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

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

Functional coupling of phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-trisphosphate receptor

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.     

Calcium-dependent clustering of inositol 1,4,5-trisphosphate receptors

Rat basophilic leukemia (RBL-2H3) cells predominantly express the type II receptor for inositol 1,4,5-trisphosphate (InsP3), which operates as an InsP3-gated calcium channel. In these cells, cross-linking the high-affinity immunoglobulin E receptor (FcepsilonR1) leads to activation of phospholipase C gamma isoforms via tyrosine kinase- and phosphatidylinositol 3-kinase-dependent pathways, release of InsP3-sensitive intracellular Ca2+ stores, and a sustained phase of Ca2+ influx. These events are accompanied by a redistribution of type II InsP3 receptors within the endoplasmic reticulum and nuclear envelope, from a diffuse pattern with a few small aggregates in resting cells to large isolated clusters after antigen stimulation. Redistribution of type II InsP3 receptors is also seen after treatment of RBL-2H3 cells with ionomycin or thapsigargin. InsP3 receptor clustering occurs within 5-10 min of stimulus and persists for up to 1 h in the presence of antigen. Receptor clustering is independent of endoplasmic reticulum vesiculation, which occurs only at ionomycin concentrations >1 microM, and maximal clustering responses are dependent on the presence of extracellular calcium. InsP3 receptor aggregation may be a characteristic cellular response to Ca2+-mobilizing ligands, because similar results are seen after activation of phospholipase C-linked G-protein-coupled receptors; cholecystokinin causes type II receptor redistribution in rat pancreatoma AR4-2J cells, and carbachol causes type III receptor redistribution in muscarinic receptor-expressing hamster lung fibroblast E36(M3R) cells. Stimulation of these three cell types leads to a reduction in InsP3 receptor levels only in AR4-2J cells, indicating that receptor clustering does not correlate with receptor down-regulation. The calcium-dependent aggregation of InsP3 receptors may contribute to the previously observed changes in affinity for InsP3 in the presence of elevated Ca2+ and/or may establish discrete regions within refilled stores with varying capacity to release Ca2+ when a subsequent stimulus results in production of InsP3.     

Hormone-evoked elementary Ca2+ signals are not stereotypic, but reflect activation of different size channel clusters and variable recruitment of channels within a cluster

Previous studies of (InsP3)-evoked elementary Ca2+ events suggested a hierarchy of signals; fundamental events ("Ca2+ blips") arising from single InsP3 receptors (InsP3Rs), and intermediate events ("Ca2+ puffs") reflecting the coordinated opening of a cluster of InsP3Rs. The characteristics of such elementary Ca2+ release signals provide insights into the functional interaction and distribution of InsP3Rs in living cells. Therefore we investigated whether elementary Ca2+ signaling is truly represented by such stereotypic release events. A histogram of >900 events revealed a wide spread of signal amplitudes (20-600 nM; mean 216 +/- 4 nM; n = 206 cells), which cannot be explained by stochastic variation of a stereotypic Ca2+ release site. We identified elementary Ca2+ release sites with consistent amplitudes (<20% difference) and locations with variable amplitudes (approximately 500% difference). Importantly, within single cells, distinct sites displayed events with significantly different mean amplitudes. Additional determinants affecting the magnitude of elementary Ca2+ release were identified to be (i) hormone concentration, (ii) day-to-day variability, and (iii) a progressively decreasing Ca2+ release during prolonged stimulation. We therefore suggest that elementary Ca2+ events are not stereotypic, instead a continuum of signals can be achieved by either recruitment of entire clusters with different numbers of InsP3Rs or by a graded recruitment of InsP3Rs within a cluster.     

Expression and regulation of types I and II inositol 1,4,5-trisphosphate receptors in rat cerebellar granule cell preparations

Previous studies have shown that as rat cerebellar granule cell cultures differentiate in the presence of 25 mM KCl, they "up-regulate" their ability to form inositol phosphates and release Ca2+ from internal stores in response to the activation of phosphoinositidase C-linked muscarinic and metabotropic receptors. Here we show that they simultaneously up-regulate their ability to respond to inositol 1,4,5-trisphosphate (InsP3) by increasing InsP3 receptor (InsP3R) expression. In contrast, if granule cells are maintained at the more physiological KCl concentration of 5 mM, most cells undergo apoptosis, although a significant number survive. The surviving cells, however, express few InsP3Rs, suggesting that an influx of Ca2+ through voltage-dependent channels is required for InsP3R up-regulation. In addition, we have determined that these cultures express two genetically distinct InsP3R types, but that only one, the type I receptor, is expressed in granule cells. Type II receptors are also present but are found exclusively in astrocytes, which are a minor contaminant of granule cell cultures. This segregation of InsP3R types explains a previous observation, showing that the muscarinic agonist carbachol causes the reduction or "down-regulation" of type I but not type II InsP3Rs.     

Single channel function of recombinant type-1 inositol 1,4,5-trisphosphate receptor ligand binding domain splice variants

In this study we describe the expression and function of the two rat type-1 inositol 1,4,5-trisphosphate receptor (InsP3R) ligand binding domain splice variants (SI+/-/SII+). Receptor protein from COS-1 cells transfected with the type-1 InsP3R expression plasmids (pInsP3R-T1, pInsP3R-T1ALT) or control DNA were incorporated into planar lipid bilayers and the single channel properties of the recombinant receptors were defined. The unitary conductance of the two splice variants were approximately 290 pS with Cs+ as charge carrier and approximately 65 pS with Ca2+ as charge carrier. Both InsP3R expression products consistently behaved like those of the native type-1 receptor isoform isolated from cerebellum in terms of their InsP3, Ca2+, and heparin sensitivity. An InsP3 receptor ligand binding domain truncation lacking the 310 amino-terminal amino acids (pInsP3R-DeltaT1ALT) formed tetrameric complexes but failed to bind InsP3 with high affinity, and did not form functional Ca2+ channels when reconstituted in lipid bilayers. These data suggest that 1) the ligand binding alternative splice site is functionally inert in terms of InsP3 binding and single channel function, and 2) the single channel properties of the expressed recombinant type-1 channel are essentially identical to those of the native channel. This work establishes a foundation from which molecular/biophysical approaches can be used to define the structure-function properties of the InsP3 receptor channel family.     

Functional interaction between InsP3 receptors and store-operated Htrp3 channels

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).     

The high affinity state of inositol 1,4,5-trisphosphate receptor is a functional state

Inositol 1,4,5-trisphosphate (InsP3) is a second messenger responsible for the rapid and discontinuous release of Ca2+ from intracellular stores. In this study, the effects of the sulfhydryl reagent thimerosal were investigated on Ca2+ mobilization and on InsP3 binding. Thimerosal was shown to release Ca2+, in a dose-dependent manner, with an EC50 of 135.8 +/- 5.2 microM, from bovine adrenal cortex microsomes. Thimerosal-induced Ca2+ release was not prevented by heparin (250 micrograms/ml), ruling out a participation of InsP3 receptor in that effect. The slow rate of thimerosal-induced Ca2+ release rather suggested an inhibition of microsomal Ca2+ ATPase. At submaximal concentration, thimerosal (100 microM) was also shown to potentiate the release of Ca2+ induced by InsP3. Dose-response experiments revealed that thimerosal enhanced the apparent affinity of InsP3 by a factor 2.21 +/- 0.28, without modifying the maximal amount of Ca2+ released by InsP3. Thimerosal also enhanced, in a dose-dependent manner, [3H]InsP3 binding to adrenal cortex microsomes (EC50 = 43.3 +/- 7.6 microM). A similar effect was also observed on [3H]InsP3 binding to solubilized receptors, suggesting a direct modification of the receptor protein by thimerosal. The effects of thimerosal on Ca2+ release and [3H]InsP3 binding were abolished in the presence of the reducing agent dithiothreitol (1 mM), suggesting a modification by thimerosal of specific thiol groups on these microsomal proteins. Scatchard analysis revealed that thimerosal (100 microM) increased InsP3 receptor affinity by 1.87 +/- 0.26-fold. Kinetic analysis indicated that this increased affinity was due to an enhancement of InsP3 association rate constant. The concomitant increases of binding affinity and Ca2+ releasing potency suggest that the high affinity state of InsP3 receptor is a functional state.     

Kinetics of Ca2+ release evoked by photolysis of caged InsP3 in rat submandibular cells

Quantitative time-resolved measurements of cytosolic Ca2+ release by photolysis of caged InsP3 have been made in single rat submandibular cells using patch clamp whole-cell recording to measure the Ca2+-activated Cl- and K+ currents. Photolytic release of InsP3 from caged InsP3 at 100 Joules caused transient inward (V(H) = 60 mV) and outward (V(H) = 0 mV) currents, which were nearly symmetric in their time course. The inward current was reduced when pipette Cl- concentration was decreased, and the outward current was suppressed by K+ channel blockers, indicating that they were carried by Cl- and K+, respectively. Intracellular pre-loading of the InsP3 receptor antagonist heparin or the Ca2+ chelator EGTA clearly prevented both inward and outward currents, indicating that activation of Ca2+-dependent Cl- and K+ currents underlies the inward and the outward currents. At low flash intensities, InsP3 caused Ca2+ release which normally activated the K+ and Cl- currents in a mono-transient manner. At higher intensities, however, InsP3 induced an additional delayed outward K+ current (I[K,(delay)]). I[K(delay)] was independent of the initial K+ current, independent of extracellular Ca2+, inhibited by TEA, and gradually prolongated by repeated flashes. The photolytic release of Ca2+ from caged Ca2+ did not mimic the I[K(delay)]. It is suggested that Ca2+ releases from the InsP3-sensitive pools in an InsP3 concentration-dependent manner. Low concentrations of InsP3 induce the transient Ca2+-dependent Cl- and K+ currents, which reflects the local Ca2+ release, whereas high concentrations of InsP3 induce a delayed Ca2+-dependent K+ current, which may reflect the Ca2+ wave propagation.     

Ca2+ spike initiation from sensitized inositol 1,4,5-trisphosphate-sensitive Ca2+ stores in megakaryocytes

Ca(2+)-mediated Ca2+ spikes were analysed in fura-2-loaded megakaryocytes. Direct Ca2+ loading using whole-cell dialysis induced an all-or-none Ca2+ spike on top of a tonic increase in cellular Ca2+ concentration ([Ca2+]i) with a latency of 3-7 s. The latency decreased with increasingly higher concentrations of Ca2+ in the dialysing solution. Spike size and its initiation did not correlate with the tonic level of [Ca2+]i. Thapsigargin completely abolished the Ca(2+)-induced spike initiation, suggesting that Ca2+ spikes originate from thapsigargin-sensitive Ca2+ pools. An inhibitor of phosphatidylinositide-specific phospholipase C (PLC), 2-nitro-4-carboxyphenyl-N,N-diphenyl-carbamate prolonged the latency without changes of spike size in most cases (6/9 cells), but abolished the spike initiation in the other cells (3/9). The results suggest that an increase in [Ca2+]i charges up the inositol-1,4,5-trisphosphate-(InsP3)- and thapsigargin-sensitive Ca2+ pools which progressively sensitize to low or slightly elevated levels of InsP3 by the action of Ca(2+)-dependent PLC until a critical Ca2+ content is reached, and then the Ca2+ spike is triggered. Thus, the limiting step of Ca2+ spike triggering is the initial filling process and the level of InsP3 in megakaryocytes.     

Functional expression and signaling properties of cloned human parathyroid hormone receptor in Xenopus oocytes. Evidence for a novel signaling pathway

Expression of human parathyroid hormone receptor (hPTHR) was obtained in Xenopus oocytes. Receptor function was detected by hormone stimulation of endogenous Ca2+-activated Cl- current. This current was blocked by injected, but not by extracellular, EGTA, confirming that the hPTHR activates cytosolic Ca2+ signaling pathways. PTH responses were acutely desensitized but were regained in 6 12 h. Injection of cAMP or analogues had no effect on either responsiveness or desensitization to hPTH. The hPTH response was more sluggish than seen with serotonin 5-hydroxytryptamine (5-HT2C) receptor. In oocytes co-expressing both hPTHR and 5-HT2C receptors, homologous desensitization was seen, but cross-desensitization was not observed. Injection of inositol 1,4,5-trisphosphate (InsP3) elicited a fast inward current similar to that induced by serotonin, and complete cross-desensitization occurred between the InsP3 and 5-HT2C responses. Desensitization by hPTH did not affect responses to either InsP3 or serotonin, but cells desensitized to injected InsP3 still responded strongly to PTH. Oocytes did not respond to either cADPR or NAADP+, but NADP+ and analogues were found to be potent inhibitors of PTH signaling. We suggest that PTH cytosolic Ca2+ signaling in oocytes either involves a novel signaling system or proceeds through a Ca2+ compartment whose responsiveness is regulated in a novel way.     

Inositol 1,4,5-trisphosphate-induced Ca2+ release is regulated by cytosolic Ca2+ in intact skeletal muscle

Microinjection of inositol 1,4,5-trisphosphate (InsP3) into intact skeletal muscle fibers isolated from frogs (Rana temporaria) increased resting cytosolic Ca2+ concentration ([Ca2+]i) as measured by double-barreled Ca2+-selective microelectrodes. In contrast, microinjection of inositol 1-phosphate, inositol 1,4-biphosphate, and inositol 1,4,5,6-tetrakisphosphate did not induce changes in [Ca2+]i. Incubation in low-Ca2+ solution, or in the presence of L-type Ca2+ channel blockers did not affect InsP3-induced release of cytosolic Ca2+. Neither ruthenium red, a blocker of ryanodine receptor Ca2+-release channels, nor cytosolic Mg2+, a known inhibitor of the Ca2+-induced Ca2+-release process, modified the InsP3-induced release of cytosolic Ca2+. However, heparin, a blocker of InsP3 receptors, inhibited InsP3-induced release of cytosolic Ca2+. Also, pretreatment with dantrolene or azumulene, two inhibitors of cytosolic Ca2+ release, reduced [Ca2+]i, and prevented InsP3 from inducing release of cytosolic Ca2+. Incubation in caffeine or lengthening of the muscle increased [Ca2+]i and enhanced the ability of InsP3 to induce release of cytosolic Ca2+. These results indicate that InsP3, at physiological concentrations, induces Ca2+ release in intact muscle fibers, and suggest that the InsP3-induced Ca2+ release is regulated by [Ca2+]i. A Ca2+-dependent effect of InsP3 on cytosolic Ca2+ release could be of importance under physiological or pathophysiological conditions associated with alterations in cytosolic Ca2+ homeostasis.     

Inositol 1,4,5-trisphosphate receptor down-regulation is activated directly by inositol 1,4,5-trisphosphate binding. Studies with binding-defective mutant receptors

Activation of certain phosphoinositidase C-linked cell surface receptors is known to cause an acceleration of the proteolysis of inositol 1,4,5-trisphosphate (InsP3) receptors and, thus, lead to InsP3 receptor down-regulation. To gain insight into this process, we examined whether or not InsP3 receptor degradation is a direct consequence of InsP3 binding by analyzing the down-regulation of exogenous wild-type and binding-defective mutant InsP3 receptors expressed in SH-SY5Y human neuroblastoma cells. Stimulation of these cells with carbachol showed that wild-type exogenous receptors could be down-regulated but that the binding-defective mutant exogenous receptors were not. Thus, InsP3 binding appears to mediate down-regulation. To validate this conclusion, a comprehensive analysis of the effects of the exogenous receptors was undertaken. This showed that exogenous receptors (i) are localized appropriately within the cell, (ii) enhance InsP3-induced Ca2+ release in permeabilized cells, presumably by increasing the number of InsP3-sensitive Ca2+ channels, (iii) have minimal effects on Ca2+ mobilization and InsP3 formation in intact cells, (iv) form heteromers with endogenous receptors, and (v) do not alter the down-regulation of endogenous receptors. In total, these data show that the introduction of exogenous receptors into SH-SY5Y cells does not compromise intracellular signaling or the down-regulatory process. We can thus conclude that InsP3 binding directly activates InsP3 receptor degradation. Because InsP3 binding induces a conformational change in the InsP3 receptor, these data suggest that this change provides the signal for accelerated proteolysis.     

Inositol trisphosphate receptors: Ca2+-modulated intracellular Ca2+ channels

The three subtypes of inositol trisphosphate (InsP3) receptor expressed in mammalian cells are each capable of forming intracellular Ca2+ channels that are regulated by both InsP3 and cytosolic Ca2+. The InsP3 receptors of many, though perhaps not all, tissues are biphasically regulated by cytosolic Ca2+: a rapid stimulation of the receptors by modest increases in Ca2+ concentration is followed by a slower inhibition at higher Ca2+ concentrations. Despite the widespread occurrence of this form of regulation and the belief that it is an important element of the mechanisms responsible for the complex Ca2+ signals evoked by physiological stimuli, the underlying mechanisms are not understood. Both accessory proteins and Ca2+-binding sites on InsP3 receptors themselves have been proposed to mediate the effects of cytosolic Ca2+ on InsP3 receptor function, but the evidence is equivocal. The effects of cytosolic Ca2+ on InsP3 binding and channel opening, and the possible means whereby the effects are mediated are discussed in this review.     

Regulation of Ca2+ release by InsP3 in single guinea pig hepatocytes and rat Purkinje neurons

The repetitive spiking of free cytosolic [Ca2+] ([Ca2+]i) during hormonal activation of hepatocytes depends on the activation and subsequent inactivation of InsP3-evoked Ca2+ release. The kinetics of both processes were studied with flash photolytic release of InsP3 and time resolved measurements of [Ca2+]i in single cells. InsP3 evoked Ca2+ flux into the cytosol was measured as d[Ca2+]i/dt, and the kinetics of Ca2+ release compared between hepatocytes and cerebellar Purkinje neurons. In hepatocytes release occurs at InsP3 concentrations greater than 0.1-0.2 microM. A comparison with photolytic release of metabolically stable 5-thio-InsP3 suggests that metabolism of InsP3 is important in determining the minimal concentration needed to produce Ca2+ release. A distinct latency or delay of several hundred milliseconds after release of low InsP3 concentrations decreased to a minimum of 20-30 ms at high concentrations and is reduced to zero by prior increase of [Ca2+]i, suggesting a cooperative action of Ca2+ in InsP3 receptor activation. InsP3-evoked flux and peak [Ca2+]i increased with InsP3 concentration up to 5-10 microM, with large variation from cell to cell at each InsP3 concentration. The duration of InsP3-evoked flux, measured as 10-90% risetime, showed a good reciprocal correlation with d[Ca2+]i/dt and much less cell to cell variation than the dependence of flux on InsP3 concentration, suggesting that the rate of termination of the Ca2+ flux depends on the free Ca2+ flux itself. Comparing this data between hepatocytes and Purkinje neurons shows a similar reciprocal correlation for both, in hepatocytes in the range of low Ca2+ flux, up to 50 microM. s-1 and in Purkinje neurons at high flux up to 1,400 microM. s-1. Experiments in which [Ca2+]i was controlled at resting or elevated levels support a mechanism in which InsP3-evoked Ca2+ flux is inhibited by Ca2+ inactivation of closed receptor/channels due to Ca2+ accumulation local to the release sites. Hepatocytes have a much smaller, more prolonged InsP3-evoked Ca2+ flux than Purkinje neurons. Evidence suggests that these differences in kinetics can be explained by the much lower InsP3 receptor density in hepatocytes than Purkinje neurons, rather than differences in receptor isoform, and, more generally, that high InsP3 receptor density promotes fast rising, rapidly inactivating InsP3-evoked [Ca2+]i transients.     

Local calcium signalling by inositol-1,4,5-trisphosphate in Purkinje cell dendrites

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

Rapid activation and partial inactivation of inositol trisphosphate receptors by inositol trisphosphate

During superfusion of permeabilized hepatocytes, submaximal concentrations of inositol 1,4,5-trisphosphate (InsP3) evoked quantal Ca2+ mobilization: a rapid acceleration in the rate of 45Ca2+ release abruptly followed by a biphasic decline to the basal rate before the InsP3-sensitive stores had fully emptied. During the fast component of the decay, the Ca2+ permeability of the stores fell rapidly by 40% (t1/2 = 250 ms) to a state indistinguishable from that evoked by preincubation with InsP3 under conditions that prevented Ca2+ mobilization. This change was accompanied by a decrease in the InsP3 dissociation rate: the response declined more quickly when InsP3 was removed during the initial stages of a response than later. We suggest that InsP3 directly causes its receptor to rapidly switch (t1/2 = 250 ms) between a low-affinity (Kd approximately 1 microM) active, and a higher-affinity (Kd approximately 100 nM) less active, conformation, and that this transition underlies the fast component of the decaying phase of Ca2+ release. Ca2+ continues to leak through the unchanging less active state of the receptor until those stores that responded initially are completely empty, accounting for the slow phase of the response. The requirements for activation of InsP3 receptors are more stringent (InsP3 and then Ca2+ binding) than those for partial inactivation (InsP3 binding); rapid inactivation is therefore likely to determine whether the cytosolic [Ca2+] reaches the threshold for regenerative Ca2+ signals.     

The role of inorganic phosphate in regulating the kinetics of inositol 1,4,5-trisphosphate-induced Ca2+ release: a putative role for endoplasmic reticulum phosphate transporters

The effects of phosphate and acylphosphonate phosphate transporter inhibitors were investigated on inositol 1,4,5-trisphosphate (InsP3)-induced Ca2+ release from cerebellar microsomes. Although neither changing the phosphate concentration nor adding phosphate transporter inhibitors affected the percentage (extent) of InsP3-induced Ca2+ release, they did, however, affect the transient kinetics of this process. InsP3-induced Ca2+ release is biphasic in nature, arising from two populations of InsP3-sensitive Ca2+ stores which either release Ca2+ in a fast or slow fashion. Altering phosphate concentration or adding phosphate transporter inhibitors appeared to affect only the fast phase component. We therefore suggest that these observations could be explained by the possibility that phosphate transporters only reside in the fast releasing InsP3-sensitive Ca2+ stores.