KRAP regulates mitochondrial Ca2+ uptake by licensing IP3 receptor activity and stabilizing ER-mitochondrial junctions.

Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are high-conductance channels that allow the regulated redistribution of Ca2+ from the endoplasmic reticulum (ER) to the cytosol and, at specialized membrane contact sites (MCSs), to other organelles. Only a subset of IP3Rs release Ca2+ to the cytosol in response to IP3. These 'licensed' IP3Rs are associated with Kras-induced actin-interacting protein (KRAP, also known as ITPRID2) beneath the plasma membrane. It is unclear whether KRAP regulates IP3Rs at MCSs. We show, using simultaneous measurements of Ca2+ concentration in the cytosol and mitochondrial matrix, that KRAP also licenses IP3Rs to release Ca2+ to mitochondria. Loss of KRAP abolishes cytosolic and mitochondrial Ca2+ signals evoked by stimulation of IP3Rs via endogenous receptors. KRAP is located at ER-mitochondrial membrane contact sites (ERMCSs) populated by IP3R clusters. Using a proximity ligation assay between IP3R and voltage-dependent anion channel 1 (VDAC1), we show that loss of KRAP reduces the number of ERMCSs. We conclude that KRAP regulates Ca2+ transfer from IP3Rs to mitochondria by both licensing IP3R activity and stabilizing ERMCSs.

Dissociation of inositol 1,4,5-trisphosphate from IP3 receptors contributes to termination of Ca2+ puffs.

Ca2+ puffs are brief, localized Ca2+ signals evoked by physiological stimuli that arise from the coordinated opening of a few clustered inositol 1,4,5-trisphosphate receptors (IP3Rs). However, the mechanisms that control the amplitude and termination of Ca2+ puffs are unresolved. To address these issues, we expressed SNAP-tagged IP3R3 in HEK cells without endogenous IP3Rs and used total internal reflection fluorescence microscopy to visualize the subcellular distribution of IP3Rs and the Ca2+ puffs that they evoke. We first confirmed that SNAP-IP3R3 were reliably identified and that they evoked normal Ca2+ puffs after photolysis of a caged analog of IP3. We show that increased IP3R expression caused cells to assemble more IP3R clusters, each of which contained more IP3Rs, but the mean amplitude of Ca2+ puffs (indicative of the number of open IP3Rs) was unaltered. We thus suggest that functional interactions between IP3Rs constrain the number of active IP3Rs within a cluster. Furthermore, Ca2+ puffs evoked by IP3R with reduced affinity for IP3 had undiminished amplitude, but the puffs decayed more quickly. The selective effect of reducing IP3 affinity on the decay times of Ca2+ puffs was not mimicked by exposing normal IP3R to a lower concentration of IP3. We conclude that distinct mechanisms constrain recruitment of IP3Rs during the rising phase of a Ca2+ puff and closure of IP3Rs during the falling phase, and that only the latter is affected by the rate of IP3 dissociation.

The store-operated Ca2+ entry complex comprises a small cluster of STIM1 associated with one Orai1 channel.

Increases in cytosolic Ca2+ concentration regulate diverse cellular activities and are usually evoked by opening of Ca2+ channels in intracellular Ca2+ stores and the plasma membrane (PM). For the many signals that evoke formation of inositol 1,4,5-trisphosphate (IP3), IP3 receptors coordinate the contributions of these two Ca2+ sources by mediating Ca2+ release from the endoplasmic reticulum (ER). Loss of Ca2+ from the ER then activates store-operated Ca2+ entry (SOCE) by causing dimers of STIM1 to cluster and unfurl cytosolic domains that interact with the PM Ca2+ channel, Orai1, causing its pore to open. The relative concentrations of STIM1 and Orai1 are important, but most analyses of their interactions use overexpressed proteins that perturb the stoichiometry. We tagged endogenous STIM1 with EGFP using CRISPR/Cas9. SOCE evoked by loss of ER Ca2+ was unaffected by the tag. Step-photobleaching analysis of cells with empty Ca2+ stores revealed an average of 14.5 STIM1 molecules within each sub-PM punctum. The fluorescence intensity distributions of immunostained Orai1 puncta were minimally affected by store depletion, and similar for Orai1 colocalized with STIM1 puncta or remote from them. We conclude that each native SOCE complex is likely to include only a few STIM1 dimers associated with a single Orai1 channel. Our results, demonstrating that STIM1 does not assemble clusters of interacting Orai channels, suggest mechanisms for digital regulation of SOCE by local depletion of the ER.

Remodeling of ER-plasma membrane contact sites but not STIM1 phosphorylation inhibits Ca2+ influx in mitosis.

Store-operated Ca2+ entry (SOCE), mediated by the endoplasmic reticulum (ER) Ca2+ sensor stromal interaction molecule 1 (STIM1) and the plasma membrane (PM) channel Orai1, is inhibited during mitosis. STIM1 phosphorylation has been suggested to mediate this inhibition, but it is unclear whether additional pathways are involved. Here, we demonstrate using various approaches, including a nonphosphorylatable STIM1 knock-in mouse, that STIM1 phosphorylation is not required for SOCE inhibition in mitosis. Rather, multiple pathways converge to inhibit Ca2+ influx in mitosis. STIM1 interacts with the cochaperone BAG3 and localizes to autophagosomes in mitosis, and STIM1 protein levels are reduced. The density of ER-PM contact sites (CSs) is also dramatically reduced in mitosis, thus physically preventing STIM1 and Orai1 from interacting to activate SOCE. Our findings provide insights into ER-PM CS remodeling during mitosis and a mechanistic explanation of the inhibition of Ca2+ influx that is required for cell cycle progression.

GPN does not release lysosomal Ca2+ but evokes Ca2+ release from the ER by increasing the cytosolic pH independently of cathepsin C.

The dipeptide glycyl-l-phenylalanine 2-naphthylamide (GPN) is widely used to perturb lysosomes because its cleavage by the lysosomal enzyme cathepsin C is proposed to rupture lysosomal membranes. We show that GPN evokes a sustained increase in lysosomal pH (pHly), and transient increases in cytosolic pH (pHcyt) and Ca2+ concentration ([Ca2+]c). None of these effects require cathepsin C, nor are they accompanied by rupture of lysosomes, but they are mimicked by structurally unrelated weak bases. GPN-evoked increases in [Ca2+]c require Ca2+ within the endoplasmic reticulum (ER), but they are not mediated by ER Ca2+ channels amplifying Ca2+ release from lysosomes. GPN increases [Ca2+]c by increasing pHcyt, which then directly stimulates Ca2+ release from the ER. We conclude that physiologically relevant increases in pHcyt stimulate Ca2+ release from the ER in a manner that is independent of IP3 and ryanodine receptors, and that GPN does not selectively target lysosomes.

P2X4 Receptors Mediate Ca2+ Release from Lysosomes in Response to Stimulation of P2X7 and H1 Histamine Receptors.

The P2X4 purinergic receptor is targeted to endolysosomes, where it mediates an inward current dependent on luminal ATP and pH. Activation of P2X4 receptors was previously shown to trigger lysosome fusion, but the regulation of P2X4 receptors and their role in lysosomal Ca2+ signaling are poorly understood. We show that lysosomal P2X4 receptors are activated downstream of plasma membrane P2X7 and H1 histamine receptor stimulation. When P2X4 receptors are expressed, the increase in near-lysosome cytosolic [Ca2+] is exaggerated, as detected with a low-affinity targeted Ca2+ sensor. P2X4-dependent changes in lysosome properties were triggered downstream of P2X7 receptor activation, including an enlargement of lysosomes indicative of homotypic fusion and a redistribution of lysosomes towards the periphery of the cell. Lysosomal P2X4 receptors, therefore, have a role in regulating lysosomal Ca2+ release and the regulation of lysosomal membrane trafficking.

All three IP3 receptor subtypes generate Ca2+ puffs, the universal building blocks of IP3-evoked Ca2+ signals.

All three subtypes of inositol 1,4,5-trisphosphate receptor (IP3R) are intracellular Ca2+ channels that are co-regulated by IP3 and Ca2+ This allows IP3Rs to evoke regenerative Ca2+ signals, the smallest of which are Ca2+ puffs that reflect the coordinated opening of a few clustered IP3Rs. We use total internal reflection microscopy (TIRF) microscopy to record Ca2+ signals in HEK cells expressing all three IP3R subtypes or a single native subtype. Ca2+ puffs are less frequent in cells expressing one IP3R subtype, commensurate with them expressing fewer IP3Rs than wild-type cells. However, all three IP3R subtypes generate broadly similar Ca2+ puffs with similar numbers of IP3Rs contributing to each. This suggests that IP3R clusters may be assembled by conserved mechanisms that generate similarly sized clusters across different IP3R expression levels. The Ca2+ puffs evoked by IP3R2 had slower kinetics and more prolonged durations, which may be due to IP3 binding with greater affinity to IP3R2. We conclude that Ca2+ puffs are the building blocks for the Ca2+ signals evoked by all IP3Rs.

IP3 receptors - lessons from analyses ex cellula.

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are widely expressed intracellular channels that release Ca2+ from the endoplasmic reticulum (ER). We review how studies of IP3Rs removed from their intracellular environment ('ex cellula'), alongside similar analyses of ryanodine receptors, have contributed to understanding IP3R behaviour. Analyses of permeabilized cells have demonstrated that the ER is the major intracellular Ca2+ store, and that IP3 stimulates Ca2+ release from this store. Radioligand binding confirmed that the 4,5-phosphates of IP3 are essential for activating IP3Rs, and facilitated IP3R purification and cloning, which paved the way for structural analyses. Reconstitution of IP3Rs into lipid bilayers and patch-clamp recording from the nuclear envelope have established that IP3Rs have a large conductance and select weakly between Ca2+ and other cations. Structural analyses are now revealing how IP3 binding to the N-terminus of the tetrameric IP3R opens the pore ∼7 nm away from the IP3-binding core (IBC). Communication between the IBC and pore passes through a nexus of interleaved domains contributed by structures associated with the pore and cytosolic domains, which together contribute to a Ca2+-binding site. These structural analyses provide evidence to support the suggestion that IP3 gates IP3Rs by first stimulating Ca2+ binding, which leads to pore opening and Ca2+ release.

A genetically encoded toolkit of functionalized nanobodies against fluorescent proteins for visualizing and manipulating intracellular signalling.

BACKGROUND: Intrabodies enable targeting of proteins in live cells, but generating specific intrabodies against the thousands of proteins in a proteome poses a challenge. We leverage the widespread availability of fluorescently labelled proteins to visualize and manipulate intracellular signalling pathways in live cells by using nanobodies targeting fluorescent protein tags. RESULTS: We generated a toolkit of plasmids encoding nanobodies against red and green fluorescent proteins (RFP and GFP variants), fused to functional modules. These include fluorescent sensors for visualization of Ca2+, H+ and ATP/ADP dynamics; oligomerising or heterodimerising modules that allow recruitment or sequestration of proteins and identification of membrane contact sites between organelles; SNAP tags that allow labelling with fluorescent dyes and targeted chromophore-assisted light inactivation; and nanobodies targeted to lumenal sub-compartments of the secretory pathway. We also developed two methods for crosslinking tagged proteins: a dimeric nanobody, and RFP-targeting and GFP-targeting nanobodies fused to complementary hetero-dimerizing domains. We show various applications of the toolkit and demonstrate, for example, that IP3 receptors deliver Ca2+ to the outer membrane of only a subset of mitochondria and that only one or two sites on a mitochondrion form membrane contacts with the plasma membrane. CONCLUSIONS: This toolkit greatly expands the utility of intrabodies and will enable a range of approaches for studying and manipulating cell signalling in live cells.

Endogenous signalling pathways and caged IP3 evoke Ca2+ puffs at the same abundant immobile intracellular sites.

The building blocks of intracellular Ca2+ signals evoked by inositol 1,4,5-trisphosphate receptors (IP3Rs) are Ca2+ puffs, transient focal increases in Ca2+ concentration that reflect the opening of small clusters of IP3Rs. We use total internal reflection fluorescence microscopy and automated analyses to detect Ca2+ puffs evoked by photolysis of caged IP3 or activation of endogenous muscarinic receptors with carbachol in human embryonic kidney 293 cells. Ca2+ puffs evoked by carbachol initiated at an estimated 65±7 sites/cell, and the sites remained immobile for many minutes. Photolysis of caged IP3 evoked Ca2+ puffs at a similar number of sites (100±35). Increasing the carbachol concentration increased the frequency of Ca2+ puffs without unmasking additional Ca2+ release sites. By measuring responses to sequential stimulation with carbachol or photolysed caged IP3, we established that the two stimuli evoked Ca2+ puffs at the same sites. We conclude that IP3-evoked Ca2+ puffs initiate at numerous immobile sites and the sites become more likely to fire as the IP3 concentration increases; there is no evidence that endogenous signalling pathways selectively deliver IP3 to specific sites.

Mutant IP3 receptors attenuate store-operated Ca2+ entry by destabilizing STIM-Orai interactions in Drosophila neurons.

Store-operated Ca2+ entry (SOCE) occurs when loss of Ca2+ from the endoplasmic reticulum (ER) stimulates the Ca2+ sensor, STIM, to cluster and activate the plasma membrane Ca2+ channel Orai (encoded by Olf186-F in flies). Inositol 1,4,5-trisphosphate receptors (IP3Rs, which are encoded by a single gene in flies) are assumed to regulate SOCE solely by mediating ER Ca2+ release. We show that in Drosophila neurons, mutant IP3R attenuates SOCE evoked by depleting Ca2+ stores with thapsigargin. In normal neurons, store depletion caused STIM and the IP3R to accumulate near the plasma membrane, association of STIM with Orai, clustering of STIM and Orai at ER-plasma-membrane junctions and activation of SOCE. These responses were attenuated in neurons with mutant IP3Rs and were rescued by overexpression of STIM with Orai. We conclude that, after depletion of Ca2+ stores in Drosophila, translocation of the IP3R to ER-plasma-membrane junctions facilitates the coupling of STIM to Orai that leads to activation of SOCE.

Structural and functional conservation of key domains in InsP3 and ryanodine receptors.

Inositol-1,4,5-trisphosphate receptors (InsP(3)Rs) and ryanodine receptors (RyRs) are tetrameric intracellular Ca(2+) channels. In each of these receptor families, the pore, which is formed by carboxy-terminal transmembrane domains, is regulated by signals that are detected by large cytosolic structures. InsP(3)R gating is initiated by InsP(3) binding to the InsP(3)-binding core (IBC, residues 224-604 of InsP(3)R1) and it requires the suppressor domain (SD, residues 1-223 of InsP(3)R1). Here we present structures of the amino-terminal region (NT, residues 1-604) of rat InsP(3)R1 with (3.6 Å) and without (3.0 Å) InsP(3) bound. The arrangement of the three NT domains, SD, IBC-ß and IBC-α, identifies two discrete interfaces (α and ß) between the IBC and SD. Similar interfaces occur between equivalent domains (A, B and C) in RyR1 (ref. 9). The orientations of the three domains when docked into a tetrameric structure of InsP(3)R and of the ABC domains docked into RyR are remarkably similar. The importance of the α-interface for activation of InsP(3)R and RyR is confirmed by mutagenesis and, for RyR, by disease-causing mutations. Binding of InsP(3) causes partial closure of the clam-like IBC, disrupting the ß-interface and pulling the SD towards the IBC. This reorients an exposed SD loop ('hotspot' (HS) loop) that is essential for InsP(3)R activation. The loop is conserved in RyR and includes mutations that are associated with malignant hyperthermia and central core disease. The HS loop interacts with an adjacent NT, suggesting that activation re-arranges inter-subunit interactions. The A domain of RyR functionally replaced the SD in full-length InsP(3)R, and an InsP(3)R in which its C-terminal transmembrane region was replaced by that from RyR1 was gated by InsP(3) and blocked by ryanodine. Activation mechanisms are conserved between InsP(3)R and RyR. Allosteric modulation of two similar domain interfaces within an N-terminal subunit reorients the first domain (SD or A domain), allowing it, through interactions of the second domain of an adjacent subunit (IBC-ß or B domain), to gate the pore.

Prostaglandin E2 Inhibits Histamine-Evoked Ca2+ Release in Human Aortic Smooth Muscle Cells through Hyperactive cAMP Signaling Junctions and Protein Kinase A.

In human aortic smooth muscle cells, prostaglandin E2 (PGE2) stimulates adenylyl cyclase (AC) and attenuates the increase in intracellular free Ca2+ concentration evoked by activation of histamine H1 receptors. The mechanisms are not resolved. We show that cAMP mediates inhibition of histamine-evoked Ca2+ signals by PGE2 Exchange proteins activated by cAMP were not required, but the effects were attenuated by inhibition of cAMP-dependent protein kinase (PKA). PGE2 had no effect on the Ca2+ signals evoked by protease-activated receptors, heterologously expressed muscarinic M3 receptors, or by direct activation of inositol 1,4,5-trisphosphate (IP3) receptors by photolysis of caged IP3 The rate of Ca2+ removal from the cytosol was unaffected by PGE2, but PGE2 attenuated histamine-evoked IP3 accumulation. Substantial inhibition of AC had no effect on the concentration-dependent inhibition of Ca2+ signals by PGE2 or butaprost (to activate EP2 receptors selectively), but it modestly attenuated responses to EP4 receptors, activation of which generated less cAMP than EP2 receptors. We conclude that inhibition of histamine-evoked Ca2+ signals by PGE2 occurs through "hyperactive signaling junctions," wherein cAMP is locally delivered to PKA at supersaturating concentrations to cause uncoupling of H1 receptors from phospholipase C. This sequence allows digital signaling from PGE2 receptors, through cAMP and PKA, to histamine-evoked Ca2+ signals.

ATP evokes Ca2+ signals in cultured foetal human cortical astrocytes entirely through G protein-coupled P2Y receptors.

Extracellular ATP plays important roles in coordinating the activities of astrocytes and neurons, and aberrant signalling is associated with neurodegenerative diseases. In rodents, ATP stimulates opening of Ca2+ -permeable channels formed by P2X receptor subunits in the plasma membrane. It is widely assumed, but not verified, that P2X receptors also evoke Ca2+ signals in human astrocytes. Here, we directly assess this hypothesis. We showed that cultured foetal cortical human astrocytes express mRNA for several P2X receptor subunits (P2X4 , P2X5 , P2X6 ) and G protein-coupled P2Y receptors (P2Y1 , P2Y2 , P2Y6 , P2Y11 ). In these astrocytes, ATP stimulated Ca2+ release from intracellular stores through IP3 receptors and store-operated Ca2+ entry. These responses were entirely mediated by P2Y1 and P2Y2 receptors. Agonists of P2X receptors did not evoke Ca2+ signals, and nor did ATP when Ca2+ release from intracellular stores and store-operated Ca2+ entry were inhibited. We conclude that ATP-evoked Ca2+ signals in cultured human foetal astrocytes are entirely mediated by P2Y1 and P2Y2 receptors, with no contribution from P2X receptors.

Sustained signalling by PTH modulates IP3 accumulation and IP3 receptors through cyclic AMP junctions.

Parathyroid hormone (PTH) stimulates adenylyl cyclase through type 1 PTH receptors (PTH1R) and potentiates the Ca(2+) signals evoked by carbachol, which stimulates formation of inositol 1,4,5-trisphosphate (IP3). We confirmed that in HEK cells expressing PTH1R, acute stimulation with PTH(1-34) potentiated carbachol-evoked Ca(2+) release. This was mediated by locally delivered cyclic AMP (cAMP), but unaffected by inhibition of protein kinase A (PKA), exchange proteins activated by cAMP, cAMP phosphodiesterases (PDEs) or substantial inhibition of adenylyl cyclase. Sustained stimulation with PTH(1-34) causes internalization of PTH1R-adenylyl cyclase signalling complexes, but the consequences for delivery of cAMP to IP3R within cAMP signalling junctions are unknown. Here, we show that sustained stimulation with PTH(1-34) or with PTH analogues that do not evoke receptor internalization reduced the potentiated Ca(2+) signals and attenuated carbachol-evoked increases in cytosolic IP3. Similar results were obtained after sustained stimulation with NKH477 to directly activate adenylyl cyclase, or with the membrane-permeant analogue of cAMP, 8-Br-cAMP. These responses were independent of PKA and unaffected by substantial inhibition of adenylyl cyclase. During prolonged stimulation with PTH(1-34), hyperactive cAMP signalling junctions, within which cAMP is delivered directly and at saturating concentrations to its targets, mediate sensitization of IP3R and a more slowly developing inhibition of IP3 accumulation.

Inositol 1,4,5-trisphosphate receptors and their protein partners as signalling hubs.

Inositol 1,4,5-trisphosphate receptors (IP3 Rs) are expressed in nearly all animal cells, where they mediate the release of Ca(2+) from intracellular stores. The complex spatial and temporal organization of the ensuing intracellular Ca(2+) signals allows selective regulation of diverse physiological responses. Interactions of IP3 Rs with other proteins contribute to the specificity and speed of Ca(2+) signalling pathways, and to their capacity to integrate information from other signalling pathways. In this review, we provide a comprehensive survey of the proteins proposed to interact with IP3 Rs and the functional effects that these interactions produce. Interacting proteins can determine the activity of IP3 Rs, facilitate their regulation by multiple signalling pathways and direct the Ca(2+) that they release to specific targets. We suggest that IP3 Rs function as signalling hubs through which diverse inputs are processed and then emerge as cytosolic Ca(2+) signals.

Golgi anti-apoptotic proteins are highly conserved ion channels that affect apoptosis and cell migration.

Golgi anti-apoptotic proteins (GAAPs) are multitransmembrane proteins that are expressed in the Golgi apparatus and are able to homo-oligomerize. They are highly conserved throughout eukaryotes and are present in some prokaryotes and orthopoxviruses. Within eukaryotes, GAAPs regulate the Ca(2+) content of intracellular stores, inhibit apoptosis, and promote cell adhesion and migration. Data presented here demonstrate that purified viral GAAPs (vGAAPs) and human Bax inhibitor 1 form ion channels and that vGAAP from camelpox virus is selective for cations. Mutagenesis of vGAAP, including some residues conserved in the recently solved structure of a related bacterial protein, BsYetJ, altered the conductance (E207Q and D219N) and ion selectivity (E207Q) of the channel. Mutation of residue Glu-207 or -178 reduced the effects of GAAP on cell migration and adhesion without affecting protection from apoptosis. In contrast, mutation of Asp-219 abrogated the anti-apoptotic activity of GAAP but not its effects on cell migration and adhesion. These results demonstrate that GAAPs are ion channels and define residues that contribute to the ion-conducting pore and affect apoptosis, cell adhesion, and migration independently.

Synthesis of inositol phosphate-based competitive antagonists of inositol 1,4,5-trisphosphate receptors.

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are intracellular Ca(2+) channels that are widely expressed in animal cells, where they mediate the release of Ca(2+) from intracellular stores evoked by extracellular stimuli. A diverse array of synthetic agonists of IP3Rs has defined structure-activity relationships, but existing antagonists have severe limitations. We combined analyses of Ca(2+) release with equilibrium competition binding to IP3R to show that (1,3,4,6)IP4 is a full agonist of IP3R1 with lower affinity than (1,4,5)IP3. Systematic manipulation of this meso-compound via a versatile synthetic scheme provided a family of dimeric analogs of 2-O-butyryl-(1,3,4,6)IP4 and (1,3,4,5,6)IP5 that compete with (1,4,5)IP3 for binding to IP3R without evoking Ca(2+) release. These novel analogs are the first inositol phosphate-based competitive antagonists of IP3Rs with affinities comparable to that of the only commonly used competitive antagonist, heparin, the utility of which is limited by off-target effects.

CaBP1, a neuronal Ca2+ sensor protein, inhibits inositol trisphosphate receptors by clamping intersubunit interactions.

Calcium-binding protein 1 (CaBP1) is a neuron-specific member of the calmodulin superfamily that regulates several Ca(2+) channels, including inositol 1,4,5-trisphosphate receptors (InsP3Rs). CaBP1 alone does not affect InsP3R activity, but it inhibits InsP3-evoked Ca(2+) release by slowing the rate of InsP3R opening. The inhibition is enhanced by Ca(2+) binding to both the InsP3R and CaBP1. CaBP1 binds via its C lobe to the cytosolic N-terminal region (NT; residues 1-604) of InsP3R1. NMR paramagnetic relaxation enhancement analysis demonstrates that a cluster of hydrophobic residues (V101, L104, and V162) within the C lobe of CaBP1 that are exposed after Ca(2+) binding interact with a complementary cluster of hydrophobic residues (L302, I364, and L393) in the ß-domain of the InsP3-binding core. These residues are essential for CaBP1 binding to the NT and for inhibition of InsP3R activity by CaBP1. Docking analyses and paramagnetic relaxation enhancement structural restraints suggest that CaBP1 forms an extended tetrameric turret attached by the tetrameric NT to the cytosolic vestibule of the InsP3R pore. InsP3 activates InsP3Rs by initiating conformational changes that lead to disruption of an intersubunit interaction between a "hot-spot" loop in the suppressor domain (residues 1-223) and the InsP3-binding core ß-domain. Targeted cross-linking of residues that contribute to this interface show that InsP3 attenuates cross-linking, whereas CaBP1 promotes it. We conclude that CaBP1 inhibits InsP3R activity by restricting the intersubunit movements that initiate gating.

Cyclic AMP directs inositol (1,4,5)-trisphosphate-evoked Ca2+ signalling to different intracellular Ca2+ stores.

Cholesterol depletion reversibly abolishes carbachol-evoked Ca(2+) release from inositol (1,4,5)-trisphosphate (IP3)-sensitive stores, without affecting the distribution of IP3 receptors (IP3R) or endoplasmic reticulum, IP3 formation or responses to photolysis of caged IP3. Receptors that stimulate cAMP formation do not alone evoke Ca(2+) signals, but they potentiate those evoked by carbachol. We show that these potentiated signals are entirely unaffected by cholesterol depletion and that, within individual cells, different IP3-sensitive Ca(2+) stores are released by carbachol alone and by carbachol combined with receptors that stimulate cAMP formation. We suggest that muscarinic acetylcholine receptors in lipid rafts deliver IP3 at high concentration to associated IP3R, stimulating them to release Ca(2+). Muscarinic receptors outside rafts are less closely associated with IP3R and provide insufficient local IP3 to activate IP3R directly. These IP3R, probably type 2 IP3R within a discrete Ca(2+) store, are activated only when their sensitivity is increased by cAMP. Sensitization of IP3R by cAMP extends the effective range of signalling by phospholipase C, allowing muscarinic receptors that are otherwise ineffective to recruit additional IP3-sensitive Ca(2+) stores.