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Calcium is a ubiquitous intracellular second messenger in all vertebrate cells (1). Intracellular resting free Ca 2ϩ concentration ([Ca 2ϩ ] i ) is acutely maintained at around 100 nM via a variety of mechanisms, and even small local rises above this base line are able to elicit dramatic cellular responses. Rises in [Ca 2ϩ ] i are generated through two primary routes (2). First, many physiological agonists activate Ca 2ϩ channels that mediate the direct entry of extracellular Ca 2ϩ . Second, [Ca 2ϩ ] i can be elevated through release of sequestered Ca 2ϩ from intracellular stores (3), most notably the endoplasmic reticulum (ER). 1 Ca 2ϩ release from intracellular stores is controlled predominantly by two related Ca 2ϩ channel families, the inositol 1,4,5-trisphosphate (InsP 3 ) receptor (InsP 3 R) and the ryanodine receptor (RyR). InsP 3 Rs couple to extracellular stimuli that drive the activation of phospholipase C and subsequent liberation of the soluble second messenger InsP 3. Activation of ER-localized InsP 3 Rs by InsP 3 in turn elicits Ca 2ϩ release into the cytosol. InsP 3 R activity can be further modulated by direct binding of Ca 2ϩ itself, and this regulation explains a form of calciuminduced calcium release that is responsible for the establishment of complex regenerative Ca 2ϩ signals within cells (2). In neurons, such Ca 2ϩ signals have been intimately linked to processes as divergent as neurodegeneration, neurite outgrowth, alterations in neuronal gene expression, and neurotransmitter release (4 -10). Factors affecting the generation and propagation of intracellular Ca 2ϩ signals therefore represent potential critical integrators of neuronal responses to complex and varied stimuli.Although Ca 2ϩ is capable of eliciting direct cellular responses such as the aforementioned gating of InsP 3 Rs, many of its effects are registered indirectly through specific Ca 2ϩ -binding proteins. Calmodulin (CaM) is a universal Ca 2ϩ -binding protein, homologues of which are present in all eukaryotes (11), and a large number of Ca 2ϩ driven alterations in cell physiology can be mapped to its Ca 2ϩ sensing activity. CaM coordinates Ca 2ϩ ions through specialized EF-hand domains, and a large number of related proteins have now been characterized. In neurons, these include the neuronal calcium sensor (NCS) family of Ca 2ϩ -binding proteins (12). More recently, a group of CaM-like proteins, termed caldendrins (13, 14) or calciumbinding proteins (CaBPs) (15) have been identified that are vertebrate-specific and display a predominantly neuronal/retinal pattern of expression (14 -16). It has since been reported that members of the CaBP family are able to interact with and modulate the activity of InsP 3 Rs (17). Uniquely, it has been suggested that CaBPs may be capable of activating the InsP 3 R independently of the natural ligand ‡ To whom all correspondence should be addressed.

We have identified a novel Ca 2؉ -dependent interaction between neuronal calcium sensor-1 (NCS-1) and the GTPase ARF1. Both of these proteins are localized to the Golgi complex, and both regulate phosphatidylinositol 4-kinase III␤ (PI(4)K␤). Spatial and temporal control of phosphatidylinositol 4-phosphate levels through activation of PI(4)K␤ is important for the recruitment of trafficking complexes to the trans-Golgi network (TGN) and vesicular traffic from this organelle. The NCS-1-ARF1 interaction and its specificity have been demonstrated through in vitro binding assays, in vitro enzyme assay, and through functional cellular assays. We show that NCS-1 can exert bidirectional effects to activate PI(4)K␤ on its own or inhibit the activation by ARF1. NCS-1 was shown to modulate the effects of expression of ARF mutants that disrupt Golgi morphology and to recruit GDPloaded ARF to the Golgi complex in a Ca 2؉ -dependent manner. We demonstrate antagonist effects of NCS-1 and ARF on constitutive and regulated exocytosis. The NCS-1-ARF1 interaction provides evidence for functional cross-talk between Ca 2؉ -dependent and ARF-dependent pathways in TGN to plasma membrane traffic.

SummaryStore-operated Ca2+ entry is a ubiquitous mechanism that prevents the depletion of endoplasmic reticulum (ER) calcium [1]. A reduction of ER calcium triggers translocation of STIM proteins, which serve as calcium sensors in the ER, to subplasmalemmal puncta where they interact with and activate Orai channels ([2–8]; reviewed in [9]). In pancreatic acinar cells, inositol 1,4,5-trisphosphate (IP3) receptors populate the apical part of the ER. Here, however, we observe that STIM1 translocates exclusively to the lateral and basal regions following ER Ca2+ loss. This finding is paradoxical because the basal and lateral regions of the acinar cells contain rough ER (RER); the size of the ribosomes that decorate RER is larger than the distance that can be spanned by a STIM-Orai complex [5, 10], and STIM1 function should therefore not be possible. We resolve this paradox and characterize ribosome-free terminals of the RER that form junctions between the reticulum and the plasma membrane in the basal and lateral regions of the acinar cells. Our findings indicate that different ER compartments specialize in different calcium-handling functions (Ca2+ release and Ca2+ reloading) and that any potential interference between Ca2+ release and Ca2+ influx is minimized by the spatial separation of the two processes.

Ca2+ entry through store-operated Ca2+ channels involves the interaction at ER–PM (endoplasmic reticulum–plasma membrane) junctions of STIM (stromal interaction molecule) and Orai. STIM proteins are sensors of the luminal ER Ca2+ concentration and, following depletion of ER Ca2+, they oligomerize and translocate to ER–PM junctions where they form STIM puncta. Direct binding to Orai proteins activates their Ca2+ channel function. It has been suggested that an additional interaction of the C-terminal polybasic domain of STIM1 with PM phosphoinositides could contribute to STIM1 puncta formation prior to binding to Orai. In the present study, we investigated the role of phosphoinositides in the formation of STIM1 puncta and SOCE (store-operated Ca2+ entry) in response to store depletion. Treatment of HeLa cells with inhibitors of PI3K (phosphatidylinositol 3-kinase) and PI4K (phosphatidylinositol 4-kinase) (wortmannin and LY294002) partially inhibited formation of STIM1 puncta. Additional rapid depletion of PtdIns(4,5)P2 resulted in more substantial inhibition of the translocation of STIM1–EYFP (enhanced yellow fluorescent protein) into puncta. The inhibition was extensive at a concentration of LY294002 (50 μM) that should primarily inhibit PI3K, consistent with a major role for PtdIns(4,5)P2 and PtdIns(3,4,5)P3 in puncta formation. Depletion of phosphoinositides also inhibited SOCE based on measurement of the rise in intracellular Ca2+ concentration after store depletion. Overexpression of Orai1 resulted in a recovery of translocation of STMI1 into puncta following phosphoinositide depletion and, under these conditions, SOCE was increased to above control levels. These observations support the idea that phosphoinositides are not essential but contribute to STIM1 accumulation at ER–PM junctions with a second translocation mechanism involving direct STIM1–Orai interactions.

Neuronal calcium sensor-1 (NCS-1) is a Ca2+ sensor protein that has been implicated in the regulation of various aspects of neuronal development and neurotransmission. It exerts its effects through interactions with a range of target proteins one of which is interleukin receptor accessory protein like-1 (IL1RAPL1) protein. Mutations in IL1RAPL1 have recently been associated with autism spectrum disorders and a missense mutation (R102Q) on NCS-1 has been found in one individual with autism. We have examined the effect of this mutation on the structure and function of NCS-1. From use of NMR spectroscopy, it appeared that the R102Q affected the structure of the protein particularly with an increase in the extent of conformational exchange in the C-terminus of the protein. Despite this change NCS-1(R102Q) did not show changes in its affinity for Ca2+ or binding to IL1RAPL1 and its intracellular localisation was unaffected. Assessment of NCS-1 dynamics indicated that it could rapidly cycle between cytosolic and membrane pools and that the cycling onto the plasma membrane was specifically changed in NCS-1(R102Q) with the loss of a Ca2+ -dependent component. From these data we speculate that impairment of the normal cycling of NCS-1 by the R102Q mutation could have subtle effects on neuronal signalling and physiology in the developing and adult brain.

Calcium signalling plays a crucial role in the control of neuronal function and plasticity. Changes in neuronal Ca2+ concentration are detected by Ca2+-binding proteins that can interact with and regulate target proteins to modify their function. Members of the neuronal calcium sensor (NCS) protein family have multiple non-redundant roles in the nervous system. Here we review recent advances in the understanding of the physiological roles of the NCS proteins and the molecular basis for their specificity.

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