InsP3 receptors: their regulation, and calcium dynamics

The ability of cAMP-dependent protein kinase (PKA) to phosphorylate type I, II, and III inositol 1,4,5-trisphosphate (InsP3) receptors was examined. The receptors either were immunopurified from cell lines and then phosphorylated with purified PKA or were phosphorylated in intact cells after activating intracellular cAMP formation. The type I receptor is a good substrate for PKA, whereas type II and III receptors are phosphorylated relatively weakly. Despite these differences, each of the receptors is phosphorylated in intact cells in response to forskolin or activation of neurohormone receptors. Detailed examination of SH-SY5Y neuroblastoma cells, which express type I receptor, reveal that minor increases in cAMP concentration are sufficient to cause maximal phosphorylation. Thus, VIP and pituitary adenylyl cyclase activating peptide (acting through Gs-coupled pituitary adenylyl cyclase activating peptide-I receptors) are potent stimuli of type I receptor phosphorylation, and remarkably, even slight increases in cAMP concentration induced by carbachol (acting through Gq-coupled muscarinic receptors) or other Ca2+ mobilizing agents are sufficient to cause phosphorylation. Finally, PKA enhances InsP3-induced Ca2+ mobilization in a range of permeabilized cell types, irrespective of whether the type I, II, or III receptor is predominant. In summary, these data show that all InsP3 receptors are phosphorylated by PKA, albeit with marked differences in stoichiometry. The ability of both Gs- and Gq-coupled cell surface receptors to effect InsP3 receptor phosphorylation by PKA suggests that this process is widespread in mammalian cells and provides multiple routes by which the cAMP signaling pathway can influence Ca2+ mobilization (Wojcikiewicz, 1998).

Cyclic nucleotide-regulated phosphorylation of the neuronal type I inositol 1,4,5-trisphosphate (IP3) receptor immunopurified from rat cerebellar membranes was examined in vitro and in rat cerebellar slices in situ. The isolated IP3 receptor protein is phosphorylated by both cAMP- and cGMP-dependent protein kinases on two distinct sites as determined by thermolytic phosphopeptide mapping, phosphopeptide 1, representing Ser-1589, and phosphopeptide 2, representing Ser-1756 in the rat protein. Phosphopeptide maps show that cAMP-dependent protein kinase (PKA) labels both sites with the same time course and same stoichiometry, whereas cGMP-dependent protein kinase (PKG) phosphorylates Ser-1756 with a higher velocity and a higher stoichiometry than Ser-1589. Synthetic decapeptides corresponding to the two phosphorylation sites [peptide 1, AARRDSVLAA (Ser-1589), and peptide 2, SGRRESLTSF (Ser-1756)] were used to determine kinetic constants for the phosphorylation by PKG and PKA, and the catalytic efficiencies were in agreement with the results obtained by in vitro phosphorylation of the intact protein. In cerebellar slices prelabeled with [32P]orthophosphate, activation of endogenous kinases by incubation in the presence of cAMP/cGMP analogs and specific inhibitors of PKG and PKA induces in both cases a 3-fold increase in phosphorylation of the IP3 receptor. Thermolytic phosphopeptide mapping of in situ labeled IP3 receptor by PKA shows labeling on the same sites (Ser-1589 and Ser-1756) as in vitro. In contrast to the findings in vitro, PKG preferentially phosphorylates Ser-1589 in situ. Because both PKG and the IP3 receptor are specifically enriched in cerebellar Purkinje cells, PKG may be an important IP3 receptor regulator in vivo (Haug, 1999).

In the central nervous system, release of Ca2+ from intracellular stores contributes to numerous functions, including neurotransmitter release and long-term potentiation and depression. The developmental profile and the regulation of inositol 1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (RyR) was examined in primary cultures of cerebellar granule cells. The expression of both receptor types increases during development. Whereas the expression of type 1 IP3R appears to be regulated by Ca2+ influx through L type channels or N-methyl-D-aspartate (NMDA) receptors, RyR levels increase independent of Ca2+. The main target of Ca2+-influx-regulating IP3R expression is the Ca2+ calmodulin-dependent protein phosphatase calcineurin, because pharmacological blockade of this protein abolishes IP3R expression. Although calcineurin has been shown to regulate the phosphorylation state of the IP3R, the effect described here is at the transcriptional level because IP3R mRNA changes in parallel with protein levels. Thus, calcineurin plays a dual role in IP3R-mediated Ca2+ signaling: it regulates IP3R function by dephosphorylation in the short-term time scale and IP3R expression over more extended periods (Genazzani, 1999).

The interactions between calmodulin, inositol 1,4,5-trisphosphate (InsP3), and pure cerebellar InsP3 receptors were characterized by using a scintillation proximity assay. In the absence of Ca2+, 125I-labeled calmodulin reversibly binds to multiple sites on InsP3 receptors and Ca2+ increases the binding by 190% +/- 10%; the half-maximal effect occurs when the Ca2+ concentration is 184 +/- 14 nM. In the absence of Ca2+, calmodulin causes a reversible, concentration-dependent (IC50 = 3.1 +/- 0.2 microM) inhibition of [3H]InsP3 binding by decreasing the affinity of the receptor for InsP3. This effect is similar at all Ca2+ concentrations, indicating that the site through which calmodulin inhibits InsP3 binding has similar affinities for calmodulin and Ca2+-calmodulin. Calmodulin (10 microM) inhibita the Ca2+ release from cerebellar microsomes evoked by submaximal (but not by maximal) concentrations of InsP3. Tonic inhibition of InsP3 receptors by the high concentrations of calmodulin within cerebellar Purkinje cells may account for their relative insensitivity to InsP3 and limit spontaneous activation of InsP3 receptors in the dendritic spines. Inhibition of InsP3 receptors by calmodulin at all cytosolic Ca2+ concentrations, together with the known redistribution of neuronal calmodulin evoked by protein kinases and Ca2+, suggests that calmodulin may also allow both feedback control of InsP3 receptors and integration of inputs from other signaling pathways (Patel, 1997).

The second messenger inositol-1,4,5-trisphosphate (InsP3) releases Ca2+ from intracellular Ca2+ stores by activating specific receptors on the membranes of these stores. In many cells, InsP3 is a global signalling molecule that liberates Ca2+ throughout the cytoplasm. However, in neurons the situation might be different, because synaptic activity may produce InsP3 at discrete locations. InsP3 signaling was characterized in postsynaptic cerebellar Purkinje neurons, which have a high level of InsP3 receptors. Repetitive activation of the synapse between parallel fibers and Purkinje cells causes InsP3-mediated Ca2+ release in the Purkinje cells. This Ca2+ release is restricted to individual postsynaptic spines, where both metabotropic glutamate receptors and InsP3 receptors are located, or to multiple spines and adjacent dendritic shafts. Focal photolysis of caged InsP3 in Purkinje cell dendrites also produces Ca2+ signals that spread only a few micrometers from the site of InsP3 production. Uncaged InsP3 produces a long-lasting depression of parallel-fiber synaptic transmission that is limited to synapses where the Ca2+ concentration is raised. Thus, in Purkinje cells InP3 acts within a restricted spatial range that allows it to regulate the function of local groups of parallel-fiber synapses (Finch, 1999).

The inositol 1,4,5-trisphosphate receptor (InsP3R) is the main calcium (Ca2+) release channel in most tissues. Three isoforms have been identified, but only types I and II InsP3R have been characterized. The functional properties of the type III InsP3R were examined because this receptor is restricted to the trigger zone from which Ca2+ waves originate and it has distinctive InsP3-binding properties. Type III InsP3R forms Ca2+ channels with single-channel currents that are similar to those of type I InsP3R; however, the open probability of type III InsP3R isoform increases monotonically with increased cytoplasmic Ca2+ concentration, whereas the type I isoform has a bell-shaped dependence on cytoplasmic Ca2+. The properties of type III InsP3R provide positive feedback as Ca2+ is released; the lack of negative feedback allows complete Ca2+ release from intracellular stores. Thus, activation of type III InsP3R in cells that express only this isoform results in a single transient, but global, increase in the concentration of cytosolic Ca2+. The bell-shaped Ca2+-dependence curve of type I InsP3R is ideal for supporting Ca2+ oscillations, whereas the properties of type III InsP3R are better suited to signal initiation (Hagar, 1998).

Calcium ions are released from intracellular stores in response to agonist-stimulated production of inositol 1,4,5-trisphosphate (InsP3), a second messenger generated at the cell membrane. Depletion of Ca2+ from internal stores triggers a capacitative influx of extracellular Ca2+ across the plasma membrane. The influx of Ca2+ can be recorded as store-operated channels (SOC) in the plasma membrane or as a current known as the Ca2+-release-activated current [I(crac)]. A critical question in cell signaling is how SOC and I(crac) sense and respond to Ca2+-store depletion: in one model, a messenger molecule is generated that activates Ca2+ entry in response to store depletion; in an alternative model, InsP3 receptors in the stores are coupled to SOC and I(crac). The mammalian Htrp3 protein forms a well defined store-operated channel and so provides a suitable system for studying the effect of Ca2+-store depletion on SOC and I(crac). Htrp3 channels stably expressed in HEK293 cells are in a tight functional interaction with the InsP3 receptors. Htrp3 channels present in the same plasma membrane patch can be activated by Ca2+ mobilization in intact cells and by InsP3 in excised patches. This activation of Htrp3 by InsP3 is lost on extensive washing of excised patches but is restored by addition of native or recombinant InsP3-bound InsP3 receptors. These results provide evidence for the coupling hypothesis, in which InsP3 receptors activated by InsP3 interact with SOC and regulate I(crac) (Kiselyov, 1998).

The interaction of intracellular free calcium ([Ca2+]i) and cAMP signaling mechanisms was examined in intact single megakaryocytes by using a combination of single-cell fluorescence microscopy to measure [Ca2+]i and flash photolysis of caged Ca2+, inositol 1,4, 5-trisphosphate (IP3), and/or cAMP in order to rapidly elevate the concentration of these compounds inside the cell. Photolysis of caged IP3 stimulates Ca2+ release from an IP3-sensitive store. The cAMP-elevating agent carbacyclin inhibits this IP3-induced rise in [Ca2+]i but does not affect the rate of Ca2+ removal from the cytoplasm after photolysis of caged Ca2+. Photolysis of caged cAMP during ADP-induced [Ca2+]i oscillations causes the [Ca2+]i oscillation to transiently cease without affecting the rate of Ca2+ uptake and/or extrusion. It has been concluded that the principal mechanism of cAMP-dependent inhibition of Ca2+ mobilization in megakaryocytes appears to be inhibition of IP3-induced Ca2+ release and not stimulation of Ca2+ removal from the cytoplasm. Two inhibitors of cAMP-dependent protein kinase, a specific peptide inhibitor of the catalytic subunit of cAMP protein kinase and KT5720, block the inhibitory effect of carbacyclin, indicating that the inhibition of IP3-induced Ca2+-release by carbacyclin is mediated by cAMP-dependent protein kinase. These results imply that cAMP protein kinase is required for inhibition of IP3-induced Ca2+-release, although it is not clear whether this inhibition requires phosphorylation of only the IP3 receptor, or if the phosphorylation of other proteins is involved (Tertyshnikova, 1998).

Repetitive transient increases in cytosolic calcium concentration (calcium spikes or calcium oscillations) are a common mode of signal transduction in receptor-mediated cell activation. Repetitive calcium spikes are initiated by phospholipase C-mediated production of inositol 1,4,5-trisphosphate (InsP3) and are thought to be generated by a positive feedback mechanism in which calcium potentiates its own release, a negative feedback mechanism by which calcium release is terminated, and a slow recovery process that defines the time interval between calcium spikes. The molecular mechanisms that terminate each calcium spike and define the spike frequency are not yet known. In intact rat basophilic leukemia cells calcium responses induced by InsP3 are diminished for a period of 30-60 s following an InsP3-induced calcium spike. The sensitivity of calcium release for InsP3 was probed by UV laser-mediated photorelease of InsP3, and calcium responses were monitored by fluorescence calcium imaging. A maximal loss in sensitivity (desensitization) is observed for InsP3 increases that result in a near maximal calcium spike and is expressed as an 80-100% reduction in the calcium response to an equal amount of InsP3, released 10 s after the first UV pulse. When the amount of released InsP3 in the second pulse is increased 2-3-fold, desensitization is overcome and a second calcium response of equal amplitude to the first is produced. A power dependence of 3.2 was measured between the amount of released InsP3 and the amplitude of the triggered calcium response, explaining how a small decrease in InsP3 sensitivity can lead to a nearly complete reduction in the calcium response. Desensitization is abolished by the addition of the calcium buffers BAPTA and EGTA and can be induced by microinjection of calcium, suggesting that it is a calcium-dependent process. Half-maximal desensitization is observed at a free calcium concentration of 290 nM and increases with a power of 3.7 with peak calcium concentration. These studies suggest that reversible desensitization of InsP3-induced calcium release serves as a "saw-tooth" parameter that controls the termination of each spike and the frequency of calcium spikes (Oancea, 1996).

Activation of intracellular Ca2+ channels by inositol 1,4,5-trisphosphate (InsP3) represents the initial Ca2+ mobilization step in response to many extracellular signals. InsP3-induced channel activation in permeabilized hepatocytes is followed by a time-dependent inactivation, which is a direct consequence of ligand binding. The inactivation by InsP3 parallels the quantal character of channel opening, giving rise to a unique process of incremental inactivation whereby discrete channel populations are inhibited at each InsP3 dose. InsP3 can induce inactivation in the absence of stored Ca2+, but the inactivation rate is enhanced by increases of cytosolic Ca2+. The inhibitory effect of InsP3 can be reversed by InsP3 washout, or by chelation of cytosolic Ca2+. Thus, InsP3 and Ca2+ act as coinhibitors of the InsP3-sensitive Ca2+ channel. Inactivation is an inherent consequence of InsP3-induced channel opening that can terminate increases of cytosolic Ca2+ (Hajnoczky, 1994).

T cell receptor stimulation triggers a physical association between the nonreceptor protein tyrosine kinase Fyn and the InsP3R, which induces tyrosine phosphorylation of the InsP3R. Fyn activates an InsP3-gated calcium channel in vitro, and tyrosine phosphorylation of the InsP3R during T cell activation is reduced in thymocytes from fyn-/- mice. Thus, activation of the InsP3R by tyrosine phosphorylation may play a role in regulating intracellular calcium (Jayaraman, 1996).

Transient rise in nuclear calcium concentration is implicated in the regulation of events controlling gene expression. Various mechanisms by which calcium is transported to the nucleus are the subject of vehement debate. Inositol 1,4,5-trisphosphate (InsP3) and inositol-1,3,4,5-tetrakisphosphate (InsP4) receptors have been located to the nucleus and their role in nuclear calcium signaling has been proposed. Outer nuclear membrane was separated from the inner membrane. The two membrane preparations were, as best as possible, devoid of cross contamination as attested by marker enzyme activity, Western blotting with antilamin antibody, and electron microscopy. InsP4 receptor and Ca(2+)-ATPase were located to the outer nuclear membrane. InsP3 receptor was located to the inner nuclear membrane. ATP or InsP4 induces nuclear calcium uptake. External free calcium concentration, in the medium bathing the nuclei, determines the choice for ATP or InsP4-mediated calcium transport. A mechanistic model is presented for nuclear calcium transport. According to this model, calcium can reach the nucleus envelope either by the action of ATP or InsP4. However, the calcium release from the nucleus envelope to the nucleoplasm is mediated by InsP3 through the activation of InsP3 receptor, which is located to the inner nuclear membrane. The action of InsP3 in this process is instantaneous and transient and is sensitive to heparin (Humbert, 1996).

The immunophilin FK506 binding protein 12 (FKBP12) is associated with and modulates the ryanodine receptor calcium release channel of skeletal muscle. Ryanodine receptor has amino acid homology and functional similarity with another intracellular Ca2+ release channel, the inositol 1,4,5-trisphosphate receptor (InsP3R). Highly purified preparations of InsP3R contain FKBP12. The complex of these two proteins is disrupted by the immunosuppressants FK506 and rapamycin, both of which are known to bind FKBP12 with high affinity. Disrupting the InsP3R-FKBP12 interaction increases Ca2+ flux through InsP3R, an effect that is reversed by added FKBP12. FKBP12 appears to be physiologically linked to InsP3R, regulating its Ca2+ conductance (Cameron, 1995).

Rat basophilic leukemia (RBL-2H3) cells predominantly express the type II receptor for inositol 1,4,5-trisphosphate (InsP3), which operates as an InsP3-gated calcium channel. In these cells, cross-linking the high-affinity immunoglobulin E receptor leads to activation of phospholipase C gamma isoforms via tyrosine kinase- and phosphatidylinositol 3-kinase-dependent pathways, release of InsP3-sensitive intracellular Ca2+ stores, and a sustained phase of Ca2+ influx. These events are accompanied by a redistribution of type II InsP3 receptors within the endoplasmic reticulum and nuclear envelope, from a diffuse pattern with a few small aggregates in resting cells to large isolated clusters after antigen stimulation. Redistribution of type II InsP3 receptors is also seen after treatment of RBL-2H3 cells with ionomycin or thapsigargin. InsP3 receptor clustering occurs within 5-10 min of stimulus and persists for up to 1 h in the presence of antigen. Receptor clustering is independent of endoplasmic reticulum vesiculation, which occurs only at ionomycin concentrations >1 microM; maximal clustering responses are dependent on the presence of extracellular calcium. InsP3 receptor aggregation may be a characteristic cellular response to Ca2+-mobilizing ligands, because similar results are seen after activation of phospholipase C-linked G-protein-coupled receptors; cholecystokinin causes type II receptor redistribution in rat pancreatoma AR4-2J cells, and carbachol causes type III receptor redistribution in muscarinic receptor-expressing hamster lung fibroblast E36(M3R) cells. Stimulation of these three cell types leads to a reduction in InsP3 receptor levels only in AR4-2J cells, indicating that receptor clustering does not correlate with receptor down-regulation. The calcium-dependent aggregation of InsP3 receptors may contribute to the previously observed changes in affinity for InsP3 in the presence of elevated Ca2+ and/or may establish discrete regions within refilled stores with varying capacities to release Ca2+ when a subsequent stimulus results in production of InsP3 (Wilson, 1998).

During early embryonic development, IP₃-Ca²⁺ signaling transduces ventral signaling at the time of dorsoventral axis formation. To identify molecules functioning upstream in this signal pathway, effects were measured of a panel of inhibitory antibodies against Galphaq/11, Galphas/olf, or Galphai/o/t/z. While all these antibodies show direct inhibition of their targets, their effects varied in terms of the redirection of ventral mesoderm to a dorsal fate. Anti-Galphas/olf antibody shows strong induction of dorsal fate; anti-Galphai/o/t/z antibody does so weakly, and anti-Galphaq/11 antibody is without effect. Injection of betaARK, a Gbetagamma inhibitor, mimics the dorsalizing effect of anti-Galphas/olf antibody, whereas injection of adenylyl cyclase inhibitors at a concentration that inhibits Galphas-coupled cAMP increase does not do so. The activation of Galphas-coupled receptor gives rise to Ca²⁺ transients. All these results suggest that activation of the Galphas-coupled receptor relays dorsoventral signal to Gbetaggamma, which then stimulates PLCbeta and then the IP₃-Ca²⁺ system. This signaling pathway may play a crucial role in transducing ventral signals (Kume, 2000).

In Xenopus, patterning of the body axis occurs by sequential inductive events. Maternal activation of the Wnt pathway is required for the initiation of axis formation, by creating a Nieuwkoop center and mediating the dorsalizing function of the Nieuwkoop center. The Spemann organizer of the Xenopus embryo can be subdivided into two discrete activities: trunk organizer and head organizer. The molecular mechanism of trunk organizer formation involves several factors secreted from the blastopore lip that act by repressing signaling by bone morphogenetic proteins (BMPs), which antagonize the organizer. The finding that anti-Galphas/olf antibody or betaARK injection induces trunk but not head organizer is similar to a previous result using anti-IP3R inhibitory antibodies. These results correlate with the facts that the secondary axes induced by Noggin, Chordin, and truncated BMP receptors often lack the anteriormost structures and that head induction requires simultaneous repression of BMP and Wnt signaling in Xenopus. The IP₃-Ca²⁺ signaling system may crosstalk with the BMP pathway, by means of a mechanism which still remains unknown (Kume, 2000).

There are other candidates for upstream factors that may activate the IP₃-Ca²⁺ signaling system. Zygotic activation of the Wnt pathway is suggested to be required for ventro-lateral mesoderm formation. Members of the Wnt-5a class of proteins do not induce ectopic dorsal axis duplication, unless coexpressed with certain members of the frizzled family, yet they do decrease cell adhesion and perturb morphogenetic movement during gastrulation in Xenopus embryos. There is evidence that the Wnt-5a class can function in a non-cell-autonomous manner to block the ability of members of the Wnt-1 class to induce a secondary axis. Overexpression of Xwnt-5a with rat frizzled-2 increases the frequencies of Ca²⁺ spikes in zebrafish. Activation of Galphas/olf-coupled receptor elicits Ca²⁺ transients of an interval that resembles one reported for Xwnt-5a. It will be of interest to determine whether Xwnt-5a is the endogenous upstream ligand that activates IP₃-Ca²⁺ signaling during embryonic development (Kume, 2000).

The downstream targets of the Ca²⁺ transients during early embryonic axis formation remain largely unknown. There is evidence that varying the frequency or intensity of Ca²⁺ transients can alter the physiological output. One well-known example of molecules modulated by frequency of Ca²⁺ is calmodulin-dependent kinase II, which regulates other enzymes dependent on Ca²⁺. The enzyme is activated to varying degrees depending on the frequency of Ca²⁺ oscillations. Varying the frequency or intensity of the Ca²⁺ rise can contribute to activation of different subsets of developmental genes. Examinations of the types of IP₃-Ca²⁺ signaling that activate separate sets of genes and, in turn, lead to a specific developmental program, are expected to elucidate the mechanism underlying the early developmental events (Kume, 2000).

A novel function of the presenilins (PS1 and PS2) in governing capacitative calcium entry (CCE), a refilling mechanism for depleted intracellular calcium stores, is reported. Abrogation of functional PS1, by either knocking out PS1 or expressing inactive PS1, markedly potentiates CCE, suggesting a role for PS1 in the modulation of CCE. In contrast, familial Alzheimer's disease (FAD)-linked mutant PS1 or PS2 significantly attenuates CCE and store depletion-activated currents. While inhibition of CCE selectively increases the amyloidogenic amyloid ß peptide (Aß42), increased accumulation of the peptide has no effect on CCE. Thus, reduced CCE is most likely an early cellular event leading to increased Aß42 generation associated with FAD mutant presenilins. These data indicate that the CCE pathway is a novel therapeutic target for Alzheimer's disease (Yoo, 2000).

It is suggested that autosomal dominant FAD mutant presenilins exert a gain of function by downregulating CCE while increasing IP₃-mediated release from the ER store, leading to diminished luminal Ca²⁺ concentration ([Ca²⁺]_ER). It is interesting to note that changes in [Ca²⁺]_ER influence a number of cellular functions, including chaperone activities and gene expression. Therefore, it is tempting to speculate that reduced CCE may also be an upstream event leading to other molecular phenotypes associated with FAD mutant presenilins, including altered unfolded protein response. Interestingly, in transgenic mice harboring spinocerebellar ataxia type 1 (SCA1) mutant gene products, TRP3, SERCA2, and IP₃-R (all components of CCE), are specifically downregulated. This suggests the potential contribution of CCE dysregulation in other neurodegenerative diseases in addition to AD. CCE involves direct physical interaction between the ER and plasma membrane constituents. According to this conformational coupling mechanism, a conformational change of the IP₃ receptor (IP₃-R) upon agonist stimulation and subsequent release of Ca²⁺ leads to the formation of a molecular complex containing IP₃-R bound to molecular constituents in the plasma membrane harboring CCE channels. This then allows extracellular Ca²⁺ to replenish the ER store. It has been postulated that the presenilins modulate the gamma-secretase activity via few possible mechanisms: the presenilins might be the gamma-secretases themselves, and serve as essential cofactors for the gamma-secretase action, or regulate intracellular trafficking of a putative gamma-secretase to the target site where relevant substrates are localized. Given a role for presenilins in governing CCE, the presenilins may also modulate proteolytic processing of APP and Notch at or near the cell surface at sites of ER-plasma membrane coupling. It is conceivable that the presenilins may regulate or directly mediate the cleavage of protein(s) involved in modulating CCE. In any event, a gain in the biological activity of the presenilins, owing to autosomal dominant FAD mutations, may attenuate CCE while increasing gamma-secretase activity. Further experimentation will be necessary to elucidate this connection. Finally, augmentation of CCE, through the identification of agonists of plasma membrane store-operated Ca²⁺ channels (e.g., TRP or as yet undiscovered CCE channels) that mediate CCE, could potentially be employed to reduce PS-associated gamma-secretase activity, and the generation of Aß as a novel therapeutic means for preventing or treating AD (Yoo, 2000).

Agonist-evoked [Ca²⁺]_i oscillations have been considered a biophysical phenomenon reflecting the regulation of the IP₃ receptor by [Ca²⁺]_i. [Ca²⁺]_i oscillations are a biochemical phenomenon emanating from regulation of Ca2+ signaling by the regulators of G protein signaling (RGS) proteins. [Ca²⁺]_i oscillations evoked by G protein-coupled receptors require the action of RGS proteins. Inhibition of endogenous RGS protein action disrupts agonist-evoked [Ca²⁺]_i oscillations by a stepwise conversion to a sustained response. Based on these findings and the effect of mutant RGS proteins and anti-RGS protein antibodies on Ca2+ signaling, it is proposed that RGS proteins within the G protein-coupled receptor complexes provide a biochemical control of [Ca²⁺]_i oscillations (Luo, 2001).

A possible model for the biochemical control of [Ca²⁺]_i oscillation by RGS proteins is based on the present work and on the regulation of RGS protein function by PIP₃ and Ca²⁺-calmodulin (CaM). In the resting state, RGS proteins in signaling complexes are active and exert tonic inhibition by converting all spontaneously generated alphaq·GTP to alphaq·GDP. The findings that (1) all recombinant RGS proteins that interact with Galphaq tested to date are potent inhibitors of Ca²⁺ signaling when infused into cells and (2) activation of IP₃-dependent signaling by the K/Q mutant, and activation of Ca²⁺ signaling by the anti-RGS protein Abs show that RGS proteins are indeed active in resting cells to exert tonic inhibition of Ca²⁺ signaling. The agonist-activated receptor simulates the GDP/GTP exchange reaction to increase the rate of generation and the steady-state concentration of alpha·GTP. At this stage, the rate of alpha·GTP generation is faster than the rate of RGS protein-assisted GTP hydrolysis, resulting in the activation of PLCbeta, hydrolysis of PIP₂ to generate IP₃, and initiation of Ca²⁺ (step 1). In the first of the four steps described here, the activated receptor may not only activate Galpha, but may also stabilize the inactive state of RGS proteins by promoting the formation of PIP₃. The rate and extent of Galpha·GTP production and inhibition of RGS protein GAP activity is a function of agonist concentration. The Galphaq·PLCbeta complex continues to produce IP₃ and release Ca²⁺ until all stores exposed to IP₃ are depleted. At low concentration of agonist, only the stores in the vicinity of signaling complexes are exposed to IP₃. At increasing agonist concentration and, thus, IP₃ production, an increased fraction of the stores is discharged by IP₃ (Luo, 2001).

At the end of Ca²⁺ release and at the peak of [Ca²⁺]_i increase, high [Ca²⁺]_i in the vicinity of the IP₃ pore inhibits the channel to reduce the Ca²⁺ permeability of the stores' membrane. To terminate the stimulated state, the activity of RGS proteins has to be restored. This can be by formation of sufficient [Ca²⁺-CaM] to relieve the inhibition of RGS protein GAP activity by PIP₃ (step 2). This will lead to binding of the Ca²⁺/CaM/RGS protein to alphaq·GTP, acceleration of GTP hydrolysis, and inhibition of PLCbeta and IP₃ production. The reduction in [IP₃] together with reduced IP₃R channel activity, reloads the stores with Ca²⁺ (step 3). It is proposed that subsequent dissociation of Ca²⁺-CaM from RGS proteins stabilizes the RGS protein conformation that binds PIP₃ (or another inhibitor) to block RGS GAP activity (step 4). An important feature of the transition between step 3 and step 4 is that Galphaq cannot activate PLCbeta until Ca²⁺-CaM dissociates from RGS proteins. This provides a plausible mechanism for regulation of oscillation frequency. For many calmodulin-regulated proteins, regulation of enzymatic activity by calmodulin shows a hysteresis behavior. Upon Ca²⁺ increase, binding of calmodulin to target proteins and their activation is fast, whereas upon reduction of Ca²⁺, dissociation of Ca²⁺-CaM and termination of the active state is slow. Such behavior may also be a feature of the interaction of Ca²⁺-CaM with RGS proteins and determines the duration of the delay between [Ca²⁺]_i spikes. At low agonist concentrations, the rates of Galphaq activation and, possibly, dissociation of Ca²⁺-CaM from RGS proteins are slow, resulting in a low frequency oscillation in [IP₃] and [Ca²⁺]_i. Increasing agonist concentration can increase [Ca²⁺]_i oscillation frequency by increasing the rates of Galphaq activation and/or dissociation of Ca²⁺-CaM from RGS proteins. At very high agonist concentration, the rate of Galphaq activation is maximal, and RGS proteins remain in the inactive, PIP₃-bound state. This would, of course, result in a sustained response (Luo, 2001).

Biochemical control of Ca²⁺ oscillations by RGS proteins can explain several features of the oscillations. (1) The mode of activation of Galphaq by the receptor and inhibition of Galphaq by Ca²⁺-CaM bound RGS proteins can generate the receptor-specific patterns of [Ca²⁺]_i spiking observed in many cell types. In fact, RGS proteins differentially interact with the muscarinic, CCK, and bombesin receptors in pancreatic acini. (2) The model can explain how several receptors respond to increasing agonist concentration with increased oscillation frequency without affecting spike amplitude. Increased rates of Galphaq activation of the same signaling complexes will result in constant amplitude but increased oscillations frequency. (3) In many cells, including pancreatic acinar cells, the shape of individual Ca²⁺ spikes is receptor specific. The shape of individual [Ca²⁺]_i spikes can be determined by the rates of Galphaq activation and periodic activation and inactivation of RGS GAP activity. (4) Slow dissociation of Ca²⁺-CaM from RGS proteins can explain oscillations in [IP₃] and maintain low [IP₃] between [Ca²⁺]_i spikes. Reduction in [IP₃] between Ca²⁺ spikes can explain how Ca²⁺ release remains inactive for long periods of time between the spikes (Luo, 2001).

It is important to note that the biochemical (by RGS proteins) and biophysical (by Ca²⁺-dependent regulation of IP₃R) regulation of [Ca²⁺]_i oscillations are not mutually exclusive. Rather, it is likely that both regulatory events determine the final shape of the oscillations. However, the results suggest that the primary oscillator is the receptor·Galphaq·RGS protein complex. Regulation of IP₃R channel activity and, probably, the Ca²⁺ pumps is necessary to aid the receptor complex in controlling the oscillations. For example, inhibition of the IP₃R channel by high local Ca²⁺ will start reuptake of Ca²⁺ into the store by the SERCA pumps prior to complete reduction in [IP₃]. By placing the oscillator at the receptor complex, the receptor governs regulation of [Ca²⁺]_i oscillations. Furthermore, in this manner the oscillations can be precisely controlled by the state of occupancy of the receptor with ligands. In other words, the mode of occupancy of the receptor with agonist will determine the type of signal transduced into the cell interior (Luo, 2001).

The most common form of Ca²⁺ signaling by Gq-coupled receptors entails activation of PLCβ2 by Gαq to generate IP₃ and evoke Ca²⁺ release from the ER. Another form of Ca²⁺ signaling by G protein-coupled receptors involves activation of Gi to release Gβγ, which activates PLCβ1. Whether Gβγ has additional roles in Ca²⁺ signaling is unknown. Introduction of Gβγ into cells activates Ca²⁺ release from the IP₃ Ca²⁺ pool and Ca² oscillations. This can be due to activation of PLCβ1 or direct activation of the IP₃R by Gβγ. Gβγ potently activates the IP₃ receptor. Thus, Gβγ-triggered [Ca²⁺]_i oscillations are not affected by inhibition of PLCβ. Coimmunoprecipitation and competition experiments with Gβγ scavengers suggest binding of Gβγ to IP₃ receptors. Furthermore, Gβγ inhibits IP₃ binding to IP₃ receptors. Notably, Gβγ activates single IP₃R channels in native ER as effectively as IP₃. The physiological significance of this form of signaling is demonstrated by the reciprocal sensitivity of Ca²⁺ signals evoked by Gi- and Gq-coupled receptors to Gβγ scavenging and PLCβ inhibition. It is proposed that gating of IP₃R by Gβγ is a new mode of Ca²⁺ signaling with particular significance for Gi-coupled receptors (Zeng, 2003).