The IP3R (inositol 1,4,5-trisphosphate receptor) releases Ca2+ from the ER (endoplasmic reticulum) store upon binding to its ligand InsP3, which is thought to be generated by activation of certain membrane-bound G-protein-coupled receptors in Drosophila. Depletion of Ca2+ in the ER store also activates SOCE (store-operated Ca2+ entry) from the extracellular milieu across the plasma membrane, leading to a second rise in cytosolic Ca2+, which is then pumped back into the ER. The role of the IP3R and SOCE in mediating Ca2+ homoeostasis in neurons, their requirement in neuronal function and effect on neuronal physiology and as a consequence behaviour, are reviewed in the present article.
Changes in cellular Ca2+ concentration affect multiple signalling processes in both excitable and non-excitable cells . Ca2+ signals can determine the nature and strength of neural connections in a circuit by specifying neurotransmitters and receptors . The extracellular medium, as well as intracellular stores, act as sources and sinks for signalling Ca2+. Most neuronal Ca2+ signals have been attributed to entry of extracellular Ca2+ through voltage-operated channels or ionotropic receptors. Intracellular Ca2+ channels are also present in both vertebrate and invertebrate neurons , but their functions are less thoroughly understood. In Drosophila, it has recently been demonstrated that IP3R (inositol 1,4,5-trisphosphate receptor)-mediated Ca2+ release and SOCE (store-operated Ca2+ entry) are both required in neurons for flight . We have been studying the release of Ca2+ from intracellular stores through IP3Rs and the role of InsP3-mediated intracellular Ca2+ release followed by SOCE in primary cultures of Drosophila larval neurons. In the present review, we discuss how IP3R-mediated and store-operated Ca2+ signalling functions in the maintenance of neuronal Ca2+ homoeostasis. These studies are relevant in the context of certain neurodegenerative diseases in vertebrates.
Signalling through Ca2+
Ca2+ exerts its effect through Ca2+-binding proteins, which in turn interact with proteins of different functions. High concentrations of cytosolic Ca2+ can precipitate phosphates and proteins. As Ca2+ cannot be metabolized or destroyed like other second messengers, the tight regulation of Ca2+ signalling is essential. Cells invest considerable energy to maintain a ~2000-fold Ca2+ barrier between the cytosol and either the ER (endoplasmic reticulum) store or the extracellular milieu. Resting cytosolic Ca2+ in most cells is ~100–400 nM, whereas that in the ER and extracellular milieu is approximately 2 mM . Highly co-ordinated oscillations of cytosolic Ca2+ can dictate cellular function and responses, and there are dedicated components of the ‘Ca2+ signalling toolkit’ that control intracellular Ca2+ oscillations. These are mainly Ca2+ channels and pumps that sequester Ca2+ into intracellular and intercellular compartments. The level of intracellular Ca2+ at any given time in the cell is determined by a balance of processes that facilitate cytoplasmic Ca2+ elevation and those which extrude excess Ca2+ from the cytosol . The ER and extracellular milieu are the primary source and sink of Ca2+, although there is also a contribution from mitochondrial and lysosomal stores in metazoan cells.
A model of existing and proposed pathways that contribute to spontaneous Ca2+ spikes and excitability in neurons
Depending on their functions, cells employ different components from the Ca2+ signalling toolkit to generate and interpret a variety of Ca2+ signals . These distinct combinations of the Ca2+ signalling toolkit impart a unique signature of Ca2+ homoeostasis that suit cell-specific physiology. External stimuli at the PM (plasma membrane) activate GPCRs (G-protein-coupled receptors), which in turn can activate a component of this Ca2+ toolkit, the IP3R, which releases Ca2+ from ER stores. Reduced store Ca2+ triggers another component of the toolkit, store-operated Ca2+ channels, which replenish Ca2+ from the extracellular milieu to the cytosol. Molecular components of SOCE were discovered recently in RNAi (RNA interference) screens in Drosophila S2 cell lines [7–10]. STIM (stromal interaction molecule) is a single-pass transmembrane molecule in the ER membrane which has an EF hand in the ER lumen for sensing luminal Ca2+ depletion. On Ca2+ depletion from the ER, STIM starts forming clusters and shuttles close to the ER–PM junction where it activates Orai channels in the PM through which Ca2+ enters the cytosol . The Ca2+ is pumped back into the ER store by the SERCA (sarcoplasmic/endoplasmic reticulum Ca2+ ATPase) pump. In vertebrates, there are three genes that encode isotypes of the IP3R, Orai and SERCA, whereas two genes encode isotypes of STIM. Different isotypes of these molecules in vertebrates have been found to effect cellular functions differentially [12–16]. The Drosophila genome encodes just one copy of genes for the IP3R (itpr), STIM (dSTIM), Orai (dOrai) and SERCA (dSERCA Ca-P60A). Thus fruitflies serve as a useful genetic model for studying the role of these molecules in the systemic physiology of a whole organism.
Requirement of IP3R function and SOCE in Drosophila neurons for motor co-ordination and rhythmic flight
Molecular genetic studies of itpr mutants have revealed a critical role for the IP3R in larval viability and adult flight [17,18]. Interestingly, for both viability and flight, IP3R function has been mapped to neuronal subsets [aminergic and IPCs (insulin-producing cells)]. Moreover, the temporal requirement for the IP3R in the context of flight is primarily during pupal development [18,20]. Imaging Ca2+ in primary neuronal cultures derived from mutant itpr third instar larvae revealed reduced Ca2+ release through the IP3R as well as compromised SOCE . A dominant-negative allele of dSERCA (Kum170), which is defective in pumping Ca2+ back into the ER store, could partially suppress cold-sensitive larval lethality and adult flight deficits of itpr mutants . Similarly, overexpression of dSTIM, which triggers Orai channel activation, could suppress adult flight deficits and correlated with increased SOCE in primary neuronal cultures . Interestingly, an Orai hypermorphic allele, dOrai2, when put in the background of a flight-defective itpr mutant, not only increased SOCE, but also increased Ca2+ release through the IP3R . Compensation of IP3R mutant phenotype in fruitfly neurons with dSERCA (Kum170), dSTIM and dOrai suggests that Ins(1,4,5)P3 signalling and SOCE function together in the context of flight circuit development and function. Moreover, flight defects in an itpr mutant were suppressed to a greater extent on introducing both Kum170 and Orai2. Neurons from these triple mutant (but flight-competent) animals continued to have reduced store Ca2+ concentrations, but exhibit robust SOCE. Increased Ca2+ release through mutant IP3Rs in the presence of a dOrai hypermorphic allele suggests a feedback loop from Orai to IP3R function, which requires further investigation. These studies show for the first time that Ca2+ release through IP3R and SOCE play an important role in neuronal Ca2+ homoeostasis during development of neuronal circuits. How this signalling is tuned to the specification of neuronal function needs systematic elucidation, in terms of the precise neurons involved and their development and function.
IP3R-mediated Ca2+ homoeostasis in neuronal excitability, neuromodulation and neural circuit formation
The effect of altered Ca2+ homoeostasis in neurons has been studied extensively in Xenopus, where spontaneous Ca2+ spikes in the cytosol shape neuronal proliferation, migration and specification of activity by modulating the expression of voltage-gated ion channels and neurotransmitters. Altered expression of a neurotransmitter leads to an associated change in expression of neurotransmitter receptors in muscles at the neuromuscular junction and thus alters the architecture of the neural circuit, leading to behavioural changes . The origin of these spontaneous Ca2+ spikes in neuronal excitability have been primarily attributed to various channels and neurotransmitter receptors in the PM. Ionotropic glutamate receptors generate Na+ and Ca2+ currents that depolarize neurons throughout development, whereas TRP (transient receptor potential) channels are involved in the generation of spontaneous Ca2+ spikes in the growth cone . A contribution from the ER store in generation of spontaneous cytosolic Ca2+ transients has also been demonstrated in Xenopus neurons, where application of caffeine could generate Ca2+ spikes with or without Ca2+ in the extracellular medium . However, so far, none of these studies has implicated IP3R-mediated Ca2+ release and SOCE in Ca2+ spike-induced neuronal excitability. Given the developmental requirement of IP3R-mediated Ca2+ signals in initiating and maintaining flight rhythms, the dynamics of Ca2+ transients at different developmental time points in normal and flight-defective itpr mutants are of considerable interest. Altered Ca2+ homoeostasis in itpr mutant neurons may lead to altered level and/or activity of voltage-gated and/or ligand-gated Ca2+ channels in the PM and expression of neuropeptides and/or neurotransmitters. Two functional neuronal domains identified in itpr mutants are the aminergic neurons and the insulin-like peptide-producing neurons, both of which are neuromodulatory. Neuromodulators play an important role in the function and formation of neural circuits such as central pattern generators for rhythmic and co-ordinated behaviour . Thus changes in Ca2+ homoeostasis in a neuromodulatory domain will first affect the cellular properties of modulatory neurons, and in turn this can lead to altered input to the central pattern generator which specifies the functioning of a motor circuit. In this context, flight-defective itpr mutants serve as a useful system to explore the neural mechanisms underlying a rhythmic motor behaviour such as insect flight, and the contribution of Ca2+ homoeostasis and its role in neuromodulatory neurons to the function of the central pattern generator for flight.
The IP3R is also important for human motor function since heterozygosity of IP3R1 is the genetic basis for SCA (spinocerebellar ataxia) 15/16 . Thus understanding the mechanism by which Ins(1,4,5)P3-mediated Ca2+ release effects neuronal circuit function in the context of motor behaviour may also help interpret its cellular role in neurodegenerative diseases such as the SCAs that arise from distorted Ca2+ homoeostasis . Importantly, the suppression of defects due to IP3R mutants by raising cellular SOCE in Drosophila, opens up new therapeutic possibilities for this class of neurodegenerative disorders.
Signalling 2011: a Biochemical Society Centenary Celebration: A Biochemical Society Focused Meeting held at the University of Edinburgh, U.K., 8–10 June 2011. Organized and Edited by Nicholas Brindle (Leicester, U.K.), Simon Cook (The Babraham Institute, U.K.), Jeff McIlhinney (Oxford, U.K.), Simon Morley (University of Sussex, U.K.), Sandip Patel (University College London, U.K.), Susan Pyne (University of Strathclyde, U.K.), Colin Taylor (Cambridge, U.K.), Alan Wallace (AstraZeneca, U.K.) and Stephen Yarwood (Glasgow, U.K.).
S.C. is supported by a Senior Research Fellowship from the Council of Scientific and Industrial Research (CSIR), India.