDEP. Its specificity was confirmed in immunohistochemical, cellular localization, Western blot and immunoprecipitation experiments, as well as by showing a statistically significant decrease of endogenous CRNDEP expression in the cells with transient shRNA-mediated knockdown of CRNDE. Endogenous CRNDEP localizes predominantly to the nucleus and its expression seems to be elevated in highly proliferating tissues, like the parabasal layer of the squamous epithelium, intestinal crypts or spermatocytes. After its artificial overexpression in HeLa cells, in a fusion with either the EGFP or DsRed Monomer fluorescent tag, CRNDEP seems to stimulate the formation of stress granules and localize to them. Although the exact role of CRNDEP is unknown, our preliminary results suggest that it may be involved in the regulation of the cell proliferation. Possibly, CRNDEP also participates in oxygen metabolism, considering our in silico results, and the correlation between its enforced overexpression and the formation of stress granules. This is the first report showing the existence of a peptide encoded by the CRNDE gene.

One of the numerous calcium-involving processes in mammalian cells is store-operated calcium entry (SOCE) -- the process in which depletion of calcium stores in the endoplasmic reticulum (ER) induces calcium influx from the extracellular space. Previously supposed to function only in non-excitable cells, SOCE is now known to play a role also in such excitable cells as neurons, muscles and neuroendocrine cells and is found in many different cell types. SOCE participates not only in processes dependent on ER calcium level but also specifically regulates some important processes such as cAMP production, T lymphocyte activation or induction of long-term potentiation. Impairment of SOCE can be an element of numerous disorders such as acute pancreatitis, primary immunodeficiency and, since it can take part in apoptosis or cell cycle regulation, SOCE may also be partially responsible for such serious disorders as Alzheimer disease and many types of cancer. Even disturbances in the 'servant' role of maintaining ER calcium level may cause serious effects because they can lead to ER homeostasis disturbance, influencing gene expression, protein synthesis and processing, and the cell cycle.

In developing muscle cells environmental stimuli transmitted by purines binding to the specific receptors are crucial proliferation regulators. C2C12 myoblasts express numerous purinergic receptors representing both main classes: P2X and P2Y. Among P2Y receptors we have found the expression of P2Y₁, P2Y₂, P2Y₄, P2Y₆ and P2Y₁₂ family members while among P2X receptors P2X₄, P2X₅ and P2X₇ were discovered. We have been able to show that activation of those receptors is responsible for ERK class kinase activity, responsible for regulation of cell proliferation pathway. We have also demonstrated that this activity is calcium dependent suggesting Ca²⁺ ions as secondary messenger between receptor and kinase regulatory system. More specifically, we do suspect that in C2C12 myoblasts calcium channels of P2X receptors, particularly P2X₅ play the main role in proliferation regulation. In further development of myoblasts into myotubes, when proliferation is gradually inhibited, the pattern of P2 receptors is changed. This phenomenon is followed by diminishing of the P2Y₂-dependent Ca²⁺ signaling, while the mRNA expression of P2Y₂ receptor reminds still on the high level. Moreover, P2X₂ receptor mRNA, absent in myoblasts appears in myotubes. These data show that differentiation of C2C12 cell line satellite myoblasts is accompanied by changes in P2 receptors expression pattern.