Effectors and the regulation of intracellular Ca2 concentration

Ca2+ is a ubiquitous intracellular messenger that controls a multitude of activities within cells (Berridge 1998; Berridge et al. 2000). Increases in [Ca2+]i can exert direct effects on Ca2+-sensitive proteins or, more commonly, exert effects via Ca2+-binding proteins such as calmodulin (Berridge et al. 2000; Chin and Means 2000). Ca2+ /calmodulin can, in turn, regulate a variety of proteins, either by direct binding or via regulation of phosphorylation status through Ca2+/calmodulin-dependent protein kinases (Corcoran and Means 2001; Soderling et al. 2001) or phosphatases (calcineurin) (Rusnak and Mertz 2000; Crabtree 2001). GPCRs can regulate [Ca2+]i through a variety of mechanisms. At the level of the plasma membrane GPCRs can directly modulate voltage-operated Ca2+ channels (Dolphin 1998; Miller 1998), or act through less direct pathways to reduce Ca2+-influx (e.g. by membrane hyperpolarization through K+-channel activation), or to promote Ca2+-efflux (e.g. by increasing plasma membrane Ca2+-ATPase activity) (Wickman and Clapham 1995; Penniston and Enyedi 1998).

Probably the most studied mechanism linking GPCRs to increases in [Ca2+]i involves the phospholipase C/inositol 1,4,5-trisphosphate (IP3) signalling pathway, where IP3 gates Ca2+-mobilization through activation of IP3 receptors in the endoplasmic reticulum (ER)

(Berridge 1993). The mobilization of Ca2+ from ER stores can, in turn, trigger another process termed store-operated (or 'capacitative') Ca2+ entry, where ER Ca2+ -store depletion in some way gates Ca2+ entry via a plasma membrane cation channel (Putney 1986; Parekh and Penner 1997). This Ca2+-signalling pathway has been most studied in non-excitable cells; however, there is increasing evidence that the GPCR-regulated phospholipase C/IP3 signalling pathway may also play an important role in neuronal cell function.

In addition to IP3 receptors, the ER may also express other proteins involved with Ca2+ -mobilization. Ryanodine receptors are the best-characterized receptor-operated Ca2+ -channels that are responsible for Ca2+-induced Ca2+-release. Thus, global or localized changes in cytoplasmic Ca2+ can activate ryanodine receptors to cause a further rise through ER Ca2+-mobilization (Verkhratsky and Shmigol 1996; Berridge etal. 2000). IP3 receptors and ryanodine receptors possess structural similarities and are regulated in analogous ways. Thus, the ability of changes in IP3 concentration to activate types 1-3 IP3 receptors can be profoundly affected by cytoplasmic Ca2+. Conversely, Ca2+ activation of type 2 ryanodine receptors is modulated by the level of cyclic ADP-ribose in the cell, which, in turn, can be regulated by cell-surface receptors (Higashida et al. 2001). Other metabolites (e.g. NAADP, sphingosine 1-phosphate) are also proposed as regulators of ER Ca2+-mobilization, however, for these putative messengers information on the Ca2+ -release mechanisms is lacking at present (Berridge et al. 2000; Rizzuto 2001).

In the CNS, Ca2+ entry, via receptor-mediated (e.g. NMDA receptor) and voltage-gated mechanisms, has been the focus of much research, and extracellular Ca2+ has long been considered to be the major source for changes in [Ca2+];. However, recent studies have suggested that intracellular Ca2+ stores may also fulfil specific neuronal functions, in particular, to play a role in activity-dependent synaptic plasticity (Emptage 1999; Rose and Konnerth 2001). Anatomical studies have shown that the ER ramifies into almost all neuronal cellular compartments, including dendritic spines. Furthermore, a number of studies have demonstrated key roles for GPCR-mediated Ca2+ mobilization in bringing about changes in synaptic efficacy. Perhaps the most complete picture has emerged for the role of group I metabotropic glutamate (mGlu) receptors (Hermans and Challiss 2001) in LTD at parallel fibre-Purkinje cell synapses in the cerebellum. Parallel fibre firing causes glutamate release and a highly localized increase in [Ca2+]i in single spines or spinodendritic domains of the Purkinje cell. The involvement of mGlu1 receptors in cerebellar LTD has been demonstrated by the knockout (Aiba et al. 1994; Conquet et al. 1994) and selective rescue (Ichise et al. 2000) of mGlu1 receptor function in transgenic mice. Further, the pathway linking mGlu1 receptor activation to PLC and IP3-mediated Ca2+ release has been shown to be essential in establishing LTD (Inoue et al. 1998; Rose and Konnerth 2001), with very recent studies highlighting the roles of specific PLC isoenzymes and classical PKCs in the pathway (Hirono et al. 2001). The crucial role of the ER as a Ca2+ source in LTD has also been elegantly demonstrated using mutant rats and mice where the ER does not ramify into dendritic spines (Miyata et al. 2000). Collectively, these studies demonstrate clear roles for ER Ca2+-stores and GPCR-mediated Ca2+ mobilization in modifying synaptic function, and highlight the potential of this source of Ca2+ to provide highly compartmentalized and temporally resolved Ca2+ signals (Rose and Konnerth 2001). These and other studies are beginning to provide an insight into the likely roles of IP3 and ryanodine receptors in the CNS.

Until recently, neurobiologists have, in the main, viewed the vast array of GPCRs expressed in the CNS and their diverse signalling with respect to playing a modulatory role upon ionotropic-mediated synaptic transmission. Thus, GPCR activation, by regulating directly or indirectly ion channel function, can modulate both pre- and post-synaptic events to alter the temporal and spatial integration of synaptic activity that in turn alters the frequency and timing of action potentials in central neurons.

It seems possible that this view will change as our understanding of GPCR-mediated signalling in neurons evolves, particularly in relation to its association with signalling complexes in discrete neuronal compartments. For example, computational analysis of GPCR signalling, both in simple neuronal systems, as well as in some mammalian CNS pathways, suggests that synaptically activated GPCRs can play a role in information transfer independent of voltage-mediated signals (Bourne and Nicoll 1993; Katz and Clemens 2001). Thus, perhaps second messenger signalling may eventually be considered to play a more direct, integral part in short-term information transfer in the CNS. However, it is also becoming increasingly clear that GPCR signalling cascades in the CNS do play a crucial role in long-term changes brought about through changes in gene transcription and protein synthesis. We emphasize this aspect of signalling in the remainder of this Chapter.

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