Regulation of the type III InsP(3) receptor by InsP(3) and calcium

It has been proposed that the inositol 1,4,5-trisphosphate receptor (InsP(3)R) type III acts as a trigger for InsP(3)-mediated calcium (Ca(2+)) signaling, because this InsP(3) isoform lacks feedback inhibition by cytosolic Ca(2+). We tested this hypothesis in RIN-m5F cells, which express predominantly the type III receptor. Extracellular ATP increases Ca(2+) in these cells, and we found that this effect is independent of extracellular Ca(2+) but is blocked by the InsP(3)R antagonist heparin. There was a dose-dependent increase in the number of cells responding to ATP and two-photon flash photolysis of caged-Ca(2+) heightened the sensitivity of RIN-m5F cells to this increase. These findings provide evidence that Ca(2+) increases the sensitivity of the InsP(3)R type III in intact cells and supports the idea that this isoform can act as a trigger for hormone-induced Ca(2+) signaling.     

Unitary Ca(2+) current through recombinant type 3 InsP(3) receptor channels under physiological ionic conditions

The ubiquitous inositol 1,4,5-trisphosphate (InsP(3)) receptor (InsP(3)R) channel, localized primarily in the endoplasmic reticulum (ER) membrane, releases Ca(2+) into the cytoplasm upon binding InsP(3), generating and modulating intracellular Ca(2+) signals that regulate numerous physiological processes. Together with the number of channels activated and the open probability of the active channels, the size of the unitary Ca(2+) current (i(Ca)) passing through an open InsP(3)R channel determines the amount of Ca(2+) released from the ER store, and thus the amplitude and the spatial and temporal nature of Ca(2+) signals generated in response to extracellular stimuli. Despite its significance, i(Ca) for InsP(3)R channels in physiological ionic conditions has not been directly measured. Here, we report the first measurement of i(Ca) through an InsP(3)R channel in its native membrane environment under physiological ionic conditions. Nuclear patch clamp electrophysiology with rapid perfusion solution exchanges was used to study the conductance properties of recombinant homotetrameric rat type 3 InsP(3)R channels. Within physiological ranges of free Ca(2+) concentrations in the ER lumen ([Ca(2+)](ER)), free cytoplasmic [Ca(2+)] ([Ca(2+)](i)), and symmetric free [Mg(2+)] ([Mg(2+)](f)), the i(Ca)-[Ca(2+)](ER) relation was linear, with no detectable dependence on [Mg(2+)](f). i(Ca) was 0.15 +/- 0.01 pA for a filled ER store with 500 microM [Ca(2+)](ER). The i(Ca)-[Ca(2+)](ER) relation suggests that Ca(2+) released by an InsP(3)R channel raises [Ca(2+)](i) near the open channel to approximately 13-70 microM, depending on [Ca(2+)](ER). These measurements have implications for the activities of nearby InsP(3)-liganded InsP(3)R channels, and they confirm that Ca(2+) released by an open InsP(3)R channel is sufficient to activate neighboring channels at appropriate distances away, promoting Ca(2+)-induced Ca(2+) release.     

Calpain-cleaved type 1 inositol 1,4,5-trisphosphate receptor (InsP(3)R1) has InsP(3)-independent gating and disrupts intracellular Ca(2+) homeostasis

The type 1 inositol 1,4,5-trisphosphate receptor (InsP(3)R1) is a ubiquitous intracellular Ca(2+) release channel that is vital to intracellular Ca(2+) signaling. InsP(3)R1 is a proteolytic target of calpain, which cleaves the channel to form a 95-kDa carboxyl-terminal fragment that includes the transmembrane domains, which contain the ion pore. However, the functional consequences of calpain proteolysis on channel behavior and Ca(2+) homeostasis are unknown. In the present study we have identified a unique calpain cleavage site in InsP(3)R1 and utilized a recombinant truncated form of the channel (capn-InsP(3)R1) corresponding to the stable, carboxyl-terminal fragment to examine the functional consequences of channel proteolysis. Single-channel recordings of capn-InsP(3)R1 revealed InsP(3)-independent gating and high open probability (P(o)) under optimal cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) conditions. However, some [Ca(2+)](i) regulation of the cleaved channel remained, with a lower P(o) in suboptimal and inhibitory [Ca(2+)](i). Expression of capn-InsP(3)R1 in N2a cells reduced the Ca(2+) content of ionomycin-releasable intracellular stores and decreased endoplasmic reticulum Ca(2+) loading compared with control cells expressing full-length InsP(3)R1. Using a cleavage-specific antibody, we identified calpain-cleaved InsP(3)R1 in selectively vulnerable cerebellar Purkinje neurons after in vivo cardiac arrest. These findings indicate that calpain proteolysis of InsP(3)R1 generates a dysregulated channel that disrupts cellular Ca(2+) homeostasis. Furthermore, our results demonstrate that calpain cleaves InsP(3)R1 in a clinically relevant injury model, suggesting that Ca(2+) leak through the proteolyzed channel may act as a feed-forward mechanism to enhance cell death.     

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

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

The type 2 inositol (1,4,5)-trisphosphate (InsP3) receptor determines the sensitivity of InsP3-induced Ca2+ release to ATP in pancreatic acinar cells

Calcium release through inositol (1,4,5)-trisphosphate receptors (InsP(3)R) is the primary signal driving digestive enzyme and fluid secretion from pancreatic acinar cells. The type 2 (InsP(3)R2) and type 3 (InsP(3)R3) InsP(3)R are the predominant isoforms expressed in acinar cells and are required for proper exocrine gland function. Both InsP(3)R2 and InsP(3)R3 are positively regulated by cytosolic ATP, but InsP(3)R2 is 10-fold more sensitive than InsP(3)R3 to this form of modulation. In this study, we examined the role of InsP(3)R2 in setting the sensitivity of InsP(3)-induced Ca(2+) release (IICR) to ATP in pancreatic acinar cells. IICR was measured in permeabilized acinar cells from wild-type (WT) and InsP(3)R2 knock-out (KO) mice. ATP augmented IICR from WT pancreatic cells with an EC(50) of 38 mum. However, the EC(50) was 10-fold higher in acinar cells isolated from InsP(3)R2-KO mice, indicating a role for InsP(3)R2 in setting the sensitivity of IICR to ATP. Consistent with this idea, heterologous expression of InsP(3)R2 in RinM5F cells, which natively express predominately InsP(3)R3, increased the sensitivity of IICR to ATP. Depletion of ATP attenuated agonist-induced Ca(2+) signaling in WT pancreatic acinar cells. This effect was more profound in acinar cells prepared from InsP(3)R2-KO mice. These data suggest that the sensitivity of IICR to ATP depletion is regulated by the particular complement of InsP(3)R expressed in an individual cell. The effects of metabolic stress on intracellular Ca(2+) signals can therefore be determined by the relative amount of InsP(3)R2 expressed in cells.     

Functional characterization of mammalian inositol 1,4,5-trisphosphate receptor isoforms

Inositol 1,4,5-trisphosphate receptors (InsP3R) play a key role in intracellular calcium (Ca2+) signaling. Three mammalian InsP3R isoforms--InsP3R type 1 (InsP3R1), InsP3R type 2 (InsP3R2), and InsP3R type 3 (InsP3R3) are expressed in mammals, but the functional differences between the three mammalian InsP3R isoforms are poorly understood. Here we compared single-channel behavior of the recombinant rat InsP3R1, InsP3R2, and InsP3R3 expressed in Sf9 cells, reconstituted into planar lipid bilayers and recorded with 50 mM Ba2+ as a current carrier. We found that: 1), for all three mammalian InsP3R isoforms the size of the unitary current is 1.9 pA and single-channel conductance is 74-80 pS; 2), in optimal recording conditions the maximal single-channel open probability for all three mammalian InsP3R isoforms is in the range 30-40%; 3), in optimal recording conditions the mean open dwell time for all three mammalian InsP3R isoforms is 7-8 ms, the mean closed dwell time is approximately 10 ms; 4), InsP3R2 has the highest apparent affinity for InsP(3) (0.10 microM), followed by InsP3R1 (0.27 microM), and then by InsP3R3 (0.40 microM); 5), InsP3R1 has a high-affinity (0.13 mM) ATP modulatory site, InsP3R2 gating is ATP independent, and InsP3R3 has a low-affinity (2 mM) ATP modulatory site; 6), ATP modulates InsP3R1 gating in a noncooperative manner (n(Hill) = 1.3); 7), ATP modulates InsP3R3 gating in a highly cooperative manner (n(Hill) = 4.1). Obtained results provide novel information about functional properties of mammalian InsP3R isoforms.     

ATP regulation of type 1 inositol 1,4,5-trisphosphate receptor channel gating by allosteric tuning of Ca(2+) activation.

Inositol 1,4,5-trisphosphate (InsP(3)) mobilizes intracellular Ca(2+) by binding to its receptor (InsP(3)R), an endoplasmic reticulum-localized Ca(2+) release channel. Patch clamp electrophysiology of Xenopus oocyte nuclei was used to study the effects of cytoplasmic ATP concentration on the cytoplasmic Ca(2+) ([Ca(2+)](i)) dependence of single type 1 InsP(3)R channels in native endoplasmic reticulum membrane. Cytoplasmic ATP free-acid ([ATP](i)), but not the MgATP complex, activated gating of the InsP(3)-liganded InsP(3)R, by stabilizing open channel state(s) and destabilizing the closed state(s). Activation was associated with a reduction of the half-maximal activating [Ca(2+)](i) from 500 +/- 50 nM in 0 [ATP](i) to 29 +/- 4 nM in 9.5 mM [ATP](i), with apparent ATP affinity = 0.27 +/- 0.04 mM, similar to in vivo concentrations. In contrast, ATP was without effect on maximum open probability or the Hill coefficient for Ca(2+) activation. Thus, ATP enhances gating of the InsP(3)R by allosteric regulation of the Ca(2+) sensitivity of the Ca(2+) activation sites of the channel. By regulating the Ca(2+)-induced Ca(2+) release properties of the InsP(3)R, ATP may play an important role in shaping cytoplasmic Ca(2+) signals, possibly linking cell metabolic state to important Ca(2+)-dependent processes.     

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

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

Spontaneous channel activity of the inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R). Application of allosteric modeling to calcium and InsP3 regulation of InsP3R single-channel gating

The InsP3R Ca2+ release channel has a biphasic dependence on cytoplasmic free Ca2+ concentration ([Ca2+]i). InsP3 activates gating primarily by reducing the sensitivity of the channel to inhibition by high [Ca2+]i. To determine if relieving Ca2+ inhibition is sufficient for channel activation, we examined single-channel activities in low [Ca2+]i in the absence of InsP3, by patch clamping isolated Xenopus oocyte nuclei. For both endogenous Xenopus type 1 and recombinant rat type 3 InsP3R channels, spontaneous InsP3-independent channel activities with low open probability Po ( approximately 0.03) were observed in [Ca2+]i < 5 nM with the same frequency as in the presence of InsP3, whereas no activities were observed in 25 nM Ca2+. These results establish the half-maximal inhibitory [Ca2+]i of the channel to be 1.2-4.0 nM in the absence of InsP3, and demonstrate that the channel can be active when all of its ligand-binding sites (including InsP3) are unoccupied. In the simplest allosteric model that fits all observations in nuclear patch-clamp studies of [Ca2+]i and InsP3 regulation of steady-state channel gating behavior of types 1 and 3 InsP3R isoforms, including spontaneous InsP3-independent channel activities, the tetrameric channel can adopt six different conformations, the equilibria among which are controlled by two inhibitory and one activating Ca2+-binding and one InsP3-binding sites in a manner outlined in the Monod-Wyman-Changeux model. InsP3 binding activates gating by affecting the Ca2+ affinities of the high-affinity inhibitory sites in different conformations, transforming it into an activating site. Ca2+ inhibition of InsP3-liganded channels is mediated by an InsP3-independent low-affinity inhibitory site. The model also suggests that besides the ligand-regulated gating mechanism, the channel has a ligand-independent gating mechanism responsible for maximum channel Po being less than unity. The validity of this model was established by its successful quantitative prediction of channel behavior after it had been exposed to ultra-low bath [Ca2+].     

ATP modulation of Ca2+ release by type-2 and type-3 inositol (1, 4, 5)-triphosphate receptors. Differing ATP sensitivities and molecular determinants of action

ATP enhances Ca(2+) release from inositol (1,4,5)-trisphosphate receptors (InsP(3)R). However, the three isoforms of InsP(3)R are reported to respond to ATP with differing sensitivities. Ca(2+) release through InsP(3)R1 is positively regulated at lower ATP concentrations than InsP(3)R3, and InsP(3)R2 has been reported to be insensitive to ATP modulation. We have reexamined these differences by studying the effects of ATP on InsP(3)R2 and InsP(3)R3 expressed in isolation on a null background in DT40 InsP(3)R knockout cells. We report that the Ca(2+)-releasing activity as well as the single channel open probability of InsP(3)R2 was enhanced by ATP, but only at submaximal InsP(3) levels. Further, InsP(3)R2 was more sensitive to ATP modulation than InsP(3)R3 under similar experimental conditions. Mutations in the ATPB sites of InsP(3)R2 and InsP(3)R3 were generated, and the functional consequences of these mutations were tested. Surprisingly, mutation of the ATPB site in InsP(3)R3 had no effect on ATP modulation, suggesting an additional locus for the effects of ATP on this isoform. In contrast, ablation of the ATPB site of InsP(3)R2 eliminated the enhancing effects of ATP. Furthermore, this mutation had profound effects on the patterns of intracellular calcium signals, providing evidence for the physiological significance of ATP binding to InsP(3)R2.     

Modulation of mammalian inositol 1,4,5-trisphosphate receptor isoforms by calcium: a role of calcium sensor region

In the accompanying article, we compared main functional properties of the three mammalian inositol 1,4,5-trisphosphate receptors (InsP3R) isoforms. In this article we focused on modulation of mammalian InsP3R isoforms by cytosolic Ca2+. We found that: 1), when recorded in the presence of 2 microM InsP3 and 0.5 mM ATP all three mammalian InsP3R isoforms display bell-shaped Ca2+ dependence in physiological range of Ca2+ concentrations (pCa 8-5); 2), in the same experimental conditions InsP3R3 is most sensitive to modulation by Ca2+ (peak at 107 nM Ca2+), followed by InsP3R2 (peak at 154 nM Ca2+), and then by InsP3R1 (peak at 257 nM Ca2+); 3), increase in ATP concentration to 5 mM had no significant effect of Ca2+ dependence of InsP3R1 and InsP3R2; 4), increase in ATP concentration to 5 mM converted Ca2+ dependence of InsP3R3 from "narrow" shape to "square" shape; 5), ATP-induced change in the shape of InsP3R3 Ca2+ dependence was mainly due to an >200-fold reduction in the apparent affinity of the Ca2+-inhibitory site; 6), the apparent Ca2+ affinity of the Ca2+ sensor region (Cas) determined in biochemical experiments is equal to 0.23 microM Ca2+ for RT1-Cas, 0.16 microM Ca2+ for RT2-Cas, and 0.10 microM Ca2+ for RT3-Cas; and 7), Ca2+ sensitivity of InsP3R1 and InsP3R3 isoforms recorded in the presence of 2 microM InsP3 and 0.5 mM ATP or 2 microM InsP3 and 5 mM ATP can be exchanged by swapping their Cas regions. Obtained results provide novel information about functional properties of mammalian InsP3R isoforms and support the importance of the Ca2+ sensor region (Cas) in determining the sensitivity of InsP3R isoforms to modulation by Ca2+.     

Inositol (1,4,5)-trisphosphate (InsP3)-gated Ca channels from cerebellum: conduction properties for divalent cations and regulation by intraluminal calcium

The conduction properties of inositol (1,4,5)-trisphosphate (InsP3)- gated calcium (Ca) channels (InsP3R) from canine cerebellum for divalent cations and the regulation of the channels by intraluminal Ca were studied using channels reconstituted into planar lipid bilayers. Analysis of single-channel recordings performed with different divalent cations present at 55 mM on the trans (intraluminal) side of the membrane revealed that the current amplitude at 0 mV and the single- channel slope conductance fell in the sequence: Ba (2.2 pA, 85 pS) > Sr (2.0 pA, 77 pS) > Ca (1.4 pA, 53 pS) > Mg (1.1 pA, 42 pS). The mean open time of the InsP3R recorded with Ca (2.9 ms) was significantly shorter than with other divalent cations (approximately 5.5 ms). The "anomalous mole fraction effect" was not observed in mixtures of divalent cations (Mg and Ba), suggesting that these channels are single- ion pores. Measurements of InsP3R activity at different intraluminal Ca levels demonstrated that Ca in the submillimolar range did not potentiate channel activity, and that very high levels of intraluminal Ca (> or = 10 mM) decreased channel open probability 5-10-fold. When InsP3R were measured with Ba as a current carrier in the presence of 110 mM cis potassium, a PBa/PK of 6.3 was estimated from the extrapolated value for the reversal potential. When the unitary current through the InsP3R at 0 mV was measured as a function of the permeant ion (Ba) concentration, the half-maximal current occurred at 10 mM trans Ba. The following conclusions are drawn from these data: (a) the conduction properties of InsP3R are similar to the properties of the ryanodine receptor, another intracellular Ca channel, and differ dramatically from the properties of voltage-gated Ca channels of the plasma membrane. (b) The estimated size of the Ca current through the InsP3R under physiological conditions is 0.5 pA, approximately four times less than the Ca current through the ryanodine receptor. (c) The potentiation of InsP3R by intraluminal Ca in the submillimolar range remains controversial. (d) A quantitative model that explains the inhibitory effects of high trans Ca on InsP3R activity was developed and the kinetic parameters of InsP3R gating were determined.     

Mode switching is the major mechanism of ligand regulation of InsP3 receptor calcium release channels

The inositol 1,4,5-trisphosphate (InsP(3)) receptor (InsP(3)R) plays a critical role in generation of complex Ca(2+) signals in many cell types. In patch clamp recordings of isolated nuclei from insect Sf9 cells, InsP(3)R channels were consistently detected with regulation by cytoplasmic InsP(3) and free Ca(2+) concentrations ([Ca(2+)](i)) very similar to that observed for vertebrate InsP(3)R. Long channel activity durations of the Sf9-InsP(3)R have now enabled identification of a novel aspect of InsP(3)R gating: modal gating. Using a novel algorithm to analyze channel modal gating kinetics, InsP(3)R gating can be separated into three distinct modes: a low activity mode, a fast kinetic mode, and a burst mode with channel open probability (P(o)) within each mode of 0.007 +/- 0.002, 0.24 +/- 0.03, and 0.85 +/- 0.02, respectively. Channels reside in each mode for long periods (tens of opening and closing events), and transitions between modes can be discerned with high resolution (within two channel opening and closing events). Remarkably, regulation of channel gating by [Ca(2+)](i) and [InsP(3)] does not substantially alter channel P(o) within a mode. Instead, [Ca(2+)](i) and [InsP(3)] affect overall channel P(o) primarily by changing the relative probability of the channel being in each mode, especially the high and low P(o) modes. This novel observation therefore reveals modal switching as the major mechanism of physiological regulation of InsP(3)R channel activity, with implications for the kinetics of Ca(2+) release events in cells.     

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

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

Inositol 1,4,5-trisphosphate receptor isoforms show similar Ca2+ release kinetics

The inositol 1,4,5-trisphosphate receptor (InsP3R) is an intracellular Ca2+ release channel which upon activation initiates many cellular functions. Multiple InsP3R subtypes are expressed in most cell types but the physiological significance of this heterogeneity is poorly understood. This study has directly compared the functional properties of the three different InsP3R isoforms by analyzing their InsP3-induced Ca2+ release (IICR) properties in cell lines which predominantly express each isoform subtype. The InsP3-dependence of the amount or extent of IICR was InsP3R isoform-specific, with the type III isoform having the lowest affinity with respect to Ca2+ release. The transient kinetics of IICR, measured using stopped-flow spectrofluorimetry, however, were similar for all three InsP3R isoforms. At maximal InsP3 concentrations (20 microM) the rate constants where between 0.8 and 1.0 s(-1) for the fast phase and 0.25-0.45 s(-1) for the slow phase. The concentration of InsP3 required to induce half-maximal rates of Ca2+ release (EC50) were also similar for the three isoforms (0.2-0.4 microM for the fast phase and 0.75-0.95 microM for the slow phase). These results indicate the InsP3R channel does not significantly differ functionally in terms of Ca2+ release rates between isoforms. The temporal and spatial features of intracellular Ca2+ signals are thus probably achieved through InsP3R isoform-specific regulation or localization rather than their intrinsic Ca2+ efflux properties.     

ATP-dependent adenophostin activation of inositol 1,4,5-trisphosphate receptor channel gating: kinetic implications for the durations of calcium puffs in cells

The inositol 1,4,5-trisphosphate (InsP(3)) receptor (InsP(3)R) is a ligand-gated intracellular Ca(2+) release channel that plays a central role in modulating cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)). The fungal metabolite adenophostin A (AdA) is a potent agonist of the InsP(3)R that is structurally different from InsP(3) and elicits distinct calcium signals in cells. We have investigated the effects of AdA and its analogues on single-channel activities of the InsP(3)R in the outer membrane of isolated Xenopus laevis oocyte nuclei. InsP(3)R activated by either AdA or InsP(3) have identical channel conductance properties. Furthermore, AdA, like InsP(3), activates the channel by tuning Ca(2+) inhibition of gating. However, gating of the AdA-liganded InsP(3)R has a critical dependence on cytoplasmic ATP free acid concentration not observed for InsP(3)-liganded channels. Channel gating activated by AdA is indistinguishable from that elicited by InsP(3) in the presence of 0.5 mM ATP, although the functional affinity of the channel is 60-fold higher for AdA. However, in the absence of ATP, gating kinetics of AdA-liganded InsP(3)R were very different. Channel open time was reduced by 50%, resulting in substantially lower maximum open probability than channels activated by AdA in the presence of ATP, or by InsP(3) in the presence or absence of ATP. Also, the higher functional affinity of InsP(3)R for AdA than for InsP(3) is nearly abolished in the absence of ATP. Low affinity AdA analogues furanophostin and ribophostin activated InsP(3)R channels with gating properties similar to those of AdA. These results provide novel insights for interpretations of observed effects of AdA on calcium signaling, including the mechanisms that determine the durations of elementary Ca(2+) release events in cells. Comparisons of single-channel gating kinetics of the InsP(3)R activated by InsP(3), AdA, and its analogues also identify molecular elements in InsP(3)R ligands that contribute to binding and activation of channel gating.     

The type III inositol 1,4,5-trisphosphate receptor preferentially transmits apoptotic Ca2+ signals into mitochondria

There are three isoforms of the inositol 1,4,5- trisphosphate receptor (InsP(3)R), each of which has a distinct effect on Ca(2+) signaling. However, it is not known whether each isoform similarly plays a distinct role in the activation of Ca(2+)-mediated events. To investigate this question, we examined the effects of each InsP(3)R isoform on transmission of Ca(2+) signals to mitochondria and induction of apoptosis. Each isoform was selectively silenced using isoform-specific small interfering RNA in Chinese hamster ovary cells, which express all three InsP(3)R isoforms. ATP-induced cytosolic Ca(2+) signaling patterns were altered, regardless of which isoform was silenced, but in a different fashion depending on the isoform. ATP also induced Ca(2+) signals in mitochondria, which were inhibited more effectively by silencing the type III InsP(3)R than by silencing either the type I or type II isoform. The type III isoform also co-localized most strongly with mitochondria. When apoptosis was induced by activation of either the extrinsic or intrinsic apoptotic pathway, induction was reduced most effectively by silencing the type III InsP(3)R. These findings provide evidence that the type III isoform of the InsP(3)R plays a special role in induction of apoptosis by preferentially transmitting Ca(2+) signals into mitochondria.     

Differential effect of PKA on the Ca2+ release kinetics of the type I and III InsP3 receptors

The effects of protein kinase A (PKA) on the inositol 1,4,5-trisphosphate (InsP(3)) receptor isoforms type I and type III were studied. The effects of PKA on the extent and rate constants for InsP(3)-induced Ca(2+) release (IICR) were different for the two isoforms. The effects of PKA on the type I isoform showed a biphasic relationship dependent upon the concentration of PKA used. At low concentrations of PKA (<50U/ml), both the extent and rate constants for IICR increased, while at higher concentrations (>200U/ml) the extent and rate constants decreased. The type III isoform showed only an increase in the extent of IICR and not in the rate constants. The effects of PKA on the type I InsP(3) receptor using single channel electrophysiological studies were also investigated. The stimulatory effect of PKA is due to an increase in conductance levels and not to a change in the mean open time of the channel.     

Novel model of calcium and inositol 1,4,5-trisphosphate regulation of InsP3 receptor channel gating in native endoplasmic reticulum

The InsP3R Ca(2+)-release channel has biphasic dependence on cytoplasmic free Ca2+ concentration ([Ca2+]i). InsP3 activates gating primarily by reducing high [Ca2+]i inhibition. To determine whether relieving Ca2+ inhibition is sufficient for activation, we examined single-channels in low [Ca2+]i in the absence of InsP3 by patch clamping isolated Xenopus oocyte nuclei. For both endogenous Xenopus type 1 and recombinant rat type 3 InsP3R channels, spontaneous InsP3-independent activities with low open probability Po (approximately 0.03) were observed in [Ca2+]i < 5 nM, whereas none were observed in 25 nM Ca2+. These results establish the half-maximal inhibitory [Ca2+]i in the absence of InsP3 and demonstrate that the channel can be active when all of its ligand-binding sites are unoccupied. In the simplest allosteric model that fits all observations in nuclear patch-clamp studies, the tetrameric channel can adopt six conformations, the equilibria among which are controlled by two inhibitory, one activating Ca(2+)-binding, and one InsP3-binding sites in a manner similar to the Monod-Wyman-Changeux model. InsP3 binding activates gating by affecting the relative affinity for Ca2+ of one of the inhibitory sites in different channel conformations, transforming it into an activating site. Ca2+ inhibition of InsP3-liganded channels is mediated by an InsP3-independent second inhibitory site.     

Mechanism of Ca2+ inhibition of inositol 1,4,5-trisphosphate (InsP3) binding to the cerebellar InsP3 receptor.

Ca2+ efficiently inhibits binding of inositol 1,4,5-trisphosphate (InsP3) to the InsP3 receptor in cerebellar membranes but not to the purified receptor. We have now investigated the mechanism of action by which Ca2+ inhibits InsP3 binding. Our results suggest that Ca2+ does not cause the stable association of a Ca(2+)-binding protein with the receptor. Instead, Ca2+ leads to the production of a soluble, heat-stable, low molecular weight substance from cerebellar membranes that competes with InsP3 for binding. This inhibitory substance probably represents endogenously generated InsP3 as judged by the fact that it co-purifies with InsP3 on anion-exchange chromatography, competes with [3H]InsP3 binding in a pattern similar to unlabeled InsP3, and is in itself capable of releasing 45Ca2+ from permeabilized cells. A potent Ca(2+)-activated phospholipase C activity producing InsP3 was found in cerebellar microsomes that exhibited a Ca2+ dependence identical to the Ca(2+)-dependent inhibition of InsP3 binding. Together these results suggest that the Ca(2+)-dependent inhibition of InsP3 binding to the cerebellar receptor is due to activation of a Ca(2+)-sensitive phospholipase C enriched in cerebellum. Nevertheless, Ca2+ probably also modulates the InsP3 receptor function by a direct interaction with the receptor that does not affect InsP3 binding but regulates InsP3-dependent channel gating.