Presenilins form ER Ca2+ leak channels, a function disrupted by familial Alzheimer's disease-linked mutations

Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disorder. Mutations in presenilins 1 and 2 (PS1 and PS2) account for approximately 40% of familial AD (FAD) cases. FAD mutations and genetic deletions of presenilins have been associated with calcium (Ca(2+)) signaling abnormalities. We demonstrate that wild-type presenilins, but not PS1-M146V and PS2-N141I FAD mutants, can form low-conductance divalent-cation-permeable ion channels in planar lipid bilayers. In experiments with PS1/2 double knockout (DKO) mouse embryonic fibroblasts (MEFs), we find that presenilins account for approximately 80% of passive Ca(2+) leak from the endoplasmic reticulum. Deficient Ca(2+) signaling in DKO MEFs can be rescued by expression of wild-type PS1 or PS2 but not by expression of PS1-M146V or PS2-N141I mutants. The ER Ca(2+) leak function of presenilins is independent of their gamma-secretase activity. Our data suggest a Ca(2+) signaling function for presenilins and provide support for the "Ca(2+) hypothesis of AD."     

Isoform-specific function of single inositol 1,4,5-trisphosphate receptor channels.

The inositol 1,4,5-trisphosphate receptor (InsP3R) family of Ca2+ release channels is central to intracellular Ca2+ signaling in mammalian cells. The InsP3R channels release Ca2+ from intracellular compartments to generate localized Ca2+ transients that govern a myriad of cellular signaling phenomena (Berridge, 1993. Nature. 361:315-325; Joseph, 1996. Cell Signal. 8:1-7; Kume et al., 1997. Science. 278:1940-1943; Berridge, 1997. Nature. 368:759-760). express multiple InsP3R isoforms, but only the function of the single type 1 InsP3R channel is known. Here the single-channel function of single type 2 InsP3R channel is defined for the first time. The type 2 InsP3R forms channels with permeation properties similar to that of the type 1 receptor. The InsP3 regulation and Ca2+ regulation of type 1 and type 2 InsP3R channels are strikingly different. Both InsP3 and Ca2+ are more effective at activating single type 2 InsP3R, indicating that single type 2 channels mobilize substantially more Ca2+ than single type 1 channels in cells. Furthermore, high cytoplasmic Ca2+ concentrations inactivate type 1, but not type 2, InsP3R channels. This indicates that type 2 InsP3R channel is different from the type 1 channel in that its activity will not be inherently self-limiting, because Ca2+ passing through an active type 2 channel cannot feed back and turn the channel off. Thus the InsP3R identity will help define the spatial and temporal nature of local Ca2+ signaling events and may contribute to the segregation of parallel InsP3 signaling cascades in mammalian cells.     

Presenilin mutations linked to familial Alzheimer's disease reduce endoplasmic reticulum and Golgi apparatus calcium levels

Presenilin-1 and -2 (PS1 and PS2) mutations, the major cause of familial Alzheimer's disease (FAD), have been causally implicated in the pathogenesis of neuronal cell death through a perturbation of cellular Ca(2+) homeostasis. We have recently shown that, at variance with previous suggestions obtained in cells expressing other FAD-linked PS mutations, PS2-M239I and PS2-T122R cause a reduction and not an increase in cytosolic Ca(2+) rises induced by Ca(2+) release from stores. In this contribution we have used different cell models: human fibroblasts from controls and FAD patients, cell lines (SH-SY5Y, HeLa, HEK293, MEFs) and rat primary neurons expressing a number of PS mutations, e.g. P117L, M146L, L286V, and A246E in PS1 and M239I, T122R, and N141I in PS2. The effects of FAD-linked PS mutations on cytosolic Ca(2+) changes have been monitored either by using fura-2 or recombinant cytosolic aequorin as the probe. Independently of the cell model or the employed probe, the cytosolic Ca(2+) increases, caused by agonist stimulation or full store depletion by drug treatment, were reduced or unchanged in cells expressing the PS mutations. Using aequorins, targeted to the endoplasmic reticulum or the Golgi apparatus, we here show that FAD-linked PS mutants lower the Ca(2+) content of intracellular stores. The phenomenon was most prominent in cells expressing PS2 mutants, and was observed also in cells expressing the non-pathogenic, "loss-of-function" PS2-D366A mutation. Taken as a whole, our findings, while confirming the capability of presenilins to modify Ca(2+) homeostasis, suggest a re-evaluation of the "Ca(2+) overload" hypothesis in AD and a new working hypothesis is presented.     

Calcium homeostasis in vascular smooth muscle cells is altered in type 2 diabetes by Bcl-2 protein modulation of InsP3R calcium release channels

This study examines the extent to which the antiapoptotic Bcl-2 proteins Bcl-2 and Bcl-x(L) contribute to diabetic Ca(2+) dysregulation and vessel contractility in vascular smooth muscle cells (VSMCs) through their interaction with inositol 1,4,5-trisphosphate receptor (InsP(3)R) intracellular Ca(2+) release channels. Measurements of intracellular ([Ca(2+)](i)) and sarcoplasmic reticulum ([Ca(2+)](SR)) calcium concentrations were made in primary cells isolated from diabetic (db/db) and nondiabetic (db/m) mice. In addition, [Ca(2+)](i) and constriction were recorded simultaneously in isolated intact arteries. Protein expression levels of Bcl-x(L) but not Bcl-2 were elevated in VSMCs isolated from db/db compared with db/m age-matched controls. In single cells, InsP(3)-evoked [Ca(2+)](i) signaling was enhanced in VSMCs from db/db mice compared with db/m. This was attributed to alterations in the intrinsic properties of the InsP(3)R itself because there were no differences between db/db and db/m in the steady-state [Ca(2+)](SR) or InsP(3)R expression levels. Moreover, in permeabilized cells the rate of InsP(3)R-dependent SR Ca(2+) release was increased in db/db compared with db/m VSMCs. The enhanced InsP(3)-dependent SR Ca(2+) release was attenuated by the Bcl-2 protein inhibitor ABT-737 only in diabetic cells. Application of ABT-737 similarly attenuated enhanced agonist-induced [Ca(2+)](i) signaling only in intact aortic and mesenteric db/db vessels. In contrast, ABT-737 had no effect on agonist-evoked contractility in either db/db or db/m vessels. Taken together, the data suggest that in type 2 diabetes the mechanism for [Ca(2+)](i) dysregulation in VSMCs involves Bcl-2 protein-dependent increases in InsP(3)R excitability and that dysregulated [Ca(2+)](i) signaling does not appear to contribute to increased vessel reactivity.     

Gamma-secretase activity of presenilin 1 regulates acetylcholine muscarinic receptor-mediated signal transduction

Familial Alzheimer's disease (FAD) presenilin 1 (PS1) mutations give enhanced calcium responses upon different stimuli, attenuated capacitative calcium entry, an increased sensitivity of cells to undergo apoptosis, and increased gamma-secretase activity. We previously showed that the FAD mutation causing an exon 9 deletion in PS1 results in enhanced basal phospholipase C (PLC) activity (Cedazo-Minguez, A., Popescu, B. O., Ankarcrona, M., Nishimura, T., and Cowburn, R. F. (2002) J. Biol. Chem. 277, 36646-36655). To further elucidate the mechanisms by which PS1 interferes with PLC-calcium signaling, we studied the effect of two other FAD PS1 mutants (M146V and L250S) and two dominant negative PS1 mutants (D257A and D385N) on basal and carbachol-stimulated phosphoinositide (PI) hydrolysis and intracellular calcium concentrations ([Ca2+]i) in SH-SY5Y neuroblastoma cells. We found a significant increase in basal PI hydrolysis in PS1 M146V cells but not in PS1 L250S cells. Both PS1 M146V and PS1 L250S cells showed a significant increase in carbachol-induced [Ca2+]i as compared with nontransfected or wild type PS1 transfected cells. The elevated carbachol-induced [Ca2+]i signals were reversed by the PLC inhibitor neomycin, the ryanodine receptor antagonist dantrolene, the general aspartyl protease inhibitor pepstatin A, and the specific gamma-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester. The cells expressing either PS1 D257A or PS1 D385N had attenuated carbachol-stimulated PI hydrolysis and [Ca2+]i responses. In nontransfected or PS1 wild type transfected cells, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester and pepstatin A also attenuated both carbachol-stimulated PI hydrolysis and [Ca2+]i responses to levels found in PS1 D257A or PS1 D385N dominant negative cells. Our findings suggest that PS1 can regulate PLC activity and that this function is gamma-secretase activity-dependent.     

Functional consequences of phosphomimetic mutations at key cAMP-dependent protein kinase phosphorylation sites in the type 1 inositol 1,4,5-trisphosphate receptor

Regulation of Ca(2+) release through inositol 1,4,5-trisphosphate receptors (InsP(3)R) has important consequences for defining the particular spatio-temporal properties of intracellular Ca(2+) signals. In this study, regulation of Ca(2+) release by phosphorylation of type 1 InsP(3)R (InsP(3)R-1) was investigated by constructing "phosphomimetic" charge mutations in the functionally important phosphorylation sites of both the S2+ and S2- InsP(3)R-1 splice variants. Ca(2+) release was investigated following expression in Dt-40 3ko cells devoid of endogenous InsP(3)R. In cells expressing either the S1755E S2+ or S1589E/S1755E S2- InsP(3)R-1, InsP(3)-induced Ca(2+) release was markedly enhanced compared with nonphosphorylatable S2+ S1755A and S2- S1589A/S1755A mutants. Ca(2+) release through the S2- S1589E/S1755E InsP(3)R-1 was enhanced approximately 8-fold over wild type and approximately 50-fold when compared with the nonphosphorylatable S2- S1589A/S1755A mutant. In cells expressing S2- InsP(3)R-1 with single mutations in either S1589E or S1755E, the sensitivity of Ca(2+) release was enhanced approximately 3-fold; sensitivity was midway between the wild type and the double glutamate mutation. Paradoxically, forskolin treatment of cells expressing either single Ser/Glu mutation failed to further enhance Ca(2+) release. The sensitivity of Ca(2+) release in cells expressing S2+ S1755E InsP(3)R-1 was comparable with the sensitivity of S2- S1589E/S1755E InsP(3)R-1. In contrast, mutation of S2+ S1589E InsP(3)R-1 resulted in a receptor with comparable sensitivity to wild type cells. Expression of S2- S1589E/S1755E InsP(3)R-1 resulted in robust Ca(2+) oscillations when cells were stimulated with concentrations of alpha-IgM antibody that were threshold for stimulation in S2- wild type InsP(3)R-1-expressing cells. However, at higher concentrations of alpha-IgM antibody, Ca(2+) oscillations of a similar period and magnitude were initiated in cells expressing either wild type or S2- phosphomimetic mutations. Thus, regulation by phosphorylation of the functional sensitivity of InsP(3)R-1 appears to define the threshold at which oscillations are initiated but not the frequency or amplitude of the signal when established.     

Huntingtin and huntingtin-associated protein 1 influence neuronal calcium signaling mediated by inositol-(1,4,5) triphosphate receptor type 1

Huntington's disease (HD) is caused by polyglutamine expansion (exp) in huntingtin (Htt). The type 1 inositol (1,4,5)-triphosphate receptor (InsP3R1) is an intracellular calcium (Ca2+) release channel that plays an important role in neuronal function. In a yeast two-hybrid screen with the InsP3R1 carboxy terminus, we isolated Htt-associated protein-1A (HAP1A). We show that an InsP3R1-HAP1A-Htt ternary complex is formed in vitro and in vivo. In planar lipid bilayer reconstitution experiments, InsP3R1 activation by InsP3 is sensitized by Httexp, but not by normal Htt. Transfection of full-length Httexp or caspase-resistant Httexp, but not normal Htt, into medium spiny striatal neurons faciliates Ca2+ release in response to threshold concentrations of the selective mGluR1/5 agonist 3,5-DHPG. Our findings identify a novel molecular link between Htt and InsP3R1-mediated neuronal Ca2+ signaling and provide an explanation for the derangement of cytosolic Ca2+ signaling in HD patients and mouse models.     

Phosphorylation of type-1 inositol 1,4,5-trisphosphate receptors by cyclic nucleotide-dependent protein kinases: a mutational analysis of the functionally important sites in the S2+ and S2- splice variants

Inositol 1,4,5-trisphosphate receptors (InsP3R) are the major route of intracellular calcium release in eukaryotic cells and as such are pivotal for stimulation of Ca2+-dependent effectors important for numerous physiological processes. Modulation of this release has important consequences for defining the particular spatio-temporal characteristics of Ca2+ signals. In this study, regulation of Ca2+ release by phosphorylation of type-1 InsP3R (InsP3R-1) by cAMP (PKA)- and cGMP (PKG)-dependent protein kinases was investigated in the two major splice variants of InsP3R-1. InsP3R-1 was expressed in DT-40 cells devoid of endogenous InsP3R. In cells expressing the neuronal, S2+ splice variant of the InsP3R-1, Ca2+ release was markedly enhanced when either PKA or PKG was activated. The sites of phosphorylation were investigated by mutation of serine residues present in two canonical phosphorylation sites present in the protein. Potentiated Ca2+ release was abolished when serine 1755 was mutated to alanine (S1755A) but was unaffected by a similar mutation of serine 1589 (S1589A). These data demonstrate that Ser-1755 is the functionally important residue for phosphoregulation by PKA and PKG in the neuronal variant of the InsP3R-1. Activation of PKA also resulted in potentiated Ca2+ release in cells expressing the non-neuronal, S2- splice variant of the InsP3R-1. However, the PKA-induced potentiation was still evident in S1589A or S1755A InsP3R-1 mutants. The effect was abolished in the double (S1589A/S1755A) mutant, indicating both sites are phosphorylated and contribute to the functional effect. Activation of PKG had no effect on Ca2+ release in cells expressing the S2- variant of InsP3R-1. Collectively, these data indicate that phosphoregulation of InsP3R-1 has dramatic effects on Ca2+ release and defines the molecular sites phosphorylated in the major variants expressed in neuronal and peripheral tissues.     

Regulation by Ca2+ and inositol 1,4,5-trisphosphate (InsP3) of single recombinant type 3 InsP3 receptor channels. Ca2+ activation uniquely distinguishes types 1 and 3 insp3 receptors

The inositol 1,4,5-trisphosphate (InsP(3)) receptor (InsP3R) is an endoplasmic reticulum-localized Ca2+ -release channel that controls complex cytoplasmic Ca(2+) signaling in many cell types. At least three InsP3Rs encoded by different genes have been identified in mammalian cells, with different primary sequences, subcellular locations, variable ratios of expression, and heteromultimer formation. To examine regulation of channel gating of the type 3 isoform, recombinant rat type 3 InsP3R (r-InsP3R-3) was expressed in Xenopus oocytes, and single-channel recordings were obtained by patch-clamp electrophysiology of the outer nuclear membrane. Gating of the r-InsP3R-3 exhibited a biphasic dependence on cytoplasmic free Ca2+ concentration ([Ca2+]i). In the presence of 0.5 mM cytoplasmic free ATP, r-InsP3R-3 gating was inhibited by high [Ca2+]i with features similar to those of the endogenous Xenopus type 1 Ins3R (X-InsP3R-1). Ca2+ inhibition of channel gating had an inhibitory Hill coefficient of approximately 3 and half-maximal inhibiting [Ca2+]i (Kinh) = 39 microM under saturating (10 microM) cytoplasmic InsP3 concentrations ([InsP3]). At [InsP3] < 100 nM, the r-InsP3R-3 became more sensitive to Ca2+ inhibition, with the InsP(3) concentration dependence of Kinh described by a half-maximal [InsP3] of 55 nM and a Hill coefficient of approximately 4. InsP(3) activated the type 3 channel by tuning the efficacy of Ca2+ to inhibit it, by a mechanism similar to that observed for the type 1 isoform. In contrast, the r-InsP3R-3 channel was uniquely distinguished from the X-InsP3R-1 channel by its enhanced Ca2+ sensitivity of activation (half-maximal activating [Ca2+]i of 77 nM instead of 190 nM) and lack of cooperativity between Ca2+ activation sites (activating Hill coefficient of 1 instead of 2). These differences endow the InsP3R-3 with high gain InsP3-induced Ca2+ release and low gain Ca2+ -induced Ca2+ release properties complementary to those of InsP3R-1. Thus, distinct Ca2+ signals may be conferred by complementary Ca2+ activation properties of different InsP3R isoforms.     

The inositol 1,4,5-trisphosphate receptors

The inositol (1,4,5)-trisphosphate receptors (InsP3R) are the intracellular calcium (Ca2+) release channels that play a key role in Ca2+ signaling in cells. Three InsP3R isoforms-InsP3R type 1 (InsP3R1), InsP3R type 2 (InsP3R2), and InsP3R type 3 (InsP3R3) are expressed in mammals. A single InsP3R isoform is expressed in Drosophila melanogaster (DmInsP3R) and Caenorhabditis elegans (CeInsP3R). The progress made during last decade towards understanding the function and the properties of the InsP3R is briefly reviewed in this chapter. The main emphasis is on studies that revealed structural determinants responsible for the ligand recognition by the InsP3R, ion permeability of the InsP3R, modulation of the InsP3R by cytosolic Ca2+, ATP and PKA phosphorylation and on the recently identified InsP3R-binding partners. The main focus is on the InsP3R1, but the recent information about properties of other InsP3R isoforms is also discussed.     

Elevated testosterone induces apoptosis in neuronal cells

Testosterone plays a crucial role in neuronal function, but elevated concentrations can have deleterious effects. Here we show that supraphysiological levels of testosterone (micromolar range) initiate the apoptotic cascade. We used three criteria, annexin V labeling, caspase activity, and DNA fragmentation, to determine that apoptotic pathways were activated by testosterone. Micromolar, but not nanomolar, testosterone concentrations increased the response in all three assays of apoptosis. In addition, testosterone induced different concentration-dependent Ca2+ signaling patterns: at low concentrations of testosterone (100 nm), Ca2+ oscillations were produced, whereas high concentrations (1-10 microm) induced a sustained Ca2+ increase. Elevated testosterone concentrations increase cell death, and this effect was abolished in the presence of either inhibitors of caspases or the inositol 1,4,5-trisphosphate receptor (InsP3R)-mediated Ca2+ release. Knockdown of InsP3R type 1 with specific small interfering RNA also abolished the testosterone-induced cell death and the prolonged Ca2+ signals. In contrast, knockdown of InsP3R type 3 modified neither the apoptotic response nor the Ca2+ signals. These results support our hypothesis that elevated testosterone alters InsP3R type 1-mediated intracellular Ca2+ signaling and that the prolonged Ca2+ signals lead to apoptotic cell death. These effects of testosterone on neurons will have long term effects on brain function.     

Cell signaling microdomain with Na,K-ATPase and inositol 1,4,5-trisphosphate receptor generates calcium oscillations

Recent studies indicate novel roles for the ubiquitous ion pump, Na,K-ATPase, in addition to its function as a key regulator of intracellular sodium and potassium concentration. We have previously demonstrated that ouabain, the endogenous ligand of Na,K-ATPase, can trigger intracellular Ca2+ oscillations, a versatile intracellular signal controlling a diverse range of cellular processes. Here we report that Na,K-ATPase and inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) form a cell signaling microdomain that, in the presence of ouabain, generates slow Ca2+ oscillations in renal cells. Using fluorescent resonance energy transfer (FRET) measurements, we detected a close spatial proximity between Na,K-ATPase and InsP3R. Ouabain significantly enhanced FRET between Na,K-ATPase and InsP3R. The FRET effect and ouabain-induced Ca2+ oscillations were not observed following disruption of the actin cytoskeleton. Partial truncation of the NH2 terminus of Na,K-ATPase catalytic alpha1-subunit abolished Ca2+ oscillations and downstream activation of NF-kappaB. Ouabain-induced Ca2+ oscillations occurred in cells expressing an InsP3 sponge and were hence independent of InsP3 generation. Thus, we present a novel principle for a cell signaling microdomain where an ion pump serves as a receptor.     

The endoplasmic reticulum gateway to apoptosis by Bcl-X(L) modulation of the InsP3R

Members of the Bcl-2 protein family modulate outer mitochondrial membrane permeability to control apoptosis. However, these proteins also localize to the endoplasmic reticulum (ER), the functional significance of which is controversial. Here we provide evidence that anti-apoptotic Bcl-2 proteins regulate the inositol 1,4,5-trisphosphate receptor (InsP(3)R) ER Ca(2+) release channel resulting in increased cellular apoptotic resistance and enhanced mitochondrial bioenergetics. Anti-apoptotic Bcl-X(L) interacts with the carboxyl terminus of the InsP(3)R and sensitizes single InsP(3)R channels in ER membranes to low [InsP(3)], enhancing Ca(2+) and InsP(3)-dependent regulation of channel activity in vitro and in vivo, reducing ER Ca(2+) content and stimulating mitochondrial energetics. The pro-apoptotic proteins Bax and tBid antagonize this effect by blocking the biochemical interaction of Bcl-X(L) with the InsP(3)R. These data support a novel model in which Bcl-X(L) is a direct effector of the InsP(3)R, increasing its sensitivity to InsP(3) and enabling ER Ca(2+) release to be more sensitively coupled to extracellular signals. As a consequence, cells are protected against apoptosis by a more sensitive and dynamic coupling of ER to mitochondria through Ca(2+)-dependent signal transduction that enhances cellular bioenergetics and preserves survival.     

Inositol 1,4,5-trisphosphate and its co-players in the concert of Ca2+ signalling--new faces in the line up

Release of Ca2+ from intracellular stores can occur by different intracellular messengers such as InsP3, cADPR and NAADP. Although in some cells messengers may operate on different stores, there are also Ca2+ stores with sensitivities for all three of these messengers. It is well documented, that InsP3- and cADPR-sensitive Ca2+ stores are involved in the activation of "store-operated Ca2+ channels" (SOCC). It has not yet been unequivocally shown, however, if Ca2+ release from stores, which respond to NAADP but not to InsP3 or cADPR, also generate signals which lead to "store-operated Ca2+ entry". Neither localization nor the mechanism of coupling to the plasma membrane of those InsP3- and cADPR-sensitive Ca2+ stores which activate SOCCs is yet clear. In this review localization and properties of InsP3-, cADPR- and NAADP-sensitive Ca2+ pools and their mutual interactions are discussed. Differential sensitivities of Ca2+ release mechanisms to InsP3, cADPR and NAADP have consequences on Ca2+ release, Ca2+ oscillations, propagation of Ca2+ waves and on activation of SOCC. Possible interaction of InsP3R and cADPR with candidates of SOCCs (TRP channels) and mechanisms involved in the regulation of SOCCs (activation-deactivation) will be elaborated.     

ATP regulation of recombinant type 3 inositol 1,4,5-trisphosphate receptor gating

A family of inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) Ca2+ release channels plays a central role in Ca2+ signaling in most cells, but functional correlates of isoform diversity are unclear. Patch-clamp electrophysiology of endogenous type 1 (X-InsP3R-1) and recombinant rat type 3 InsP3R (r-InsP3R-3) channels in the outer membrane of isolated Xenopus oocyte nuclei indicated that enhanced affinity and reduced cooperativity of Ca2+ activation sites of the InsP3-liganded type 3 channel distinguished the two isoforms. Because Ca2+ activation of type 1 channel was the target of regulation by cytoplasmic ATP free acid concentration ([ATP](i)), here we studied the effects of [ATP]i on the dependence of r-InsP(3)R-3 gating on cytoplasmic free Ca2+ concentration ([Ca2+]i. As [ATP]i was increased from 0 to 0.5 mM, maximum r-InsP3R-3 channel open probability (Po) remained unchanged, whereas the half-maximal activating [Ca2+]i and activation Hill coefficient both decreased continuously, from 800 to 77 nM and from 1.6 to 1, respectively, and the half-maximal inhibitory [Ca2+]i was reduced from 115 to 39 microM. These effects were largely due to effects of ATP on the mean closed channel duration. Whereas the r-InsP3R-3 had a substantially higher Po than X-InsP3R-1 in activating [Ca2+]i (< 1 microM) and 0.5 mM ATP, the Ca2+ dependencies of channel gating of the two isoforms became remarkably similar in the absence of ATP. Our results suggest that ATP binding is responsible for conferring distinct gating properties on the two InsP3R channel isoforms. Possible molecular models to account for the distinct regulation by ATP of the Ca2+ activation properties of the two channel isoforms and the physiological implications of these results are discussed. Complex regulation by ATP of the types 1 and 3 InsP3R channel activities may enable cells to generate sophisticated patterns of Ca2+ signals with cytoplasmic ATP as one of the second messengers.     

The InsP3 receptor: its role in neuronal physiology and neurodegeneration

The InsP3 receptor is a ligand-gated channel that releases Ca2+ from intracellular stores in a variety of cell types, including neurons. Genetic studies from vertebrate and invertebrate model systems suggest that coordinated rhythmic motor functions are most susceptible to changes in Ca2+ release from the InsP3 receptor. In many cases, the InsP3 receptor interacts with other signaling mechanisms that control levels of cytosolic Ca2+, suggesting that the maintenance of Ca2+ homeostasis in normal cells could be controlled by the activity of the InsP3R. In support of this idea, recent studies show that altered InsP3 receptor activity can be partially responsible for Ca2+ dyshomeostasis seen in many neurodegenerative conditions. These observations open new avenues for carrying out genetic and drug screens that target InsP3R function in neurodegenerative conditions.     

Regulatory and spatial aspects of inositol trisphosphate-mediated calcium signals

Hormones that act to release Ca2+ from intracellular stores initiate a signaling cascade that culminates in the production of inositol 1,4,5-trisphosphate (InsP3). The Ca2+ response mediated by InsP3 is not a sustained increase in the cytosolic Ca2+ concentration, but rather a series of periodic spikes that manifest as waves in larger cells. In vitro studies have determined that the key positive feedback parameter driving spikes and waves is a highly localized direct Ca(2+)-activation of InsP3-gated Ca2+ channels. Advances in fluorescent Ca2+ imaging have facilitated the resolution of individual positive feedback units. These studies have revealed that there are several modes of channel coupling underlying global Ca2+ signals; single channel openings or Ca2+ "blips," synchronized clusters of channels or Ca2+ "puffs," and cell wide calcium waves. It appears that the channel clusters that produce Ca2+ puffs are synchronized by the highly localized positive feedback that was predicted by the in vitro studies of channel regulation. Localization of InsP3-induced Ca2+ signals has been shown to be important for activation of several cellular processes including uni-directional salt flow and mitochondrial activation.     

The store-operated calcium entry pathways in human carcinoma A431 cells: functional properties and activation mechanisms

Activation of phospholipase C (PLC)-mediated signaling pathways in nonexcitable cells causes the release of Ca2+ from intracellular Ca2+ stores and activation of Ca2+ influx across the plasma membrane. Two types of Ca2+ channels, highly Ca2+-selective ICRAC and moderately Ca2+-selective ISOC, support store-operated Ca2+ entry process. In previous patch-clamp experiments with a human carcinoma A431 cell line we described store-operated Imin/ICRACL plasma membrane Ca2+ influx channels. In the present paper we use whole-cell and single-channel recordings to further characterize store-operated Ca2+ influx pathways in A431 cells. We discovered that (a) ICRAC and ISOC are present in A431 cells; (b) ICRAC currents are highly selective for divalent cations and fully activate within 150 s after initiation of Ca2+ store depletion; (c) ISOC currents are moderately selective for divalent cations (PBa/PCs = 14.5) and require at least 300 s for full activation; (d) ICRAC and ISOC currents are activated by PLC-coupled receptor agonists; (e) ISOC currents are supported by Imin/ICRACL channels that display 8.5-10 pS conductance for sodium; (f) ICRAC single channel conductance for sodium is estimated at 0.9 pS by the noise analysis; (g) Imin/ICRACL channels are activated in excised patches by an amino-terminal fragment of InsP3R1 (InsP3R1N); and (h) InsP3 binding to InsP3R1N is necessary for activation of Imin/ICRACL channels. Our findings provide novel information about store-operated Ca2+ influx pathways in A431 cells.