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Intimate Plasma Membrane–ER Interactions Underlie Capacitative Calcium Entry: “Kissin' Cousins”

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... However, SOC is also activated by store depletion in the absence of any increase in IP 3 . The nature of the signal which links the internal Ca 2ϩ store to the plasma membrane, and the molecule that mediates Ca 2ϩ influx across the plasma membrane, is not yet known in any non-excitable cell type (1,2). ...
... We have also detected INAD-like transcripts in HSG cells. 2 Further studies will be required to determine whether an INAD-like protein is also involved in the localization and regulation of the signaling complex associated with mammalian Trp protein(s). ...
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Trp1 has been proposed as a component of the store-operated Ca(2+) entry (SOC) channel. However, neither the molecular mechanism of SOC nor the role of Trp in this process is yet understood. We have examined possible molecular interactions involved in the regulation of SOC and Trp1 and report here for the first time that Trp1 is assembled in signaling complex associated with caveolin-scaffolding lipid raft domains. Endogenous hTrp1 and caveolin-1 were present in low density fractions of Triton X-100-extracted human submandibular gland cell membranes. Depletion of plasma membrane cholesterol increased Triton X-100 solubility of Trp1 and inhibited carbachol-stimulated Ca(2+) signaling. Importantly, thapsigargin stimulated Ca(2+) influx, but not internal Ca(2+) release, and inositol 1,4,5-triphosphate (IP(3))-stimulated I(soc) were also attenuated. Furthermore, both anti-Trp1 and anti-caveolin-1 antibodies co-immunoprecipitated hTrp1, caveolin-1, Galpha(q/11), and IP(3) receptor-type 3 (IP(3)R3). These results demonstrate that caveolar microdomains provide a scaffold for (i) assembly of key Ca(2+) signaling proteins into a complex and (ii) coordination of the molecular interactions leading to the activation of SOC. Importantly, we have shown that Trp1 is also localized in this microdomain where it interacts with one or more components of this complex, including IP(3)R3. This finding is potentially important in elucidating the physiological function of Trp.
... ER Ca 2ϩ store depletion by IP 3 , according to the capacitative model of Ca 2ϩ homeostasis, opens plasma membrane Ca 2ϩ channels, inducing Ca 2ϩ influx into the cell (165). The subsequent refilling of ER Ca 2ϩ stores terminates this Ca 2ϩ influx, even in the continued presence of agonists. ...
... The subsequent refilling of ER Ca 2ϩ stores terminates this Ca 2ϩ influx, even in the continued presence of agonists. Storeoperated Ca 2ϩ channels (SOC) are responsible for this Ca 2ϩ entry (165). In mouse TBC, LCFA-mediated IP 3 release leads to the opening of SOC channels (47). ...
Article
An attraction for palatable foods rich in lipids is shared by rodents and humans. Over the last decade, the mechanisms responsible for this specific eating behavior have been actively studied, and compelling evidence implicates a taste component in the orosensory detection of dietary lipids [i.e., long-chain fatty acids (LCFA)], in addition to textural, olfactory, and postingestive cues. The interactions between LCFA and specific receptors in taste bud cells (TBC) elicit physiological changes that affect both food intake and digestive functions. After a short overview of the gustatory pathway, this review brings together the key findings consistent with the existence of a sixth taste modality devoted to the perception of lipids. The main steps leading to this new paradigm (i.e., chemoreception of LCFA in TBC, cell signaling cascade, transfer of lipid signals throughout the gustatory nervous pathway, and their physiological consequences) will be critically analyzed. The limitations to this concept will also be discussed in the light of our current knowledge of the sense of taste. Finally, we will analyze the recent literature on obesity-related dysfunctions in the orosensory detection of lipids (“fatty” taste?), in relation to the overconsumption of fat-rich foods and the associated health risks.
... Calreticulin also affects the function of SERCA2b and the InsP 3 R (Camacho andLechleiter 1995, Jouaville et al. 1999), both of which may be structurally coupled to SOCs (Lockwich et al. 2001). The structural relationship between the ER/SR calcium release channels and SOCs of the plasma membrane has been a matter of controversy, however, substantial evidence points to structural coupling between the two channels (Putney 1999(Putney , 2005. In view of the heterogeneity of the ER/SR (Meldolesi andPozzan 1998, Petersen et al. 2001), it is intuitive that not all of the ER is coupled to the plasma membrane, however morphological data supporting this notion are scarce. ...
... Calreticulin also affects the function of SERCA2b and the InsP 3 R (Camacho andLechleiter 1995, Jouaville et al. 1999), both of which may be structurally coupled to SOCs (Lockwich et al. 2001). The structural relationship between the ER/SR calcium release channels and SOCs of the plasma membrane has been a matter of controversy, however, substantial evidence points to structural coupling between the two channels (Putney 1999(Putney , 2005. In view of the heterogeneity of the ER/SR (Meldolesi andPozzan 1998, Petersen et al. 2001), it is intuitive that not all of the ER is coupled to the plasma membrane, however morphological data supporting this notion are scarce. ...
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Calreticulin is a Ca2+-binding protein of the ER/SR, from where it acts as a chaperone, and affectscalcium homeostasis, gene expression and cell adhesion. Cell adhesion to the extracellular matrix cangenerate transmembrane signals important for cell survival and migration. In a variety of cell types,integrin stimulation by ECM proteins, such as fibronectin, leads to changes in intracellular proteintyrosine phosphorylation. Tyrosine phosphorylation leads to the co-localization of focal adhesion kinase,vinculin and paxillin at focal contacts. Interaction between focal adhesion kinase and paxillin is criticalfor the activation of signaling cascades involved in cell survival and motility. Fibroblasts either over- orunderexpressing calreticulin show differences in their adhesive properties, which are related to thecalmodulin/CaMKII pathway. Inhibition of these pathways causes the weekly adhesive calreticulinunderexpressing cells to behave like the calreticulin overexpressers, through increased spreading andincreased levels of focal adhesion kinase, paxillin and fibronectin. We propose that calreticulin, via itsCa2+-homeostatic effects, may affect fibronectin synthesis and matrix assembly by modulating fibronectingene expression, and by influencing formation of cellular adhesions, both of which are instrumental inmatrix assembly and remodelling. Interestingly, it appears that besides the calmodulin/CaMKII pathway,differential calreticulin expression also modulates the c-src pathway.
... second messengers such as cGMP or inositol 1,4,5-trisphosphate (IP 3 ) or mediators such as the calcium influx factor, which are either generated with, or in response to, the release of Ca 2ϩ from the internal Ca 2ϩ store; (ii) recruitment of channels into the plasma membrane by a process involving vesicle fusion; (iii) a physical interaction between the SOC channel in the plasma membrane and the IP 3 receptor (IP 3 R) in the internal Ca 2ϩ store membrane, i.e. the conformational-coupling hypothesis (1,2,5,6). A major hurdle in establishing the mechanism of activation for SOCE has been the lack of information regarding the identity of the SOCE channel protein(s). ...
... Recently, mammalian homologues of the Drosophila trp gene have been suggested to encode the SOCE channel protein (2,3,7). Seven different trps have been cloned. ...
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Transient receptor potential protein 1 (Trp1) has been proposed as a component of the store-operated Ca(2+) entry (SOCE) channel. However, the exact mechanism by which Trp1 is regulated by store depletion is not known. Here, we examined the role of the Trp1 C-terminal domain in SOCE by expressing hTrp1alpha lacking amino acids 664-793 (DeltaTrp1alpha) or full-length hTrp1alpha in the HSG (human submandibular gland) cell line. Both carbachol (CCh) and thapsigargin (Tg) activated sustained Ca(2+) influx in control (nontransfected), DeltaTrp1alpha-, and Trp1alpha-expressing cells. Sustained [Ca(2+)](i), following stimulation with either Tg or CCh in DeltaTrp1alpha-expressing cells, was about 1.5-2-fold higher than in Trp1alpha-expressing cells and 4-fold higher than in control cells. Importantly, (i) basal Ca(2+) influx and (ii) Tg- or CCh-stimulated internal Ca(2+) release were similar in all the cells. A similar increase in Tg-stimulated Ca(2+) influx was seen in cells expressing Delta2Trp1alpha, lacking the C-terminal domain amino acid 649-793, which includes the EWKFAR sequence. Further, both inositol 1,4,5-trisphosphate receptor-3 and caveolin-1 were immunoprecipitated with DeltaTrp1alpha and Trp1alpha. In aggregate, these data suggest that (i) the EWKFAR sequence does not contribute significantly to the Trp1-associated increase in SOCE, and (ii) the Trp1 C-terminal region, amino acids 664-793, is involved in the modulation of SOCE.
... Although the pathway between agonist-receptor binding and the opening of Ca 2+ channels in the ER is largely understood [16], the signaling events that lead to SOC activation are under active investigation. Putney, who originally proposed that store depletion caused Ca 2+ influx [8], has recently reviewed the different models of SOC activation [17]. A vesicle-mediated model of SOC ...
Article
Direct microinjection of the clostridial neurotoxins botulinum neurotoxin A light chain or tetanus neurotoxin into cells of a human embryonic kidney cell line significantly reduced calcium entry after depletion of internal calcium stores by cyclopiazonic acid, a reversible inhibitor of the sarcoplasmic-endoplasmic reticular calcium-ATPases. Botulinum neurotoxin A light chain specifically hydrolyzes a synaptosomal-associated protein of 25 kilodaltons (SNAP-25), and tetanus neurotoxin specifically hydrolyzes synaptobrevin-2 (vesicle-associated membrane protein 2, VAMP-2) and cellubrevin (vesicle-associated membrane protein 3, VAMP-3). Since these substrate proteins are required for vesicle docking and fusion, inhibition of store-operated calcium entry by botulinum neurotoxin A light chain and tetanus neurotoxin supports a model in which vesicle fusion is a prerequisite for activation of store-operated calcium entry. Brefeldin A, a fungal metabolite that interferes with vesicle traffic, partially reduced calcium entry following store depletion. The size of the reserve pool of vesicles or parallel vesicle recycling pathways employing brefeldin A-sensitive and brefeldin A-insensitive ADP-ribosylation factors may explain the failure of brefeldin A to completely inhibit store-operated calcium entry.
... This proposal has been supported by the observation that disrupting physically the interaction between the reticulum and the cell membrane impairs the transduction of calcium signal elicited by potassium to the mitochondria, and therefore steroidogenesis in H295R cells (154). It is noteworthy that this organization of calcium influx, involving the local transfer of calcium entering the cell directly into the endoplasmic reticulum, is somehow reminiscent of the early "capacitative" model proposed by Putney (155), before revisiting it for taking into account results obtained with thapsigargin (156,157), and it is quite intriguing that the α1H isoform of T channels, which is the most abundant in glomerulosa cells, has been proposed to mediate a capacitative calcium entry in non-excitable cancer cells (158). ...
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Intracellular calcium plays a crucial role in modulating a variety of functions such as muscle contraction, hormone secretion, gene expression or cell growth. Calcium signaling has been however shown to be more complex than initially thought. Indeed, it is confined within cell microdomains and different calcium channels are associated with different functions, as shown by various channelopathies. Sporadic mutations on voltage-operated L-type calcium channels in adrenal glomerulosa cells have been shown recently to be the second most prevalent genetic abnormalities present in human aldosterone-producing adenoma. The observed modification of the threshold of activation of the mutated channels not only provides an explanation for this gain of function but reminds us on the importance of maintaining adequate electrophysiological characteristics to make channels able to exert specific cellular functions. Indeed, the contribution to steroid production of the various calcium channels expressed in adrenocortical cells is not equal and the reason has been investigated for a long time. Given the very negative resting potential of these cells, and the small membrane depolarization induced by their physiological agonists, low threshold T-type calcium channels are particularly well suited for responding under these conditions and conveying calcium into the cell, at the right place for controlling steroidogenesis. In contrast, high threshold L-type channels are normally activated by much stronger cell depolarizations. The fact that dihydropyridine calcium antagonists, specific for L-type channels, are poorly efficient for reducing aldosterone secretion either in vivo or in vitro, strongly supports the view that these two types of channels differently affect steroid biosynthesis. Whether a similar analysis is transposable to fasciculata cells and cortisol secretion is one of the questions addressed in the present review. No similar mutations on L-type or T-type channels have been described yet to affect cortisol secretion or to be linked to the development of Cushing syndrome, but several evidences suggest that the function of T channels is also crucial in fasciculata cells. Putative molecular mechanisms and cellular structural organization making T channels a privileged entry for the “steroidogenic calcium” are also discussed.
... Although it has been widely accepted that the key event initiating the opening of storeoperated Ca 2ϩ channels (SOCs) in the plasma membrane is the decrease in the concentration of Ca 2ϩ in the lumen of the ER, neither the mechanism that couples these two events nor the structures of SOCs are well understood (2). The results of recent experiments indicate that an essential prerequisite for the activation of SOCs is the close association between regions of the ER and the plasma membrane (3). It is proposed that this association is maintained by cytoskeletal elements such as the F-actin (4). ...
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The roles of the heterotrimeric G-protein, G(i2), in regulating the actin cytoskeleton and the activation of store-operated Ca(2+) channels in rat hepatocytes were investigated. Galpha(i2) was principally associated with the plasma membrane and microsomes. Both F-actin and Galpha(i2) were detected by Western blot analysis in a purified plasma membrane preparation, the supernatant and pellet obtained by treating the plasma membrane with Triton X-100, and after depolymerization and repolymerization of F-actin in the Triton X-100-insoluble pellet. Actin in the Triton X-100-soluble supernatant co-precipitated with Galpha(i2) using either anti-Galpha(i2) or anti-actin antibodies. The principally cortical location of F-actin in hepatocytes cultured for 0.5 h changed to a pericanalicular distribution over a further 3.5 h. Some Galpha(i2) co-localized with F-actin at the plasma membrane. Pretreatment with pertussis toxin ADP-ribosylated 70-80% of Galpha(i2) in the plasma membrane and microsomes, prevented the redistribution of F-actin, caused redistribution and fragmentation of the endoplasmic reticulum, and inhibited vasopressin-stimulated Ca(2+) inflow. It is concluded that (i) a significant portion of hepatocyte Galpha(i2) associates with, and regulates the arrangement of, cortical F-actin and the endoplasmic reticulum and (ii) either or both of these regulatory roles are likely to be required for normal vasopressin activation of Ca(2+) inflow.
... The store-operated Ca 2+ influx pathway is coupled to the Ca 2+ content of the store such that the empty store transmits a signal to the plasma membrane to open Ca 2+ channels (Putney, 1999). The best characterized store-operated Ca 2+ current is the Ca 2+ -release-activated Ca 2+ current (I CRAC ) (Hoth and Penner, 1992). ...
... Two major general hypotheses for the mechanism of CCE have been formulated. The first postulates that the endoplasmic reticulum, as it becomes depleted of Ca 2+ , liberates a factor that induces the opening of the CCE channel, the second proposes some form of interaction of endoplasmic reticulum proteins with the plasma membrane CCE Ca 2+ channels (Putney, 1999). In more detail, two main mechanisms are being discussed: (1) a secretion process in which store depletion would trigger the fusion of membrane units, originating from the ER and containing the CCE channels, with the plasma membrane (Patterson et al., 1999;Yao et al., 1999); ...
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Referee: Guiseppe Inesi, M.D., Ph.D., Professor and Chairman, School of Medicine, Dept. of Biochemistry and Molecular Biology, Univeristy of Maryland, Baltimore.
... Despite these data, the activation mechanisms of SOCE are still unclear. However, three main hypothesis are proposed to explain SOC channels activity regulation: one related to a diffusible messenger termed CIF (Calcium Influx Factor) [44]), the second involves a conformational coupling between the plasma mem- brane [45] and the ER and the last one supporting an exocytosisrelated translocation of SOC channels [24,25,27,28,29]. It is known that the two last mechanisms involve cytoskeleton rearrangement [46] as cytoskeleton has been shown to modulate SOCE [47,48]. ...
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Neuroendocrine differentiation (NED) is a hallmark of advanced androgen-independent prostate cancer, for which no successful therapy exists. NED tumour cells escape apoptotic cell death by alterations of Ca(2+) homeostasis where the store-operated Ca(2+) entry (SOCE) is known to be a key event. We have previously shown that the downregulation of Orai1 protein representing the major molecular component of endogenous SOCE in human prostate cancer cells, and constituting the principal source of Ca(2+) influx used by the cell to trigger apoptosis, contributes to the establishment of an apoptosis-resistant phenotype (Cell Death Dis. 2010 Sep 16;1:e75.). Here, we report for the first time that the decrease of SOCE during NED may be caused by alternative NED-induced mechanism involving cytoskeleton reorganisation. NED induced by androgen deprivation resulted in a decrease of SOCE due to cortical F-actin over-polymerization which inhibits thapsigargin-induced SOCE. The disruption of F-actin polymerization by Cytochalasin D in NED cells restored SOCE, while the induction of F-actin polymerization by jasplakinolide or calyculin A diminished SOCE without changing the expression of key SOCE players: Orai1, STIM1, and TRPC1. Our data suggest that targeting cytoskeleton-induced pathways of malignant cells together with SOCE-involved channels may prove a useful strategy in the treatment of advanced prostate cancer.
... The alternative suggestion is that the ER InsPjR and the plasm a m em brane Ca^^ channels interact directly, giving rise to the conformational coupling model. While there is considerable experimental evidence to support the diffusable messenger model, recently published findings support the idea of a close interaction between ER Ca^^ stores and plasma membrane channels (reviewed by Putney, 1999b). A brief discussion of the sim plest model of conform ational coupling is considered below (for a more detailed review of capacitative entry see Barritt (1999)). ...
Thesis
Phosphatidylinositol transfer proteins (PtdIns-TPs) have historically been described as proteins that constitutively catalyse the exchange of phospholipid monomers between membrane bilayers, thereby facilitating lipid distribution within cells. Recent data have identified an acute requirement for PtdIns-TPs during several essential cellular processes. The various PtdIns-TPs, including PITPα, PITPβ, retinal degeneration B (rdgB) and PYK2 amino-terminal domain interacting (Nir) proteins, can be divided into two structural families. The small, soluble PITP isoforms contain only a PtdIns transfer domain and have been implicated in phosphoinositide signalling and vesicle trafficking. In contrast, the rdgB proteins, which include the Nir proteins, contain an amino-terminal PITP-like domain, an acidic, Ca2+-binding domain, six putative transmembrane domains, and a conserved carboxy-terminal domain. Although the rdgB protein was originally identified as an invertebrate phototransduction protein, the biological function of rdgB proteins in vertebrates is unclear. This thesis describes the molecular cloning and characterisation of a novel rdgB protein, rdgBβ. The 38kDa, mammalian rdgBβ (MrdgBβ) protein contains an amino-terminal PITP-like domain and a short carboxy-terminal domain. Cytogenetic analysis reveals that the human rdgBβ gene is localised on chromosome 17q23. In contrast to other rdgB-like proteins, MrdgBβ contains neither transmembrane motifs nor the conserved carboxy-terminal domain. Northern analysis has demonstrated that MrdgBβ mRNA is ubiquitously expressed. Furthermore, immunofluorescence analysis of ectopic MrdgBβ showed cytoplasmic localisation. The phospholipid-binding specificity and transfer activities of MrdgBβ, relative to other PtdIns-TPs, have been addressed using radiolabelled- and pyrene-labelled phospholipid transfer assays. The functional significance of the results of these assays is discussed. While early reports found functional degeneracy in vitro, the work presented in this thesis supports more recent results, which suggest that different members of the PtdIns-TP family have distinct functions in vivo.
... A TMEM16 protein family member Ist2 regulates ER morphology and ER-plasma membrane association in yeast 44 while another member -ANO8 facilitates this in mammalian cells 45 . ER-PM tethering is crucial for cellular calcium homeostasis in mammalian cells 46 . As ER fragmentation is linked to persistent increases in [Ca 2+ ] c , this observation further suggests a role for ANO5 in ER-mediated [Ca 2+ ] c homeostasis. ...
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Autosomal recessive mutations in Anoctamin 5 (ANO5/TMEM16E), a member of the transmembrane 16 (TMEM16) family of Ca2+-activated ion channels and phospholipid scramblases, cause adult-onset muscular dystrophies (limb girdle muscular dystrophy 2L (LGMD2L) and Miyoshi Muscular Dystrophy (MMD3). However, the molecular role of ANO5 is unclear and ANO5 knockout mouse models show conflicting requirements of ANO5 in muscle. To study the role of ANO5 in human muscle cells we generated a myoblast line from a MMD3-patient carrying the c.2272C>T mutation, which we find causes the mutant protein to be degraded. The patient myoblasts exhibit normal myogenesis, but are compromised in their plasma membrane repair (PMR) ability. The repair deficit is linked to the poor ability of the endoplasmic reticulum (ER) to clear cytosolic Ca2+ increase caused by focal plasma membrane injury. Expression of wild-type ANO5 or pharmacological prevention of injury-triggered cytosolic Ca2+ overload enable injured patient muscle cells to repair. A homology model of ANO5 shows that several of the known LGMD2L/MMD3 patient mutations line the transmembrane region of the protein implicated in its channel activity. These results point to a role of cytosolic Ca2+ homeostasis in PMR, indicate a role for ANO5 in ER-mediated cytosolic Ca2+ uptake and identify normalization of cytosolic Ca2+ homeostasis as a potential therapeutic approach to treat muscular dystrophies caused by ANO5 deficit.
... In addition, in the presence of either Ca 2ϩ -free buffer or the K Ca channel blocker iberiotoxin, responses to RYN in both adult and fetal MCA were eliminated. This strongly suggests coupling between the RYN receptor and plasmalemmal K Ca channel and the L-type Ca 2ϩ channel (14,29) and may be analogous to the coupling between the Ins(1,4,5)P 3 receptor and plasma membrane transient receptor potential family of plasma membrane channel proteins (6,8,30,31). ...
Article
To test the hypothesis that sarcoplasmic reticulum (SR) Ca(2+) stores play a key role in norepinephrine (NE)-induced contraction of fetal and adult cerebral arteries and that Ca(2+) stores change with development, we performed the following study. In main branch middle cerebral arteries (MCA) from near-term fetal ( approximately 140 days) and nonpregnant adult sheep, we measured NE-induced contraction and intracellular Ca(2+) concentration ([Ca(2+)](i)) in the absence and presence of different blockers. In adult MCA, after thapsigargin (10(-6) M), the NE-induced responses of tension and [Ca(2+)](i) were 37 +/- 5 and 47 +/- 7%, respectively, of control values (P < 0.01 for each). In the fetal artery, in contrast, this treatment resulted in no significant changes from control. When this was repeated in the absence of extracellular Ca(2+), adult MCA increases in tension and [Ca(2+)](i) were 32 +/- 5 and 13 +/- 3%, respectively, of control. Fetal cerebral arteries, however, showed essentially no response. Ryanodine (RYN, 3 x 10(-6) to 10(-5) M) resulted in increases in tension and [Ca(2+)](i) in both fetal and adult MCA similar to that seen with NE. For both adult and fetal MCA, the increased tension and [Ca(2+)](i) responses to RYN were essentially eliminated in the presence of zero extracellular Ca(2+). These findings provide evidence that in fetal MCA, in contrast to those in the adult, SR Ca(2+) stores are of less importance in NE-induced contraction, with such contraction being almost wholly dependent on Ca(2+) flux via plasma membrane L-type Ca(2+) channels. In addition, they suggest that in both adult and fetal MCA, the RYN receptor is coupled to the plasma membrane Ca(2+)-activated K(+) channel and/or L-type Ca(2+) channel.
... A voltage-independent influx of calcium, called capacitative influx (Putney and McKay 1999), is also induced in adrenal glomerulosa cells by Ang II (Ambroz and Catt 1992). This influx involves particular channels, the store-operated calcium channels (SOCs), that are activated, probably through conformational coupling (Irvine 1990;Putney 1999), upon intracellular store emptying by inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3 -mediated) or by SERCA pump-inhibiting agents such as thapsigargin. ...
Article
Angiotensin II (Ang II) is, with extracellular potassium, the principal regulator of the biosynthesis of aldosterone, the main mineralocorticoid in man. Because various cardiovascular pathological states are often associated with inappropriate levels of circulating aldosterone, it is of importance to understand the intracellular molecular mechanisms leading to aldosterone output in the adrenal zona glomerulosa cell. The present chapter reviews the current knowledge on the events that are triggered by angiotensin II, from the interaction with membrane receptors to the final output of aldosterone. The initial signaling processes involve complex changes in intracellular phospholipid and calcium homeostasis resulting from effects of Ang II on various effector enzymes (phospholipases) and calcium channels, as well as on intracellular calcium stores. Concomitantly, a series of kinase pathways (protein kina se C, mitogenactivated protein kinase, tyrosine kinases, etc.) are activated. These intracellular signals then mediate a variety of localized responses along the entire cascade of events leading from uptake of cholesterol, the precursor of all steroids, to cholesterol supply to the mitochondrial enzymatic machinery that will process cholesterol to aldosterone. Indeed , Ang II increases HDL-cholesterol import at the cell surface, cholesterol ester hydrolysis in lipid droplets , scavenger receptor class B type I(HDL receptor), steroidogenic acute regulatory (StAR) protein or aldosterone synthase gene expression in the nucleus, or StAR-mediated cholesterol importation into mitochondria. In addition, recent work has shown that aldosterone can also be synthesized and act in nonclassic steroidogenic tissues such as brain, vessels and, most importantly, the heart, where it may have deleterious effects in some pathological situations such as heart failure or myocardial infarction
... So far the precise regulatory functions of Rac/Cdc42 in Ca 2ϩ responses are unclear. At least three models have been proposed for signaling capacitative Ca 2ϩ entry (7,49). First, it was suggested that a diffusible signaling factor (calcium influx factor) is generated and released from the endoplasmic reticulum (50). ...
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Using large clostridial cytotoxins as tools, the role of Rho GTPases in activation of RBL 2H3 hm1 cells was studied. Clostridium difficile toxin B, which glucosylates Rho, Rac, and Cdc42 and Clostridium sordellii lethal toxin, which glucosylates Rac and Cdc42 but not Rho, inhibited the release of hexosaminidase from RBL cells mediated by the high affinity antigen receptor (FcepsilonRI). Additionally, toxin B and lethal toxin inhibited the intracellular Ca(2+) mobilization induced by FcepsilonRI-stimulation and thapsigargin, mainly by reducing the influx of extracellular Ca(2+). In patch clamp recordings, toxin B and lethal toxin inhibited the calcium release-activated calcium current by about 45%. Calcium release-activated calcium current, the receptor-stimulated Ca(2+) influx, and secretion were inhibited neither by the Rho-ADP-ribosylating C3-fusion toxin C2IN-C3 nor by the actin-ADP-ribosylating Clostridium botulinum C2 toxin. The data indicate that Rac and Cdc42 but not Rho are not only involved in late exocytosis events but are also involved in Ca(2+) mobilization most likely by regulating the Ca(2+) influx through calcium release-activated calcium channels activated via FcepsilonRI receptor in RBL cells.
... Finally, evidence is emerging from animal studies that stimulus-specific Ca 2ϩ signals may occur without any detectable changes in cellular Ca 2ϩ levels. It is well known that Ca 2ϩ influx associated with filling of intracellular stores can occur without measurable alteration of cytosolic Ca 2ϩ (Putney, 1999). Such capacitative Ca 2ϩ entry is likely to be an essential process in the generation of repetitive Ca 2ϩ elevations (Berridge, 1995). ...
... Although it has been reported from Xenopus that ectodermal cells at these stages can establish cell to cell communication through gap junctions (Slack and Palmer, 1969;Guthrie et al., 1988), both these and our previous data (Moreau et al., 1994;Leclerc et al., 1997) suggest that the localised signals may occur via a near simultaneous opening of LTCs, mediated by a membrane depolarisation. However, we cannot exclude the possibility that Ca 2+ transients result from a depletion of intracellular Ca 2+ stores, which in turn activates a Ca 2+ entry mechanism at the plasma membrane similar to the capacitative Ca 2+ entry described in many nonexcitable cell types (for reviews see Putney, 1999;Barritt, 1999). (2) From mid-gastrula (stage 11.5), the nature of the Ca 2+ transients changed. ...
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Through the injection of f-aequorin (a calcium-sensitive bioluminescent reporter) into the dorsal micromeres of 8-cell stage Xenopus laevis embryos, and the use of a Photon Imaging Microscope, distinct patterns of calcium signalling were visualised during the gastrulation period. We present results to show that localised domains of elevated calcium were observed exclusively in the anterior dorsal part of the ectoderm, and that these transients increased in number and amplitude between stages 9 to 11, just prior to the onset of neural induction. During this time, however, no increase in cytosolic free calcium was observed in the ventral ectoderm, mesoderm or endoderm. The origin and role of these dorsal calcium-signalling patterns were also investigated. Calcium transients require the presence of functional L-type voltage-sensitive calcium channels. Inhibition of channel activation from stages 8 to 14 with the specific antagonist R(+)BayK 8644 led to a complete inhibition of the calcium transients during gastrulation and resulted in severe defects in the subsequent formation of the anterior nervous system. BayK treatment also led to a reduction in the expression of Zic3 and geminin in whole embryos, and of NCAM in noggin-treated animal caps. The possible role of calcium transients in regulating developmental gene expression is discussed.
... The final hit to the theory came from direct measurement of mitochondrial Ca 2+ content by electron microscopic techniques, which showed that, in healthy cells, these organelles contain minute amounts of Ca 2+ (ref. 3). The apparent contradiction between the data obtained in intact cells and those obtained from study of isolated organelles was explained on the basis of the low affinity of the mitochondrial Ca 2+ carrier (at physiological concentrations of Mg 2+ ions, the half-maximal rate of Ca 2+ uptake occurs at Ca 2+ concentrations above 10 µM). ...
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The CD95 protein delivers crucial signals for lymphocyte death, and may also negatively regulate T-lymphocyte activation by preventing the influx of calcium ions from the cell's exterior. The block in calcium-ion influx occurs through the activation of acidic sphingomyelinase and the release of ceramide, a metabolite that can also induce cell death.
... Ainsi, le cholestérol peut se détacher du feuillet externe de la membrane d'un organite et s'insérer dans le feuillet externe d'un autre quand ceux-ci sont extrêmement proches. En effet, il existe dans les cellules des proximités très étroites entre deux membranes d'organites distincts, comme le RE et la membrane plasmique (Putney, 1999) ou bien les endosomes de recyclage précoces et le TGN (Ioannou, 2000;Maxfield et Wustner, 2002). Ce type de transport peut être assisté par des protéines de transfert de lipides comme la protéine STAR (Ioannou, 2001), qui transfère le cholestérol du feuillet externe vers le feuillet interne de la mitochondrie. ...
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Summary The plasmic membrane of the polarized epithelial cells comprises two distinct domains, the apical domain and the basolateral domain. Each domain has a determined composition in lipids and proteins, enabling them to ensure of the specific functions. The molecular mechanisms responsible for the sorting and the targeting of transmembrane proteins towards the apical pole are still badly known. The apical membrane is enriched in glycosphingolipides and cholesterol which form microdomains called “rafts”. In experiments, the rafts can be isolated in the form of DRM (detergent-resistant membranes) defined by their resistance to a nonionic detergent, the Triton X-100. It was proposed that the rafts recruit apical proteins on the level of the trans-golgien network and are used as platform for their targeting with the apical pole. Indeed the proteins anchored by the glycosylphosphatidyl-inositol are resistant to the Triton and are in general localised with the apical membrane. On the other hand, the majority of apical transmembrane proteins are soluble in the Triton, although they are resistant to the action of mild detergents like Lubrol WX. The objective of work of thesis was to study the role of the rafts in the apical transmembrane protein targeting and to include/understand the differential effect of the Triton and Lubrol on their solubilization. The nucleotides pyrophosphatases NPP1 (basolateral) and NPP3 (apical) expressed in a stable way in cells MDCK were used as models. NPP3 is insoluble in Lubrol and partially insoluble in the Triton, while NPP1 is primarily solubilized. The study of the localization and the sensitivity to the detergents of mutants and chimeraes a combining of the cytoplasmic, transmembrane and extracellular domains of NPP3 and NPP1, showed that there was not strict correlation between apical targeting and resistance to the detergents. The resistance of NPP3 to solubilization by Lubrol is acquired precociously during its biosynthesis, independently of its final destination. This resistance depends on amino acids charged positively located in the cytoplasmic tail, close to the membrane. In order to include/understand the selectivity of the Triton and Lubrol in the extraction of proteins and the membrane lipids, the lipidic composition of the DRM obtained after extraction by the Triton and Lubrol were compared. The DRM extracted by the Triton and Lubrol are cholesterol enriched what corresponds to the definition of the rafts. However, the DRM Triton are impoverished in lipids of the internal layer while the DRM Lubrol are enriched in phosphatidylethanolamine. The DRM Lubrol are also enriched in protein associated with the internal layer with the membrane. In conclusion, this work shows that the resistance of apical protein NPP3 to the extraction by Lubrol, and partly by the Triton, is an intrinsic property which probably corresponds to an adaptation of protein to the lipidic composition of the apical domain, but that this property does not determine its polarized targeting. Moreover, this work shows that the detergents are very interesting tools to study the interactions between the membrane proteins and lipids, but that there probably does not exist detergent able to insulate in a strict way of the membrane microdomains such as are defined the rafts. Our results suggest that the layer interns rafts is enriched in phosphatidylethanolamine and cholesterol, that it is partly solubilized by the Triton, which would destabilize transmembrane proteins and would involve their extraction. Keywords : detergent, raft,, apical targeting, cholesterol, membrane microdomain, inner membrane leaflet, phosphatidylethanolamine.
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Patch-clamp experiments combined with indo-1 measurement of free intracellular Ca²⁺ concentration ([Ca²⁺]i) were used to determine the homeostatic systems involved in the maintenance of resting [Ca²⁺]i and the clearance of Ca²⁺ transients following activation of voltage-gated Ca²⁺ channels in neurones from rat superior cervical ganglion (SCG). In these neurones, the Ca²⁺ buffering capacity was estimated to be ≈ 1000 at [Ca²⁺]i close to rest and ≈ 250 at [Ca²⁺]i ≈ 1 μM and to involve at least two buffering systems with different affinities for Ca²⁺. Removal of extracellular Ca²⁺ led to a decrease in [Ca²⁺]i that was mimicked by the addition of La³⁺. This decrease in [Ca²⁺]i was more pronounced after inhibition of the endoplasmic reticulum Ca²⁺ uptake system (SERCA) and subsequent depletion of the intracellular stores. Inhibition of the plasma membrane Ca2+ pump (PMCA) by intracellular carboxyeosin or extracellular alkalisation (pH 9) both increased resting [Ca²⁺]i and prolonged the recovery of Ca²⁺ transients at peak [Ca²⁺]i < 500 nM. For [Ca²⁺]i loads > 500 nM, recovery showed an additional plateau phase that was abolished in carbonyl cyanide m-chlorohydrazone (CCCP) or on omitting Na⁺ from the intracellular solution. Inhibition of the plasma membrane Na⁺/Ca²⁺ exchanger (NCX) and of SERCA had a small but significant effect on the rate of decay of these larger Ca²⁺ transients. However, neither of these mechanisms appeared to contribute substantially to the recovery from rises in [Ca²⁺]i below 1 μM. In conclusion, resting [Ca²⁺]i is maintained as the result of a passive Ca²⁺ influx regulated by a large Ca²⁺ buffering system, Ca²⁺ extrusion via a PMCA and Ca²⁺ transport from the intracellular stores. PMCA is also the principal Ca²⁺ extrusion system at low Ca²⁺ loads, with additional participation of the NCX and intracellular organelles at high [Ca²⁺]i.
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The coupling mechanism between endoplasmic reticulum (ER) calcium ion (Ca²⁺) stores and plasma membrane (PM) store-operated channels (SOCs) is crucial to Ca²⁺ signaling but has eluded detection. SOCs may be functionally related to the TRP family of receptor-operated channels. Direct comparison of endogenous SOCs with stably expressed TRP3 channels in human embryonic kidney (HEK293) cells revealed that TRP3 channels differ in being store independent. However, condensed cortical F-actin prevented activation of both SOC and TRP3 channels, which suggests that ER-PM interactions underlie coupling of both channels. A cell-permeant inhibitor of inositol trisphosphate receptor (InsP3R) function, 2-aminoethoxydiphenyl borate, prevented both receptor-induced TRP3 activation and store-induced SOC activation. It is concluded that InsP3Rs mediate both SOC and TRP channel opening and that the InsP3R is essential for maintaining coupling between store emptying and physiological activation of SOCs.
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This chapter discusses that virtually all cell functions are regulated by the changes in the cytosolic free Ca2+ concentration. Ca2+ signals within cells can be local or global, can involve waves, oscillations, or even more-complex patterns, and can be modulated in terms of both amplitude and frequency. In mammals, many glial cells contain an elaborate endoplasmic reticulum. Associated with this membrane network are pumps for Ca2+ uptake, that, as in other cells, are inhibited by thapsigargin and cyclopiazonic acid. The membranous network also contains Ca2+-binding proteins for Ca2+ storage and channels for Ca2+ induced calcium release. These include the ubiquitous IP3 receptors and, in many cells, the ryanodine receptors. The chapter reviews that in glia, the major mechanism for Ca2+ release from internal stores involves activation of inositol 1,4,5-trisphosphate (IP3) gated Ca2+ release channels (IP3 receptors). It discusses that the regulation of IP3 receptors is complex with alternative splicing of at least three different isoforms, posttranslational modification of IP3R by phosphorylation, and interaction with adenine nucleotides, calcium, and immunophilins or FK506 binding proteins. The mitochondria and calcium storage proteins also function as high-capacity storage sites for calcium. Other calcium binding proteins play important roles in cell signaling, maintenance of the cytoskeleton, and apoptosis.
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Calcium signals have been implicated in the regulation of many diverse cellular processes. The problem of how information from extracellular signals is delivered with specificity and fidelity using fluctuations in cytosolic Ca2+ concentration remains unresolved. The capacity of cells to generate Ca2+ signals of sufficient spatial and temporal complexity is the primary constraint on their ability to effectively encode information through Ca2+. Over the past decade, a large body of literature has dealt with some basic features of Ca2+-handling in cells, as well as the multiplicity and functional diversity of intracellular Ca2+ stores and extracellular Ca2+ influx pathways. In principle, physiologists now have the necessary information to attack the problem of function- and agonist-specificity in Ca2+ signal transduction. This review explores the data indicating that Ca2+ release from diverse sources, including many types of intracellular stores, generates Ca2+ signals with sufficient complexity to regulate the vast number of cellular functions that have been reported as Ca2+-dependent. Some examples where such complexity may relate to neuroendocrine regulation of hormone secretion/synthesis are discussed. We show that the functional and spatial heterogeneity of Ca2+ stores generates Ca2+ signals with sufficient spatiotemporal complexity to simultaneously control multiple Ca2+-dependent cellular functions in neuroendocrine systems.
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The impact of calcium signalling on so many areas of cell biology reflects the crucial role of calcium signals in the control of diverse cellular functions. Despite the precision with which spatial and temporal details of calcium signals have been resolved, a fundamental aspect of the generation of calcium signals — the activation of 'store-operated channels' (SOCs) — remains a molecular and mechanistic mystery. Here we review new insights into the exchange of signals between the endoplasmic reticulum (ER) and plasma membrane that result in activation of calcium entry channels mediating crucial long-term calcium signals.
Chapter
Mutations in genes encoding the presenilins cause up to 50% of the early-onset cases of familial Alzheimer's disease. Recent studies have shown that both PS1 and PS2 play an essential role in the -secretase cleavage of amyloid β protein precursor and selected other integral membrane proteins, including Notch. Common molecular consequences of presenilin-linked Familial Alzheimer's disease mutations in the presenilins have been shown to disrupt intracellular Ca2+ homeostasis. We studied the effects of Familial Alzheimer's disease mutations on a common Ca2+-regulatory pathway, known as store-operated or capacitative Ca2+ entry, which is triggered by an intracellular Ca2+ store depletion.
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This chapter presents an overview of the properties and functions of store-operated calcium channels (SOCs). A large number of Ca2+ imaging studies indicate that SOCs are ubiquitous in nonexcitable cells, where they comprise the main Ca2+ entry pathway, and some studies suggest that they may exist in excitable cells as well. Patch-clamp methods have offered the most detailed information about the biophysical properties of these channels. The first and best characterized SOC is the Ca2+ release-activated Ca2+ (CRAC) channel, expressed in mast cells, T lymphocytes, and related cell lines. While much is known about the basic biophysical properties of these channels, a critical issue remains about the identification of the molecular mechanism(s) underlying their activity. The chapter discusses those aspects of SOCs that may be especially pertinent to the search for a molecular mechanism. It highlights CRAC channels as a prototypic example of an SOC, with the expectation that many of the issues related to the molecular mechanism will apply to other SOCs as well. The chapter also discusses the known biological functions of CRAC channels and some of the intrinsic properties that are most important for carrying out these roles.
Chapter
Calcium is one of the most versatile messengers in biological systems, translating developmental and environmental cues into cellular responses. The endoplasmic reticulum(ER) constitutes one of the most important calcium holding organelles in higher eukaryotes. Upon stimulation, calcium is released from theER into the cytosol where it may trigger downstream effectors. However, the release of calcium may also affect internal ERfunctions, such as protein folding and secretion. It is therefore important not only to view cytosolic calcium signals as isolated events, but also in context to the organellar calcium status. In animals, the calcium levels of the ER can be sensed by other calcium resources, such as the plasma membrane, which may allow calcium uptake or release depending on the overall demand in the cell. In this chapter we have tried to convey the diverse aspects of calcium and its potential impact on different ERprocesses, and organellar communications, in plants.
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La membrane plasmique des cellules épithéliales polarisées comporte deux domaines distincts, le domaine apical et le domaine basolatéral. Chaque domaine a une composition en lipides et en protéines déterminée, leur permettant d'assurer des fonctions spécifiques. Les mécanismes moléculaires responsables du tri et de l'adressage des protéines transmembranaires vers le pôle apical sont encore mal connus. La membrane apicale est enrichie en glycosphingolipides et en cholestérol qui forment des microdomaines appelés « rafts ». Expérimentalement, les rafts peuvent être isolés sous forme de DRM (detergent-resistant membranes) définis par leur résistance à un détergent non ionique, le Triton X-100. Il a été proposé que les rafts recrutent les protéines apicales au niveau du réseau trans-golgien et servent de plateforme pour leur adressage au pôle apical. Effectivement les protéines ancrées par le glycosylphosphatidyl-inositol sont résistantes au Triton et sont localisées en général à la membrane apicale. En revanche, la plupart des protéines transmembranaires apicales sont solubles dans le Triton, bien qu'elles soient résistantes à l'action de détergents plus doux comme le Lubrol WX. L'objectif des travaux de thèse a été d'étudier le rôle des rafts dans l'adressage apical de protéines transmembranaires et de comprendre l'effet différentiel du Triton et du Lubrol sur leur solubilisation. Les nucléotides pyrophosphatases NPP1 (basolatérale) et NPP3 (apicale) exprimées de façon stable dans les cellules MDCK ont servi de modèles. NPP3 est insoluble dans le Lubrol et partiellement insoluble dans le Triton, tandis que NPP1 est essentiellement solubilisée. L'étude de la localisation et de la sensibilité aux détergents de mutants et de chimères combinant des domaines cytoplasmiques, transmembranaires et extracellulaires de NPP3 et NPP1, a montré qu'il n'existait pas de corrélation stricte entre l'adressage apical et la résistance aux détergents. La résistance de NPP3 à la solubilisation par le Lubrol est acquise précocement au cours de sa biosynthèse, indépendamment de sa destination finale. Cette résistance dépend d'acides aminés chargés positivement situés dans la queue cytoplasmique, proches de la membrane. Afin de comprendre la sélectivité du Triton et du Lubrol dans l'extraction des protéines et des lipides membranaires, la composition lipidique des DRM obtenus après extraction par le Triton et le Lubrol a été comparée. Les DRM extraits par le Triton et le Lubrol sont enrichis en cholestérol ce qui correspond à la définition des rafts. Cependant, les DRM Triton sont appauvris en lipides du feuillet interne tandis que les DRM Lubrol sont enrichis en phosphatidyléthanolamine. Les DRM Lubrol sont également enrichis en protéines associées au feuillet interne de la membrane. En conclusion, ces travaux montrent que la résistance de la protéine apicale NPP3 à l'extraction par le Lubrol, et en partie par le Triton, est une propriété intrinsèque qui correspond probablement à une adaptation de la protéine à la composition lipidique du domaine apical, mais que cette propriété ne détermine pas son adressage polarisé. De plus, ces travaux montrent que les détergents sont des outils très intéressants pour étudier les interactions entre les protéines et les lipides membranaires, mais qu'il n'existe probablement pas de détergent capable d'isoler de façon stricte des microdomaines membranaires tels que sont définis les rafts. Nos résultats suggèrent que le feuillet interne des rafts est enrichi en phosphatidyléthanolamine et en cholestérol, qu'il est en partie solubilisé par le Triton, ce qui déstabiliserait les protéines transmembranaires et entraînerait leur extraction. Mots clés: détergent, raft, , ciblage apical, cholestérol, microdomaine membranaire, feuillet interne de la membrane, phosphatidyléthanolamine.
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Enterochromaffin-like (ECL) cells are neuroendocrine cells of the gastric mucosa that are important for the peripheral regulation of gastric acid secretion. Following stimulation with gastrin, histamine is released from ECL cells in a calcium-dependent manner, inducing synthesis and secretion of gastric acid in parietal cells. In this thesis, importance and generation of the calcium signal necessary for gastrin-induced histamine secretion were investigated, as well as the expression of the endocytic proteins amphiphysin and dynamin. Activation of InsP3 receports but not calcium release from intracellular stores was nessecary for the increase in [Ca2+]i. Calcium influx could be inhibited by nimodipine, indicating involvement of voltage-activated L-type calcium channels. All known dynamin and amphiphysin isoforms could be detected on mRNA and protein levels. Furthermore, a novel splice form of amphiphysin-2 was found to be expressed in ECL cells.
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This review discusses multiple ways in which the endoplasmic reticulum participates in and is influenced by signal transduction pathways. The endoplasmic reticulum provides a Ca2+ store that can be mobilized either by calcium-induced calcium release or by the diffusible messenger inositol 1,4,5-trisphosphate. Depletion of endoplasmic reticulum Ca2+ stores provides a signal that activates surface membrane Ca2+ channels, a process known as capacitative calcium entry. Depletion of endoplasmic reticulum stores can also signal long-term cellular responses such as gene expression and programmed cell death or apoptosis. In addition to serving as a source of cellular signals, the endoplasmic reticulum is also functionally and structurally modified by the Ca2+ and protein kinase C pathways. Elevated cytoplasmic Ca2+ causes a rearrangement and fragmentation of endoplasmic reticulum membranes. Protein kinase C activation reduces the storage capacity of the endoplasmic reticulum Ca2+ pool. In some cell types, protein kinase C inhibits capacitative calcium entry. Protein kinase C activation also protects the endoplasmic reticulum from the structural effects of high cytoplasmic Ca2+. The emerging view is one of a complex network of pathways through which the endoplasmic reticulum and the Ca2+ and protein kinase C signaling pathways interact at various levels regulating cellular structure and function.
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During the last 2 years, our laboratory has worked on the elucidation of the molecular basis of capacitative calcium entry (CCE) into cells. Specifically, we tested the hypothesis that CCE channels are formed of subunits encoded in genes related to the Drosophila trp gene. The first step in this pursuit was to search for mammalian trp genes. We found not one but six mammalian genes and cloned several of their cDNAs, some in their full length. As assayed in mammalian cells, overexpression of some mammalian Trps increases CCE, while expression of partial trp cDNAs in antisense orientation can interfere with endogenous CCE. These findings provided a firm connection between CCE and mammalian Trps. This article reviews the known forms of CCE and highlights unanswered questions in our understanding of intracellular Ca2+ homeostasis and the physiological roles of CCE.
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Calcium influx in electrically non-excitable cells is regulated by the filling state of intracellular calcium stores. Depletion of stores activates plasma membrane channels that are voltage-independent and highly selective for Ca2+ ions. We report here that the activation of plasma membrane Ca2+ currents induced by depletion of Ca2+ stores requires a diffusible cytosolic factor that washes out with time when dialyzing cells in the whole-cell configuration of the patch-clamp technique. The activation of calcium release-activated calcium current (ICRAC) by ionomycin- or inositol 1,4,5-trisphosphate-induced store depletion is blocked by guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) and guanyl-5'-yl imidodiphosphate, non-hydrolyzable analogs of GTP, suggesting the involvement of a GTP-binding protein. The inhibition by GTP gamma S occurs at a step prior to the activation of ICRAC and is prevented by the addition of GTP. We conclude that the activation mechanism of depletion-induced Ca2+ influx encompasses a GTP-dependent step, possibly involving an as yet unidentified small GTP-binding protein.
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The N-ethylmaleimide-sensitive fusion protein (NSF) and the soluble NSF attachment proteins (SNAPs) appear to be essential components of the intracellular membrane fusion apparatus. An affinity purification procedure based on the natural binding of these proteins to their targets was used to isolate SNAP receptors (SNAREs) from bovine brain. Remarkably, the four principal proteins isolated were all proteins associated with the synapse, with one type located in the synaptic vesicle and another in the plasma membrane, suggesting a simple mechanism for vesicle docking. The existence of numerous SNARE-related proteins, each apparently specific for a single kind of vesicle or target membrane, indicates that NSF and SNAPs may be universal components of a vesicle fusion apparatus common to both constitutive and regulated fusion (including neurotransmitter release), in which the SNAREs may help to ensure vesicle-to-target specificity.
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Recent evidence indicates that several members of the Na+-coupled transporter family are regulated, and this regulation in part occurs by redistribution of transporters between intracellular locations and the plasma membrane. We elucidate components of this process for both wild-type and mutant GABA transporters (GAT1) expressed in Xenopus oocytes using a combination of uptake assays, immunoblots, and electrophysiological measurements of membrane capacitance, transport-associated currents, and GAT1-specific charge movements. At low GAT1 expression levels, activators of protein kinase C (PKC) induce redistribution of GAT1 from intracellular vesicles to the plasma membrane; at higher GAT1 expression levels, activators of PKC fail to induce this redistribution. However, coinjection of total rat brain mRNA with GAT1 permits PKC-mediated modulation at high transporter expression levels. This effect of brain mRNA on modulation is mimicked by coinjection of syntaxin 1a mRNA and is eliminated by injecting synaptophysin or syntaxin antisense oligonucleotides. Additionally, botulinum toxins, which inactivate proteins involved in vesicle release and recycling, reduce basal GAT1 expression and prevent PKC-induced translocation. Mutant GAT1 proteins, in which most or all of a leucine heptad repeat sequence was removed, display altered basal distribution and lack susceptibility to modulation by PKC, delineating one region of GAT1 necessary for its targeting. Thus, functional regulation of GAT1 in oocytes occurs via components common to transporters and to trafficking in both neural and non-neural cells, and suggests a relationship between factors that control neurotransmitter secretion and the components necessary for neurotransmitter uptake.
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The inositol 1,4,5-trisphosphate receptor (InsP3R) plays a key role in intracellular Ca2+ signaling. InsP3R is activated by InsP3 produced from phosphatidylinositol 4,5-bisphosphate (PIP2) by phospholipase C cleavage. Using planar lipid bilayer reconstitution technique, we demonstrate here that rat cerebellar InsP3R forms a stable inhibitory complex with endogenous PIP2. Disruption of InsP3R-PIP2 interaction by specific anti-PIP2 monoclonal antibody resulted in 3-4-fold increase in InsP3R activity and 10-fold shift in apparent affinity for InsP3. Exogenously added PIP2 blocks InsP3 binding to InsP3R and inhibits InsP3R activity. Similar results were obtained with a newly synthesized water soluble analog of PIP2, dioctanoyl-(4,5)PIP2, indicating that insertion of PIP2 into membrane is not required to exert its inhibitory effects on the InsP3R. We hypothesize that the functional link between InsP3R and PIP2 described in the present report provides a basis for a local, rapid, and efficient coupling between phospholipase C activation, PIP2 hydrolysis, and intracellular Ca2+ wave initiation in neuronal and non-neuronal cells.
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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 signalling 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). We show here that 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. Our results provide evidence for the coupling hypothesis, in which InsP3 receptors activated by InsP3 interact with SOC and regulate I(crac).
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In the phospholipase C signaling system, Ca2+ is mobilized from intracellular stores by an action of inositol 1,4,5-trisphosphate. The depletion of intracellular calcium stores activates a calcium entry mechanism at the plasma membrane called capacitative calcium entry. The signal for activating the entry is unknown but likely involves either the generation or release, or both, from the endoplasmic reticulum of some diffusible signal. Recent research has focused on mammalian homologues of the Drosophila TRP protein as potential candidates for capacitative calcium entry channels.. This review summarizes current knowledge about the nature of capacitative calcium entry signals, as well as the potential role of mammalian TRP proteins as capacitative calcium entry channel molecules. BioEssays 21:38–46, 1999. Published 1999 John Wiley & Sons, Inc.
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Recombinant v- and t-SNARE proteins reconstituted into separate lipid bilayer vesicles assemble into SNAREpins-SNARE complexes linking two membranes. This leads to spontaneous fusion of the docked membranes at physiological temperature. Docked unfused intermediates can accumulate at lower temperatures and can fuse when brought to physiological temperature. A supply of unassembled v- and t-SNAREs is needed for these intermediates to form, but not for the fusion that follows. These data imply that SNAREpins are the minimal machinery for cellular membrane fusion.
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A wide variety of hormones and neurotransmitters activate cellular responses by mobilizing cellular Ca2+. In general, this Ca2+ mobilization response is comprised of a release of Ca2+ from intracellular stores, as well as increased entry of Ca2+ into the cytoplasm from the extracellular space. The mechanism for release of intracellular Ca2+ results from the Ca2(+)-mobilizing actions of a second messenger, D-myo-inositol 1,4,5-trisphosphate. Inositol polyphosphates appear also to be involved in the activation of Ca2+ entry, but the mechanism by which this is accomplished is less clear. According to the capacitative model for Ca2+ entry, the depletion of the agonist-regulated intracellular Ca2+ pool by the action of D-myo-inositol 1,4,5-trisphosphate is somehow coupled to the activation of Ca2+ entry. The evidence for this model comes from the demonstration, by diverse strategies, that the same Ca2+ entry mechanism normally activated by Ca2(+)-mobilizing agonists can be equally well triggered by depletion of the intracellular Ca2+ pool, even in the absence of receptor activation or elevated cellular levels of inositol polyphosphates.
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The release of Ca2+ from intracellular stores by sub-optimal doses of inositol trisphosphate has been shown to be dose-related ('quantal'), and a simple model is proposed here to account for this phenomenon. It is suggested that there is a regulatory Ca2(+)-binding site on, or associated with, the luminal domain of the InsP3 receptor, which allosterically controls Ca2+ efflux, and the affinity for Ca2+ of that site is modulated by InsP3 binding to the cytoplasmic domain of the receptor; a similar mechanism applied to the ryanodine receptor might also explain some aspects of Ca2(+)-induced Ca2+ release. The stimulated entry of Ca2+ into a cell which occurs upon activation of inositide-linked receptors has been variously and confusingly proposed to be regulated by InsP3, InsP4, and/or a 'capacitative' Ca2+ pool; the mechanism of InsP3 receptor action suggested here is shown to lead to a potential reconciliation of all these conflicting proposals.
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A model is proposed for the mechanism by which activation of surface membrane receptors causes sustained Ca2+ entry into cells from the extracellular space. Reassessment of previously published findings on the behavior of receptor-regulated intracellular Ca2+ pools leads to the conclusion that when such pools are empty, a pathway from the extracellular space to the pool is opened; conversely when the pool is filled, the pathway is closed and it becomes relatively stable to depletion by low Ca2+ media or chelating agents. The biphasic nature of agonist-activated Ca2+-mobilization is thus seen as an initial emptying of the intracellular Ca2+ pool by inositol (1,4,5) trisphosphate, followed by rapid entry of Ca2+ into the pool and, in the continued presence of inositol (1,4,5) trisphosphate, into the cytosol. On withdrawal of agonist, inositol (1,4,5) trisphosphate is then rapidly degraded, the pathway from the pool to the cytosol is closed, and rapid entry from the outside continues until the Ca2+ content of the pool reaches a level that inactivates Ca2+ entry. This capacitative model allows for Ca2+ release and Ca2+ entry to be controlled by a single messenger, inositol (1,4,5) trisphosphate.
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The whole-cell patch-clamp technique was used to study the effect of primaquine, an inhibitor of vesicular transport, on the calcium-release-activated current (Icrac) in rat megakaryocytes. Addition of primaquine, before emptying of internal Ca2+ stores by ionomycin, prevented the development of Icrac, with a half-maximal concentration of near 100 microM. Maximal inhibition (> or = 83%) was observed at 0.6-1 mM primaquine. At 1 mM, chloroquine, a related compound which is less effective at blocking vesicular secretion, had no effect on Icrac. Primaquine (0.8 mM) added after sustained activation of Icrac caused a gradual block of current, with maximal inhibition of 50% observed after 2-3 min. At 1 mM, internal guanosine 5'-[gamma-thio]triphosphate reduced Icrac by 65 +/- 13%. Neither 1 mM GTP nor 2 mM guanosine 5'-[beta-thio]diphosphate had any significant effect on Icrac. The recognized role of GTPases in the regulation of vesicular trafficking, together with block of Icrac activation by primaquine, provide evidence that the channels carrying Icrac may be stored in a vesicular membrane compartment and transferred to the plasma membrane following store depletion.
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In Drosophila, the store-operated Ca2+ channel, TRP, is required in photoreceptor cells for a sustained response to light. Here, we show that TRP forms a complex with phospholipase C-beta (NORPA), rhodopsin (RH1), calmodulin, and the PDZ domain containing protein INAD. Proteins with PDZ domains have previously been shown to cluster ion channels in vitro. We show that in InaD mutant flies, TRP is no longer spatially restricted to its normal subcellular compartment, the rhabdomere. These results provide evidence that a PDZ domain protein is required, in vivo, for anchoring of an ion channel to a signaling complex. Furthermore, disruption of this interaction results in retinal degeneration. We propose that the TRP channel is linked to NORPA and RH1 to facilitate feedback regulation of these upstream signaling molecules.
Article
Receptor-activated Ca2+ channels (RACCs) play a central role in regulation of the functions of animal cells. Together with voltage-operated Ca2+ channels (VOCCs) and ligand-gated non-selective cation channels, RACCs provide a variety of pathways by which Ca2+ can be delivered to the cytoplasmic space and the endoplasmic reticulum (ER) in order to initiate or maintain specific types of intracellular Ca2+ signal. Store-operated Ca2+ channels (SOCs), which are activated by a decrease in Ca2+ in the ER, are a major subfamily of RACCs. A careful analysis of the available data is required in order to discern the different types of RACCs (differentiated chiefly on the basis of ion selectivity and mechanism of activation) and to properly develop hypotheses for structures and mechanisms of activation. Despite much intensive research, the structures and mechanisms of activation of RACCs are only now beginning to be understood. In considering the physiological functions of the different RACCs, it is useful to consider the specificity for Ca2+ of each type of cation channel and the rate at which Ca2+ flows through a single open channel; the locations of the channels on the plasma membrane (in relation to the ER, cytoskeleton and other intracellular units of structure and function); the Ca2+-responsive enzymes and proteins; and the intracellular buffers and proteins that control the distribution of Ca2+ in the cytoplasmic space. RACCs which are non-selective cation channels can deliver Ca2+ directly to specific regions of the cytoplasmic space, and can also admit Na+, which induces depolarization of the plasma membrane, the opening of VOCCs and the subsequent inflow of Ca2+. SOCs appear to deliver Ca2+ specifically to the ER, thereby maintaining oscillating Ca2+ signals.
Article
The elusive coupling between endoplasmic reticulum (ER) Ca2+ stores and plasma membrane (PM) "store-operated" Ca2+ entry channels was probed through a novel combination of cytoskeletal modifications. Whereas coupling was unaffected by disassembly of the actin cytoskeleton, in situ redistribution of F-actin into a tight cortical layer subjacent to the PM displaced cortical ER and prevented coupling between ER and PM Ca2+ entry channels, while not affecting inositol 1,4,5-trisphosphate-mediated store release. Importantly, disassembly of the induced cortical actin layer allowed ER to regain access to the PM and reestablish coupling of Ca2+ entry channels to Ca2+ store depletion. Coupling is concluded to be mediated by a physical "secretion-like" mechanism involving close but reversible interactions between the ER and the PM.
Article
Depletion of Ca2+ stores in Xenopus oocytes activated entry of Ca2+ across the plasma membrane, which was measured as a current I(soc) in subsequently formed cell-attached patches. I(soc) survived excision into inside-out configuration. If cell-attached patches were formed before store depletion, I(soc) was activated outside but not inside the patches. I(soc) was potentiated by microinjection of Clostridium C3 transferase, which inhibits Rho GTPase, whereas I(soc) was inhibited by expression of wild-type or constitutively active Rho. Activation of I(soc) was also inhibited by botulinum neurotoxin A and dominant-negative mutants of SNAP-25 but was unaffected by brefeldin A. These results suggest that oocyte I(soc) is dependent not on aqueous diffusible messengers but on SNAP-25, probably via exocytosis of membrane channels or regulatory molecules.
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In the present work, we studied the interaction and effect of several IP3 receptor (IP3R) constructs on the gating of the store-operated (SOC) hTrp3 channel. Full-length IP3R coupled to silent hTrp3 channels in intact cells but did not activate them until stores were depleted of Ca2+. By contrast, constructs containing the IP3-binding domain activated silent hTrp3 channels in unstimulated cells and restored gating of hTrp3 by IP3 in excised plasma membrane patches. We conclude that the N-terminal domain of the IP3R functions as a gate and is sufficient for activation of SOCs. The sensing and transduction domains of the IP3R are required to maintain SOCs in an inactive state.
  • K Kiselyov
  • X Xu
  • G Mozhayeva
  • T Kuo
  • I Pessah
  • G Mignery
  • X Zhu
  • L Birnbaumer
  • S Muallem
Kiselyov, K., Xu, X., Mozhayeva, G., Kuo, T., Pessah, I., Mignery, G., Zhu, X., Birnbaumer, L., and Muallem, S. (1998). Nature 396, 478-482.