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.

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.

Lysosomes shape Ins(1,4,5)P3-evoked Ca2+ signals by selectively sequestering Ca2+ released from the endoplasmic reticulum.

Most intracellular Ca(2+) signals result from opening of Ca(2+) channels in the plasma membrane or endoplasmic reticulum (ER), and they are reversed by active transport across these membranes or by shuttling Ca(2+) into mitochondria. Ca(2+) channels in lysosomes contribute to endo-lysosomal trafficking and Ca(2+) signalling, but the role of lysosomal Ca(2+) uptake in Ca(2+) signalling is unexplored. Inhibition of lysosomal Ca(2+) uptake by dissipating the H(+) gradient (using bafilomycin A1), perforating lysosomal membranes (using glycyl-L-phenylalanine 2-naphthylamide) or lysosome fusion (using vacuolin) increased the Ca(2+) signals evoked by receptors that stimulate inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] formation. Bafilomycin A1 amplified the Ca(2+) signals evoked by photolysis of caged Ins(1,4,5)P(3) or by inhibition of ER Ca(2+) pumps, and it slowed recovery from them. Ca(2+) signals evoked by store-operated Ca(2+) entry were unaffected by bafilomycin A1. Video-imaging with total internal reflection fluorescence microscopy revealed that lysosomes were motile and remained intimately associated with the ER. Close association of lysosomes with the ER allows them selectively to accumulate Ca(2+) released by Ins(1,4,5)P(3) receptors.

Structural organization of signalling to and from IP3 receptors.

In the 30 years since IP3 (inositol 1,4,5-trisphosphate) was first shown to release Ca2+ from intracellular stores, the importance of spatially organized interactions within IP3-regulated signalling pathways has been universally recognized. Recent evidence that addresses three different levels of the structural determinants of IP3-evoked Ca2+ signalling is described in the present review. High-resolution structures of the N-terminal region of the IP3R (IP3 receptor) have established that the two essential phosphate groups of IP3 bind to opposite sides of the IP3-binding site, pulling its two domains together. This conformational change is proposed to disrupt an interaction between adjacent subunits within the tetrameric IP3R that normally holds the channel in a closed state. Similar structural changes are thought to allow gating of ryanodine receptors. cAMP increases the sensitivity of IP3Rs and thereby potentiates the Ca2+ signals evoked by receptors that stimulate IP3 formation. We speculate that both IP3 and cAMP are delivered to IP3Rs within signalling junctions, wherein the associated IP3Rs are exposed to a saturating concentration of either messenger. The concentration-dependent effects of extracellular stimuli come from recruitment of junctions rather than from a graded increase in the activity of individual junctions. IP3Rs within 'IP3 junctions' respond directly to receptors that stimulate phospholipase C, whereas extra-junctional IP3Rs are exposed to suboptimal concentrations of IP3 and open only when they are sensitized by cAMP. These results highlight the importance of selective delivery of diffusible messengers to IP3Rs. The spatial organization of IP3Rs also allows them to direct Ca2+ to specific intracellular targets that include other IP3Rs, mitochondria and Ca2+-regulated channels and enzymes. IP3Rs also interact functionally with lysosomes because Ca2+ released by IP3Rs, but not that entering cells via store-operated Ca2+ entry pathways, is selectively accumulated by lysosomes. This Ca2+ uptake shapes the Ca2+ signals evoked by IP3 and it may regulate lysosomal behaviour.

Analysis of IP3 receptors in and out of cells.

BACKGROUND: Inositol 1,4,5-trisphosphate receptors (IP3R) are expressed in almost all animal cells. Three mammalian genes encode closely related IP3R subunits, which assemble into homo- or hetero-tetramers to form intracellular Ca2+ channels. SCOPE OF THE REVIEW: In this brief review, we first consider a variety of complementary methods that allow the links between IP3 binding and channel gating to be defined. How does IP3 binding to the IP3-binding core in each IP3R subunit cause opening of a cation-selective pore formed by residues towards the C-terminal? We then describe methods that allow IP3, Ca2+ signals and IP3R mobility to be examined in intact cells. A final section briefly considers genetic analyses of IP3R signalling. MAJOR CONCLUSIONS: All IP3R are regulated by both IP3 and Ca2+. This allows them to initiate and regeneratively propagate intracellular Ca2+ signals. The elementary Ca2+ release events evoked by IP3 in intact cells are mediated by very small numbers of active IP3R and the Ca2+-mediated interactions between them. The spatial organization of these Ca2+ signals and their stochastic dependence on so few IP3Rs highlight the need for methods that allow the spatial organization of IP3R signalling to be addressed with single-molecule resolution. GENERAL SIGNIFICANCE: A variety of complementary methods provide insight into the structural basis of IP3R activation and the contributions of IP3-evoked Ca2+ signals to cellular physiology. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signaling.

IP3 receptors: some lessons from DT40 cells.

Inositol-1,4,5-trisphosphate receptors (IP3Rs) are intracellular Ca2+ channels that are regulated by IP3 and Ca2+ and are modulated by many additional signals. These properties allow them to initiate and, via Ca2+-induced Ca2+ release, regeneratively propagate Ca2+ signals evoked by receptors that stimulate formation of IP3. The ubiquitous expression of IP3R highlights their importance, but it also presents problems when attempting to resolve the behavior of defined IP3R. DT40 cells are a pre-B-lymphocyte cell line in which high rates of homologous recombination afford unrivalled opportunities to disrupt endogenous genes. DT40-knockout cells with both alleles of each of the three IP3R genes disrupted provide the only null-background for analysis of homogenous recombinant IP3R. We review the properties of DT40 cells and consider three areas where they have contributed to understanding IP3R behavior. Patch-clamp recording from the nuclear envelope and Ca2+ release from intracellular stores loaded with a low-affinity Ca2+ indicator address the mechanisms leading to activation of IP(3)R. We show that IP3 causes intracellular IP3R to cluster and re-tune their responses to IP3 and Ca2+, better equipping them to mediate regenerative Ca2+ signals. Finally, we show that DT40 cells reliably count very few IP3R into the plasma membrane, where they mediate about half the Ca2+ entry evoked by the B-cell antigen receptor.

From parathyroid hormone to cytosolic Ca2+ signals.

PTHR1 (type 1 parathyroid hormone receptors) mediate the effects of PTH (parathyroid hormone) on bone remodelling and plasma Ca2+ homoeostasis. PTH, via PTHR1, can stimulate both AC (adenylate cyclase) and increases in [Ca2+]i (cytosolic free Ca2+ concentration), although the relationship between the two responses differs between cell types. In the present paper, we review briefly the mechanisms that influence coupling of PTHR1 to different intracellular signalling proteins, including the G-proteins that stimulate AC or PLC (phospholipase C). Stimulus intensity, the ability of different PTH analogues to stabilize different receptor conformations ('stimulus trafficking'), and association of PTHR1 with scaffold proteins, notably NHERF1 and NHERF2 (Na+/H+ exchanger regulatory factor 1 and 2), contribute to defining the interactions between signalling proteins and PTHR1. In addition, cAMP itself can, via Epac (exchange protein directly activated by cAMP), PKA (protein kinase A) or by binding directly to IP3Rs [Ins(1,4,5)P3 receptors] regulate [Ca2+]i. Epac leads to activation of PLCϵ, PKA can phosphorylate and thereby increase the sensitivity of IP3Rs and L-type Ca2+ channels, and cAMP delivered at high concentrations to IP3R2 from AC6 increases the sensitivity of IP3Rs to InsP3. The diversity of these links between PTH and [Ca2+]i highlights the versatility of PTHR1. This versatility allows PTHR1 to evoke different responses when stimulated by each of its physiological ligands, PTH and PTH-related peptide, and it provides scope for development of ligands that selectively harness the anabolic effects of PTH for more effective treatment of osteoporosis.

Timescales of IP(3)-evoked Ca(2+) spikes emerge from Ca(2+) puffs only at the cellular level.

The behavior of biological systems is determined by the properties of their component molecules, but the interactions are usually too complex to understand fully how molecular behavior generates cellular behavior. Ca(2+) signaling by inositol trisphosphate receptors (IP(3)R) offers an opportunity to understand this relationship because the cellular behavior is defined largely by Ca(2+)-mediated interactions between IP(3)R. Ca(2+) released by a cluster of IP(3)R (giving a local Ca(2+) puff) diffuses and ignites the behavior of neighboring clusters (to give repetitive global Ca(2+) spikes). We use total internal reflection fluorescence microscopy of two mammalian cell lines to define the temporal relationships between Ca(2+) puffs (interpuff intervals, IPI) and Ca(2+) spikes (interspike intervals) evoked by flash photolysis of caged IP(3). We find that IPI are much shorter than interspike intervals, that puff activity is stochastic with a recovery time that is much shorter than the refractory period of the cell, and that IPI are not periodic. We conclude that Ca(2+) spikes do not arise from oscillatory dynamics of IP(3)R clusters, but that repetitive Ca(2+) spiking with its longer timescales is an emergent property of the dynamics of the whole cluster array.

Regulation of inositol 1,4,5-trisphosphate receptors by cAMP independent of cAMP-dependent protein kinase.

In HEK cells stably expressing type 1 receptors for parathyroid hormone (PTH), PTH causes a sensitization of inositol 1,4,5-trisphosphate receptors (IP(3)R) to IP(3) that is entirely mediated by cAMP and requires cAMP to pass directly from type 6 adenylyl cyclase (AC6) to IP(3)R2. Using DT40 cells expressing single subtypes of mammalian IP(3)R, we demonstrate that high concentrations of cAMP similarly sensitize all IP(3)R isoforms to IP(3) by a mechanism that does not require cAMP-dependent protein kinase (PKA). IP(3) binding to IP(3)R2 is unaffected by cAMP, and sensitization is not mediated by the site through which ATP potentiates responses to IP(3). In single channel recordings from excised nuclear patches of cells expressing IP(3)R2, cAMP alone had no effect, but it increased the open probability of IP(3)R2 activated by a submaximal concentration of IP(3) alone or in combination with a maximally effective concentration of ATP. These results establish that cAMP itself increases the sensitivity of all IP(3)R subtypes to IP(3). For IP(3)R2, this sensitization results from cAMP binding to a novel site that increases the efficacy of IP(3). Using stably expressed short hairpin RNA to reduce expression of the G-protein, G alpha(s), we demonstrate that attenuation of AC activity by loss of G alpha(s) more substantially reduces sensitization of IP(3)R by PTH than does comparable direct inhibition of AC. This suggests that G alpha(s) may also specifically associate with each AC x IP(3)R complex. We conclude that all three subtypes of IP(3)R are regulated by cAMP independent of PKA. In HEK cells, where IP(3)R2 selectively associates with AC6, G alpha(s) also associates with the AC x IP(3)R signaling junction.

Synthetic partial agonists reveal key steps in IP3 receptor activation.

Inositol 1,4,5-trisphosphate receptors (IP(3)Rs) are ubiquitous intracellular Ca2+ channels. IP(3) binding to the IP(3)-binding core (IBC) near the N terminus initiates conformational changes that lead to opening of a pore. The mechanisms underlying this process are unresolved. We synthesized 2-O-modified IP(3) analogs that are partial agonists of IP(3)R. These are similar to IP(3) in their interactions with the IBC, but they are less effective than IP(3) in rearranging the relationship between the IBC and the N-terminal suppressor domain (SD), and they open the channel at slower rates. IP(3)R with a mutation in the SD occupying a position similar to the 2-O substituent of the partial agonists has a reduced open probability that is similar for full and partial agonists. Bulky or charged substituents from either the ligand or the SD therefore block obligatory coupling of the IBC and the SD. Analysis of DeltaG for ligand binding shows that IP(3) is recognized by the IBC and conformational changes then propagate entirely via the SD to the pore.

Activation of IP(3) receptors by synthetic bisphosphate ligands.

Ca(2+) release by d-myo-inositol 1,4,5-trisphosphate receptors (IP(3)Rs) is widely considered to require the vicinal 4,5-bisphosphate motif of IP(3), with P-5 and P-4 engaging the alpha and beta domains of the binding site; using synthesis and mutagenesis we show that the adenine of synthetic glyconucleotides, through an interaction with Arg504, can replace the interaction of either P-1 or P-5 with the alpha-domain producing, respectively, the most potent bisphosphate agonist yet synthesised and the first agonist of IP(3)R without a vicinal bisphosphate motif; this will stimulate new approaches to IP(3)R ligand design.

How does intracellular Ca2+ oscillate: by chance or by the clock?

Ca2+ oscillations have been considered to obey deterministic dynamics for almost two decades. We show for four cell types that Ca2+ oscillations are instead a sequence of random spikes. The standard deviation of the interspike intervals (ISIs) of individual spike trains is similar to the average ISI; it increases approximately linearly with the average ISI; and consecutive ISIs are uncorrelated. Decreasing the effective diffusion coefficient of free Ca2+ using Ca2+ buffers increases the average ISI and the standard deviation in agreement with the idea that individual spikes are caused by random wave nucleation. Array-enhanced coherence resonance leads to regular Ca2+ oscillations with small standard deviation of ISIs.

Lack of receptor-selective effects of either RGS2, RGS3 or RGS4 on muscarinic M3- and gonadotropin-releasing hormone receptor-mediated signalling through G alpha q/11.

Termination of signalling by G-protein-coupled receptors requires inactivation of the G alpha-subunits of heterotrimeric G-proteins and the re-association of G alpha- and G betagamma-subunits. Inactivation of G alpha-subunits is achieved by the hydrolysis of bound GTP by an intrinsic GTPase activity, which is considerably enhanced by GTPase activating proteins. Regulators of G-protein signalling (RGS) proteins are a large family of GTPase activating proteins, many of which have structures indicating roles beyond GTPase activating protein activity and suggesting that the identity of the RGS protein recruited may also be critical to other aspects of signalling. There is some evidence of selective effects of RGS proteins against different G-protein-coupled receptors coupling to the same signalling pathways and growing evidence of physical interactions between RGS proteins and G-protein-coupled receptors. However, it is unclear as to how common such interactions are and the circumstances under which they are functionally relevant. Here we have examined potential selectivity of RGS2, 3 and 4 against signalling mediated by G alpha q/11-coupled muscarinic M3 receptors and gonadotropin-releasing hormone in an immortalised mouse pituitary cell line. Despite major structural differences between these two receptor types and agonist-dependent phosphorylation of the muscarinic M3- but not gonadotropin-releasing hormone receptor, signalling by both receptors was similarly inhibited by expression of either RGS2 or RGS3, whereas RGS4 has little effect. Thus, at least in these circumstances, RGS protein-dependent inhibition of signalling is not influenced by the nature of the G-protein-coupled receptor through which the signalling is mediated.

Ca2+ signals initiate at immobile IP3 receptors adjacent to ER-plasma membrane junctions.

IP3 receptors (IP3Rs) release Ca2+ from the ER when they bind IP3 and Ca2+. The spatial organization of IP3Rs determines both the propagation of Ca2+ signals between IP3Rs and the selective regulation of cellular responses. Here we use gene editing to fluorescently tag endogenous IP3Rs, and super-resolution microscopy to determine the geography of IP3Rs and Ca2+ signals within living cells. We show that native IP3Rs cluster within ER membranes. Most IP3R clusters are mobile, moved by diffusion and microtubule motors. Ca2+ signals are generated by a small population of immobile IP3Rs. These IP3Rs are licensed to respond, but they do not readily mix with mobile IP3Rs. The licensed IP3Rs reside alongside ER-plasma membrane junctions where STIM1, which regulates store-operated Ca2+ entry, accumulates after depletion of Ca2+ stores. IP3Rs tethered close to ER-plasma membrane junctions are licensed to respond and optimally placed to be activated by endogenous IP3 and to regulate Ca2+ entry.

Cyclic AMP Recruits a Discrete Intracellular Ca2+ Store by Unmasking Hypersensitive IP3 Receptors.

Inositol 1,4,5-trisphosphate (IP3) stimulates Ca2+ release from the endoplasmic reticulum (ER), and the response is potentiated by 3',5'-cyclic AMP (cAMP). We investigated this interaction in HEK293 cells using carbachol and parathyroid hormone (PTH) to stimulate formation of IP3 and cAMP, respectively. PTH alone had no effect on the cytosolic Ca2+ concentration, but it potentiated the Ca2+ signals evoked by carbachol. Surprisingly, however, the intracellular Ca2+ stores that respond to carbachol alone could be both emptied and refilled without affecting the subsequent response to PTH. We provide evidence that PTH unmasks high-affinity IP3 receptors within a discrete Ca2+ store. We conclude that Ca2+ stores within the ER that dynamically exchange Ca2+ with the cytosol maintain a functional independence that allows one store to be released by carbachol and another to be released by carbachol with PTH. Compartmentalization of ER Ca2+ stores adds versatility to IP3-evoked Ca2+ signals.

A systematic study of C-glucoside trisphosphates as myo-inositol trisphosphate receptor ligands. Synthesis of beta-C-glucoside trisphosphates based on the conformational restriction strategy.

Beta-C-glucoside trisphosphates having a C2 side chain (3,7-anhydro-2-deoxy-D-glycero-D-gulo-octitol 1,5,6-trisphosphate, 11) and a C3 side chain (4,8-anhydro-2,3-dideoxy-D-glycero-D-gulo-nonanitol 1,6,7-trisphosphate, 12) were designed as structurally simplified analogues of a potent D-myo-inositol 1,4,5-trisphosphate (IP3) receptor ligand, adenophostin A. Construction of the beta-C-glucosidic structure, which was the key to their synthesis, was achieved by two different methods based on the conformational restriction strategy: (1) radical cyclization with a temporary connecting silicon tether and (2) silane reduction of glyconolactols having an anomeric allyl substituent. Using these methods, the target beta-C-glycoside trisphosphates 11 and 12 were successfully synthesized. A structure-activity relationship was established on a series of C-glucoside trisphosphates, including the previously synthesized related compounds, which were a C-glycosidic analogue 3 of adenophostin A, its uracil congener 5, alpha-C-glucoside trisphosphates 7-9 having a C1, C2, or C3 side chain, and the beta-C-glucoside trisphosphates 10-12 having a C1, C2, or C3 side chain. The O-glycosidic linkage of adenophostin A and its analogues proved to be replaced by the chemically and biologically more stable C-glycosidic linkage. The alpha-C2-glucoside trisphosphate 8 stimulates Ca2+ release with a potency similar to that of IP3 in spite of its simplified structure, indicating a better fit to the receptor than the beta-C-glucoside trisphosphates and also the alpha-congeners having a shorter or longer C1 side chain, which was supported by molecular modeling using the ligand binding domain of the IP3 receptor.

Synthesis of adenophostin A analogues conjugating an aromatic group at the 5'-position as potent IP3 receptor ligands.

Previous structure-activity relationship studies of adenophostin A, a potent IP(3) receptor agonist, led us to design the novel adenophostin A analogues 5a-c, conjugating an aromatic group at the 5'-position to develop useful IP(3) receptor ligands. The common key intermediate, a D-ribosyl alpha-D-glucoside 10alpha, was stereoselectively synthesized by a glycosidation with the 1-sulfinylglucoside donor 11, which was conformationally restricted by a 3,4-O-cyclic diketal protecting group. After introduction of an aromatic group at the 5-position of the ribose moiety, an adenine base was stereoselectively introduced at the anomeric beta-position to form 7a-c, where the tetra-O-i-butyryl donors 9a-c were significantly more effective than the corresponding O-acetyl donor. Thus, the target compounds 5a-c were synthesized via phosphorylation of the 2', 3' ', and 4' '-hydroxyls. The potencies of compounds 5a-c for Ca(2+) release were shown to be indistinguishable from that of adenophostin A, indicating that bulky substitutions at the 5'-position of adenophostin A are well-tolerated in the receptor binding. This biological activity of 5a-c can be rationalized by molecular modeling using the ligand binding domain of the IP(3) receptor.

Guanophostin A: Synthesis and evaluation of a high affinity agonist of the D-myo-inositol 1,4,5-trisphosphate receptor.

Guanophostin A, the guanosine counterpart of the inositol 1,4,5-trisphosphate receptor agonist adenophostin A, has been synthesized and is the first synthetic adenophostin A-like analogue to be equipotent to its parent in stimulating intracellular Ca2+ release; its nucleotide moiety is proposed to interact with the receptor binding core by guanine base cation-pi stacking with Arg504 and hydrogen bonding with Glu505 and interaction of the ribosyl 2'-phosphate group with the helix-dipole of alpha6.

Different phospholipase-C-coupled receptors differentially regulate capacitative and non-capacitative Ca2+ entry in A7r5 cells.

Several receptors, including those for AVP (Arg8-vasopressin) and 5-HT (5-hydroxytryptamine), share an ability to stimulate PLC (phospholipase C) and so production of IP3 (inositol 1,4,5-trisphosphate) and DAG (diacylglycerol) in A7r5 vascular smooth muscle cells. Our previous analysis of the effects of AVP on Ca2+ entry [Moneer, Dyer and Taylor (2003) Biochem. J. 370, 439-448] showed that arachidonic acid released from DAG stimulated NO synthase. NO then stimulated an NCCE (non-capacitative Ca2+ entry) pathway, and, via cGMP and protein kinase G, it inhibited CCE (capacitative Ca2+ entry). This reciprocal regulation ensured that, in the presence of AVP, all Ca2+ entry occurred via NCCE to be followed by a transient activation of CCE only when AVP was removed [Moneer and Taylor (2002) Biochem. J. 362, 13-21]. We confirm that, in the presence of AVP, all Ca2+ entry occurs via NCCE, but 5-HT, despite activating PLC and evoking release of Ca2+ from intracellular stores, stimulates Ca2+ entry only via CCE. We conclude that two PLC-coupled receptors differentially regulate CCE and NCCE. We also address evidence that, in some A7r5 cells lines, AVP fails either to stimulate NCCE or inhibit CCE [Brueggemann, Markun, Barakat, Chen and Byron (2005) Biochem. J. 388, 237-244]. Quantitative PCR analysis suggests that these cells predominantly express TRPC1 (transient receptor potential canonical 1), whereas cells in which AVP reciprocally regulates CCE and NCCE express a greater variety of TRPC subtypes (TRPC1=6>2>3).

Rapid functional assays of recombinant IP3 receptors.

Functional assays of inositol 1,4,5-trisphosphate receptors (IP3R) currently use 45Ca2+ release methods, fluorescent Ca2+ indicators within either the ER or cytosol, or electrophysiological analyses of IP3R in the nuclear envelope or artificial bilayers. None of the methods is presently amenable to the rapid, high-throughput quantitative analyses of IP3R function needed to address the structural determinants of IP3R behavior. We use a low-affinity Ca2+ indicator (Mag-fluo-4) to measure free [Ca2+] within the ER of permeabilized DT40 cells expressing only rat type 1 IP(3)R, and establish that the indicator is capable of reliably reporting the Ca(2+) release evoked by IP3. A 96-well fluorescence plate reader equipped for automated fluid additions (FlexStation, Molecular Devices) is used to monitor IP3-evoked Ca2+ release. The method allows quick and economical functional assays of recombinant IP3R in small volumes (< or = 100 microl).