Alternative splicing converts STIM2 from an activator to an inhibitor of store-operated calcium channels

Store-operated calcium entry (SOCE) regulates a wide variety of essential cellular functions. SOCE is mediated by STIM1 and STIM2, which sense depletion of ER Ca2+ stores and activate Orai channels in the plasma membrane. Although the amplitude and dynamics of SOCE are considered important determinants of Ca2+-dependent responses, the underlying modulatory mechanisms are unclear. In this paper, we identify STIM2??, a highly conserved alternatively spliced isoform of STIM2, which, in contrast to all known STIM isoforms, is a potent inhibitor of SOCE. Although STIM2?? does not by itself strongly bind Orai1, it is recruited to Orai1 channels by forming heterodimers with other STIM isoforms. Analysis of STIM2?? mutants and Orai1-STIM2?? chimeras suggested that it actively inhibits SOCE through a sequence-specific allosteric interaction with Orai1. Our results reveal a previously unrecognized functional flexibility in the STIM protein family by which alternative splicing creates negative and positive regulators of SOCE to shape the amplitude and dynamics of Ca2+ signals.open

Calcium signaling : Molecular mechanisms and cellular consequences

Cells exploit calcium (Ca2+) signaling to transmit information. In a multicellular organism, each cell must be able recognize, process, and respond to information received from the surrounding environment. In this thesis I investigate molecular mechanisms and cellular consequences of Ca2+ signaling. Ouabain is an endogenous hormone, and ligand of the Na,K-ATPase, that has previously been shown to induce Ca2+ oscillations in renal cells. Here we report that the N-terminal tail of the Na,K-ATPase alpha-subunit binds directly to the N-terminus of the inositol 1,4,5-trisphosphate receptor (InsP3R). Three amino acid residues in the Na,K-ATPase N-terminal tail, LKK, conserved in most species, are essential for this binding to occur. Over-expression of a peptide encoding for the N-terminal tail impaired ouabain-triggered Ca2+ oscillations. Thus we have identified a well conserved Na,K-ATPase-motif that binds to the InsP3R and is vital for intracellular Ca2+ signaling. The role of Na,K-ATPase signaling during dendritogenesis was examined by treating embryonic cortical rat neurons with ouabain. We report that Na,K-ATPase signal transduction triggers dendritic growth as well as a transcriptional program dependent on CREB and CRE-mediated gene expression, primarily regulated via Ca2+/calmodulin-dependent protein kinases. This signaling cascade also involves intracellular Ca2+ oscillations and sustained phosphorylation of mitogen-activated protein kinases. These results suggest a novel role for the Na,K-ATPase as a modulator of dendritic growth in developing neurons. We explored the Ca2+ signaling properties of differentiating mouse embryonic stem cells. Spontaneous Ca2+ activity was shown to be present in neural progenitors derived from mouse embryonic stem cells. This Ca2+ activity was dependent on influx of extracellular Ca2+ through plasma membrane channels, since removal of extracellular Ca2+ from the medium and inhibition of voltage-dependent channels blocked the signaling event. Cross-correlation analysis revealed that the spontaneous Ca2+ activity was more synchronous in sub-populations of the neural progenitors than in the undifferentiated mouse embryonic stem cells. A significant reduction Ca2+ activity was observed when cells were challenged with gap junction blockers. Inhibiting the spontaneous Ca2+ activity significantly reduced the number of dividing cells. In conclusion, this thesis presents novel data on a conserved Na,K-ATPasemotif important for the N-terminal signaling activity and demonstrates a role of Na,K-ATPase in dendritic growth in developing cortical neurons. Further, spontaneous Ca2+ activity in neural progenitors derived from embryonic stem cells is dependent on extracellular Ca2+ and is important for cell division

Intracellular calcium release modulates polycystin-2 trafficking

Abstract Background Polycystin-2 (PC2), encoded by the gene that is mutated in autosomal dominant polycystic kidney disease (ADPKD), functions as a calcium (Ca2+) permeable ion channel. Considerable controversy remains regarding the subcellular localization and signaling function of PC2 in kidney cells. Methods We investigated the subcellular PC2 localization by immunocytochemistry and confocal microscopy in primary cultures of human and rat proximal tubule cells after stimulating cytosolic Ca2+ signaling. Plasma membrane (PM) Ca2+ permeability was evaluated by Fura-2 manganese quenching using time-lapse fluorescence microscopy. Results We demonstrated that PC2 exhibits a dynamic subcellular localization pattern. In unstimulated human or rat proximal tubule cells, PC2 exhibited a cytosolic/reticular distribution. Treatments with agents that in various ways affect the Ca2+ signaling machinery, those being ATP, bradykinin, ionomycin, CPA or thapsigargin, resulted in increased PC2 immunostaining in the PM. Exposing cells to the steroid hormone ouabain, known to trigger Ca2+ oscillations in kidney cells, caused increased PC2 in the PM and increased PM Ca2+ permeability. Intracellular Ca2+ buffering with BAPTA, inositol 1,4,5-trisphosphate receptor (InsP3R) inhibition with 2-aminoethoxydiphenyl borate (2-APB) or Ca2+/Calmodulin-dependent kinase inhibition with KN-93 completely abolished ouabain-stimulated PC2 translocation to the PM. Conclusions These novel findings demonstrate intracellular Ca2+-dependent PC2 trafficking in human and rat kidney cells, which may provide new insight into cyst formations in ADPKD.</p

Phosphorylation of residues inside the SNARE complex suppresses secretory vesicle fusion

Membrane fusion is essential for eukaryotic life, requiring SNARE proteins to zipper up in an α-helical bundle to pull two membranes together. Here, we show that vesicle fusion can be suppressed by phosphorylation of core conserved residues inside the SNARE domain. We took a proteomics approach using a PKCB knockout mast cell model and found that the key mast cell secretory protein VAMP8 becomes phosphorylated by PKC at multiple residues in the SNARE domain. Our data suggest that VAMP8 phosphorylation reduces vesicle fusion in vitro and suppresses secretion in living cells, allowing vesicles to dock but preventing fusion with the plasma membrane. Markedly, we show that the phosphorylation motif is absent in all eukaryotic neuronal VAMPs, but present in all other VAMPs. Thus, phosphorylation of SNARE domains is a general mechanism to restrict how much cells secrete, opening the door for new therapeutic strategies for suppression of secretion