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.

Capacitative Calcium Entry (CCE) in neurons seems to depend, as in non-excitatory cells, on endoplasmic reticulum calcium sensors STIM1 or STIM2. We show localization of STIM1 in the mouse brain by immunohistochemistry with a specific antibody. STIM1 immunoreactivity has wide, but not uniform, distribution throughout the brain and is observed in neuropil and cells. The most intensive immunoreactivity is observed in Purkinje neurons of cerebellum. High/moderate levels of immunostaining are found in hippocampus, cerebral cortex and in cortico-medial amygdala, low in thalamus and basolateral amygdala. Co-staining with anti-NeuN antibody identify STIM1 immunopositive cells as neurons. Real time PCR demonstrates that Stim2 expression is 7-fold higher than that of Stim1 in hippocampus and 3-fold in other regions. Immunoblotting confirms that levels of STIMs vary in different brain regions. The data show that STIM1 and STIM2 are present in the brain, thus both can be involved in CCE, depending on neuronal type.

Skeletal myoblasts grown in vitro and induced to differentiate either form differentiated multinucleated myotubes or give rise to quiescent, undifferentiated "reserve cells" that share several characteristics with muscle satellite cells. The mechanism of determination of reserve cells is poorly understood. We find that the expression level of the metalloprotease disintegrin ADAM12 is much higher in proliferating C2C12 myoblasts and in reserve cells than in myotubes. Inhibition of ADAM12 expression in differentiating C2C12 cultures by small interfering RNA is accompanied by lower expression levels of both quiescence markers (retinoblastoma-related protein p130 and cell cycle inhibitor p27) and differentiation markers (myogenin and integrin alpha7A isoform). Overexpression of ADAM12 in C2C12 cells under conditions that promote cell cycle progression leads to upregulation of p130 and p27, cell cycle arrest, and downregulation of MyoD. Thus, enhanced expression of ADAM12 induces a quiescence-like phenotype and does not stimulate differentiation. We also show that the region extending from the disintegrin to the transmembrane domain of ADAM12 and containing cell adhesion activity as well as the cytoplasmic domain of ADAM12 are required for ADAM12-mediated cell cycle arrest, while the metalloprotease domain is not essential. Our results suggest that ADAM12-mediated adhesion and/or signaling may play a role in determination of the pool of reserve cells during myoblast differentiation.