Lee P. Haynes

Neuronal Ca2+-sensor proteins: multitalented regulators of neuronal function

Many aspects of neuronal activity are regulated by Ca2+ signals. The transduction of temporally and spatially distinct Ca2+ signals requires the action of Ca2+-sensor proteins including various EF-hand-containing Ca2+-binding proteins. The neuronal Ca2+ sensor (NCS) protein family and the related Ca2+-binding proteins (CaBPs) have begun to emerge as key players in neuronal function. Many of these proteins are expressed predominantly or only in neurons, sometimes with cell-specific patterns of expression. Their ability to associate with membranes either constitutively or in response to elevated Ca2+ concentration allows the NCS proteins to discriminate between different spatial and temporal patterns of Ca2+ signals. Recent work has established several physiological roles of these proteins, including diverse actions on gene expression, ion channel function, membrane traffic of ion channels and receptors, and the control of apoptosis.

Role of phosphoinositides in STIM1 dynamics and store-operated calcium entry

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.

Calcium-binding Protein 1 Is an Inhibitor of Agonist-evoked, Inositol 1,4,5-Trisphosphate-mediated Calcium Signaling

Intracellular calcium signals are responsible for initiating a spectrum of physiological responses. The caldendrins/calcium-binding proteins (CaBPs) represent mammal-specific members of the CaM superfamily. CaBPs display a restricted pattern of expression in neuronal/retinal tissues, suggesting a specialized role in Ca2+ signaling in these cell types. Recently, it was reported that a splice variant of CaBP1 functionally interacts with inositol 1,4,5-trisphosphate (InsP3) receptors to elicit channel activation in the absence of InsP3 (Yang, J., McBride, S., Mak, D.-O. D., Vardi, N., Palczewski, K., Haeseleer, F., and Foskett, J. K. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7711–7716). These data indicate a new mode of InsP3 receptor modulation and hence control of intracellular Ca2+ concentration ([Ca2+]i) in neuronal tissues. We have analyzed the biochemistry of the long form splice variant of CaBP1 (L-CaBP1) and show that, in vitro, a recombinant form of the protein is able to bind Ca2+ with high affinity and undergo a conformational change. We also describe the localization of endogenous and overexpressed L-CaBP1 in the model neuroendocrine PC12 cell system, where it was associated with the plasma membrane and Golgi complex in a myristoylation-dependent manner. Furthermore, we show that overexpressed L-CaBP1 is able to substantially suppress rises in [Ca2+]i in response to physiological agonists acting on purinergic receptors and that this inhibition is due in large part to blockade of release from intracellular Ca2+ stores. The related protein neuronal calcium sensor-1 was without effect on the [Ca2+]i responses to agonist stimulation. Measurement of [Ca2+] within the ER of permeabilized PC12 cells demonstrated that LCaBP1 directly inhibited InsP3-mediated Ca2+ release. Expression of L-CaBP1 also inhibited histamine-induced [Ca2+]i oscillations in HeLa cells. Together, these data suggest that L-CaBP1 is able to specifically regulate InsP3 receptor-mediated alterations in [Ca2+]i during agonist stimulation.

Ribosome-free Terminals of Rough ER Allow Formation of STIM1 Puncta and Segregation of STIM1 from IP3 Receptors

Summary Store-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.

The Diversity of Calcium Sensor Proteins in the Regulation of Neuronal Function

Calcium signaling in neurons as in other cell types mediates changes in gene expression, cell growth, development, survival, and cell death. However, neuronal Ca(2+) signaling processes have become adapted to modulate the function of other important pathways including axon outgrowth and changes in synaptic strength. Ca(2+) plays a key role as the trigger for fast neurotransmitter release. The ubiquitous Ca(2+) sensor calmodulin is involved in various aspects of neuronal regulation. The mechanisms by which changes in intracellular Ca(2+) concentration in neurons can bring about such diverse responses has, however, become a topic of widespread interest that has recently focused on the roles of specialized neuronal Ca(2+) sensors. In this article, we summarize synaptotagmins in neurotransmitter release, the neuronal roles of calmodulin, and the functional significance of the NCS and the CaBP/calneuron protein families of neuronal Ca(2+) sensors.

Differential dynamics of Rab3A and Rab27A on secretory granules

We have assessed the dynamics of the association of Rab3A and Rab27A with secretory granules at various stages of their life in PC12 cells. Endogenous Rab3A colocalised with the secretory granule marker secretogranin II (SGII) and expressed EGFP-Rab3A and ECFP-Rab27A colocalised with one another. The extent of colocalisation between EGFP-Rab3A or EGFP-Rab27 and SGII increased after longer times post transfection suggesting that these Rab proteins are preferentially recruited to newly synthesised granules. Following the release of immature secretory granules from the trans-Golgi network, Rab3A and Rab27A became associated with the immature granules after a lag period of around 20 minutes. Rab dynamics on granules were analysed in fluorescence recovery after photobleaching (FRAP) experiments. The recovery profile of EGFP-Rab27A was comparable to that of ppANF-EGFP, whereas the recovery profile of EGFP-Rab3A was significantly faster, indicating that Rab3A but not Rab27A might be rapidly exchanged between granules and cytosol. Inhibition of heat-shock protein 90 with 10 μM geldanamycin did not affect the exchange process or regulated exocytosis. Rab dynamics during stimulation with 300 μM ATP were analysed in live cells. Loss of granular ppANF-EGFP fluorescence was seen at the cell periphery after stimulation but only limited changes in EGFP-Rab3A and EGFP-Rab27A fluorescence was observed, indicating that the Rab proteins do not immediately dissociate or disperse on stimulation. The data suggest potentially distinct roles for Rab3A and Rab27A and we suggest that the finding that young secretory granules have a higher capacity for binding Rab3A and Rab27A is functionally important for preferential exocytosis from these granules.

Understanding the physiological roles of the neuronal calcium sensor proteins

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.

Structural and functional deficits in a neuronal calcium sensor-1 mutant identified in a case of autistic spectrum disorder.

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.

cAMP inhibits migration, ruffling and paxillin accumulation in focal adhesions of pancreatic ductal adenocarcinoma cells: effects of PKA and EPAC.

We demonstrated that increasing intracellular cAMP concentrations result in the inhibition of migration of PANC-1 and other pancreatic ductal adenocarcinoma (PDAC) cell types. The rise of cAMP was accompanied by rapid and reversible cessation of ruffling, by inhibition of focal adhesion turnover and by prominent loss of paxillin from focal adhesions. All these phenomena develop rapidly suggesting that cAMP effectors have a direct influence on the cellular migratory apparatus. The role of two primary cAMP effectors, exchange protein activated by cAMP (EPAC) and protein kinase A (PKA), in cAMP-mediated inhibition of PDAC cell migration and migration-associated processes was investigated. Experiments with selective activators of EPAC and PKA demonstrated that the inhibitory effect of cAMP on migration, ruffling, focal adhesion dynamics and paxillin localisation is mediated by PKA, whilst EPAC potentiates migration.

ATP depletion induces translocation of STIM1 to puncta and formation of STIM1–ORAI1 clusters: translocation and re-translocation of STIM1 does not require ATP

Depletion of the endoplasmic reticulum (ER) calcium store triggers translocation of stromal interacting molecule one (STIM1) to the sub-plasmalemmal region and formation of puncta—structures in which STIM1 interacts and activates calcium channels. ATP depletion induced the formation of STIM1 puncta in PANC1, RAMA37, and HeLa cells. The sequence of events triggered by inhibition of ATP production included a rapid decline of ATP, depletion of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and a slow calcium leak from the ER followed by formation of STIM1 puncta. STIM1 puncta induced by ATP depletion were co-localized with clusters of ORAI1 channels. STIM1–ORAI1 clusters that developed as a result of ATP depletion were very poor mediators of Ca2+ influx. Re-translocation of STIM1 from puncta back to the ER was observed during total ATP depletion. We can therefore conclude that STIM1 translocation and re-translocation as well as formation of STIM1–ORAI1 clusters occur in an ATP-independent fashion and under conditions of PI(4,5)P2 depletion.