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 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.     

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.     

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

Many hormones and neurotransmitters raise intracellular calcium (Ca(2+)) by generating InsP(3) and activating the inositol 1,4, 5-trisphosphate receptor (InsP(3)R). Multiple isoforms with distinct InsP(3) binding properties () have been identified (). The type III InsP(3)R lacks Ca(2+)-dependent inhibition, a property that makes it ideal for signal initiation (). Regulation of the type III InsP(3)R by InsP(3) and ATP was explored in detail using planar lipid bilayers. In comparison to the type I InsP(3)R, the type III InsP(3)R required a higher concentration of InsP(3) to reach maximal channel activity (EC(50) of 3.2 microM versus 0.5 microM for the types III and I InsP(3)R, respectively). However, the type III InsP(3)R did reach a 2.5-fold higher level of activity. Although activation by InsP(3) was isoform-specific, regulation by ATP was similar for both isoforms. In the presence of 2 microM InsP(3), low ATP concentrations (<6 mM) increased the open probability and mean open time. High ATP concentrations (>6 mM) decreased channel activity. These results illustrate the complex nature of type III InsP(3)R regulation. Enhanced channel activity in the presence of high InsP(3) may be important during periods of prolonged stimulation, whereas allosteric modulation by ATP may help to modulate intracellular Ca(2+) signaling.     

Functional characterization of the type 1 inositol 1,4,5-trisphosphate receptor coupling domain SII(+/-) splice variants and the Opisthotonos mutant form

The type 1 inositol (1,4,5)-trisphosphate receptor (InsP3R1) plays a critical role in Ca2+ signaling in cells. Neuronal and nonneuronal isoforms of the InsP3R1 differ by alternative splicing in the coupling domain of the InsP3R1 (SII site). Deletion of 107 amino acids from the coupling domain of the InsP3R1 results in epileptic-like behaviors in opisthotonos (opt) spontaneous mouse mutant. Using Spodoptera frugiperda cells expression system, we compared single-channel behavior of recombinant InsP3R1-SII(+), InsP3R1-SII(-), and InsP3R1-opt channels in planar lipid bilayers. The main results of our study are: 1) the InsP3R1-SII(-) has a higher conductance (94 pS) and the InsP3R1-opt has a lower conductance (64 pS) than the InsP3R1-SII(+) (81 pS); 2) the bell-shaped Ca2+-dependence peaks at 200-300 nM Ca2+ for all three InsP3R1 isoforms; 3) the bell-shaped Ca2+-dependence is wider for the InsP3R1-SII(+) and narrower for the InsP3R1-SII(-) and InsP3R1-opt; 4) the apparent affinity for ATP is sixfold lower for the InsP3R1-SII(-) (1.4 mM) and 20-fold lower for the InsP3R1-opt (5.3 mM) than for the InsP3R1-SII(+) (0.24 mM); 5) the InsP3R1-SII(-) is approximately twofold more active than the InsP3R1-SII(+) in the absence of ATP. Obtained results provide novel information about the molecular determinants of the InsP3R1 function.     

Novel regulation of calcium inhibition of the inositol 1,4,5-trisphosphate receptor calcium-release channel

The inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R), a Ca2+-release channel localized to the endoplasmic reticulum, plays a critical role in generating complex cytoplasmic Ca2+ signals in many cell types. Three InsP3R isoforms are expressed in different subcellular locations, at variable relative levels with heteromultimer formation in different cell types. A proposed reason for this diversity of InsP3R expression is that the isoforms are differentially inhibited by high cytoplasmic free Ca2+ concentrations ([Ca2+]i), possibly due to their different interactions with calmodulin. Here, we have investigated the possible roles of calmodulin and bath [Ca2+] in mediating high [Ca2+]i inhibition of InsP3R gating by studying single endogenous type 1 InsP3R channels through patch clamp electrophysiology of the outer membrane of isolated Xenopus oocyte nuclei. Neither high concentrations of a calmodulin antagonist nor overexpression of a dominant-negative Ca2+-insensitive mutant calmodulin affected inhibition of gating by high [Ca2+]i. However, a novel, calmodulin-independent regulation of [Ca2+]i inhibition of gating was revealed: whereas channels recorded from nuclei kept in the regular bathing solution with [Ca2+] approximately 400 nM were inhibited by 290 muM [Ca2+]i, exposure of the isolated nuclei to a bath solution with ultra-low [Ca2+] (<5 nM, for approximately 300 s) before the patch-clamp experiments reversibly relieved Ca2+ inhibition, with channel activities observed in [Ca2+]i up to 1.5 mM. Although InsP3 activates gating by relieving high [Ca2+]i inhibition, it was nevertheless still required to activate channels that lacked high [Ca2+]i inhibition. Our observations suggest that high [Ca2+]i inhibition of InsP3R channel gating is not regulated by calmodulin, whereas it can be disrupted by environmental conditions experienced by the channel, raising the possibility that presence or absence of high [Ca2+]i inhibition may not be an immutable property of different InsP3R isoforms. Furthermore, these observations support an allosteric model in which Ca2+ inhibition of the InsP3R is mediated by two Ca2+ binding sites, only one of which is sensitive to InsP3.     

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.     

Identification of intracellular calcium pools. Selective modification by thapsigargin

Recent studies have identified inositol 1,4,5-tris-phosphate(InsP3)-sensitive and -insensitive Ca2+ pools and a GTP-dependent mechanism that transfers Ca2+ between them. Here, the Ca2+ pump-inhibitory sesquiterpene lactone, thapsigargin, is shown to distinguish these two Ca2+ pools and identify a third Ca2+ pumping pool unresponsive to InsP3 or GTP. Using saponin-permeabilized DDT1MF-2 smooth muscle cells, approximately 75% of total intracellular ATP-dependent Ca2+ accumulation is blocked by thapsigargin with an IC50 of 30 nM. In contrast, 1 mM vanadate or 5 microM A23187 block 100% of Ca2+ accumulation. The thapsigargin-responsive Ca2+ pool corresponds exactly to that released by 10 microM InsP3 in the presence of 10 microM GTP. Indeed, addition of InsP3 with GTP has no effect on Ca2+ accumulated in the presence of 3 microM thapsigargin whereas A23187 releases all the remaining Ca2+. Added after maximal Ca2+ uptake, thapsigargin induces only slow Ca2+ release consistent with blockade of pumping activity. Unlike InsP3, the action of thapsigargin is entirely heparin insensitive. The large increment in Ca2+ uptake caused by 12 mM oxalate is completely reversed by thapsigargin, indicating that thapsigargin functions on an oxalate-permeable pool. Moreover, the still larger uptake induced by GTP in the presence of oxalate is also completely reversed by either thapsigargin or InsP3. The results indicate that thasigargin blocks Ca2+ uptake into two discrete pools: the InsP3-sensitive, oxalate-permeable Ca2+ pool and the InsP3-insensitive, oxalate-impermeable Ca2+ pool that can be "recruited" into the InsP3-sensitive pool by GTP-dependent Ca2+ translocation (Ghosh, T. K., Mullaney, J.M., Tarazi, F.I., and Gill, D.L. (1989) Nature 340, 236-239). Additionally, a third Ca2+ pool is defined, unreleasable by InsP3 or GTP, and containing a thapsigargin-insensitive Ca2+ pump.     

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+].     

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.     

The inositol 1,4,5-trisphosphate receptors

The inositol (1,4,5)-trisphosphate receptors (InsP3R) are the intracellular calcium (Ca2+) release channels that play a key role in Ca2+ signaling in cells. Three InsP3R isoforms-InsP3R type 1 (InsP3R1), InsP3R type 2 (InsP3R2), and InsP3R type 3 (InsP3R3) are expressed in mammals. A single InsP3R isoform is expressed in Drosophila melanogaster (DmInsP3R) and Caenorhabditis elegans (CeInsP3R). The progress made during last decade towards understanding the function and the properties of the InsP3R is briefly reviewed in this chapter. The main emphasis is on studies that revealed structural determinants responsible for the ligand recognition by the InsP3R, ion permeability of the InsP3R, modulation of the InsP3R by cytosolic Ca2+, ATP and PKA phosphorylation and on the recently identified InsP3R-binding partners. The main focus is on the InsP3R1, but the recent information about properties of other InsP3R isoforms is also discussed.     

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.     

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.     

Epidermal growth factor stimulates the rapid accumulation of inositol (1,4,5)-trisphosphate and a rise in cytosolic calcium mobilized from intracellular stores in A431 cells

Exposure of A431 human epidermoid carcinoma cells to epidermal growth factor (EGF), bradykinin, and histamine resulted in a time- and concentration-dependent accumulation of the inositol phosphates (InsP) inositol monophosphate, inositol bisphosphate, and inositol trisphosphate (InsP3). Maximal concentrations of EGF (316 ng/ml; approximately 50 nM), bradykinin (1 microM), and histamine (1 mM) resulted in 3-, 6-, and 3-fold increases, respectively, in the amounts of inositol phosphates formed over a 10-min period. The K0.5 values for stimulation were approximately 10 nM, 3 nM, and 10 microM for EGF, bradykinin, and histamine, respectively. EGF and bradykinin stimulated the rapid accumulation of the two isomers of InsP3, Ins(1,3,4)P3, and Ins(1,4,5)P3 as determined by high performance liquid chromatography analysis; maximal accumulation of Ins(1,4,5)P3 occurred within 15 s. EGF and bradykinin also stimulated a rapid (maximal levels attained within 30 s after addition of hormone) and a sustained 4- and 6-fold rise, respectively, in cytosolic free Ca2+ levels as measured by Fura-2 fluorescence. EGF and bradykinin also produced a rapid, although transient, 3- and 5-fold increase, respectively, in cytosolic free Ca2+ after chelation of extracellular Ca2+ with 3 mM EGTA. These data are consistent with the idea that EGF elevates intracellular Ca2+ levels in A431 cells, at least in part, as a result of the rapid formation of Ins(1,4,5)P3 and the consequential release of Ca2+ from intracellular stores.     

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.     

Kinetics of intracellular calcium release by inositol 1,4,5-trisphosphate and extracellular ATP in porcine cultured aortic endothelial cells.

Quantitative, time-resolved measurements have been made of intracellular Ca ion release by inositol 1,4,5-trisphosphate (InsP3) and extracellular ATP in porcine aortic endothelial cells in tissue culture. Intracellular free [Ca] was detected with the calcium dye fluo-3 and InsP3 released intracellularly by photolysis of 'caged' InsP3 in whole-cell voltage-clamped aortic endothelial cells. A rise of [Ca] was recorded at InsP3 concentrations greater than 0.2 microM. The timecourse at low InsP3 concentrations comprised a delay of mean 300 ms (range 266-330 ms), a peak in 2-3 s before declining with a half-time of 5-10 s. The delay and time-to-peak decreased with increasing concentrations of InsP3 over the range 0.2-5 microM. At very high concentrations of InsP3 (> 5 microM), the delay in the Ca response was short, always less than 20 ms. The results are consistent with a direct binding and gating action of InsP3 on the Ca channel of the cellular store. Following InsP3 action there is a refractoriness of the InsP3 Ca release process which recovers with a timecourse of half-time about 30 s. A comparison can be made between the timecourse of InsP3 and extracellular ATP actions. High concentrations of ATP (500 microM) acted with a delay of mean 1.8 s (range 1.2-2.5 s), whereas even moderate concentrations of InsP3 acted much more quickly, suggesting that there are slow coupling steps before or during the production of InsP3 in response to extracellular ATP. Both ATP and InsP3 evoked an increase in membrane conductance to K+, probably via Ca.     

A new Na/Ca exchanger splicing pattern identified in situ leads to a functionally active 70kDa NH(2)-terminal protein

Deiters' cells function as supporting cells for the sensory-motor outer hair cells of the mammalian cochlea and are interconnected by gap junctions. Here the electrical and Ca2+ responses of Deiters' cells evoked by purinergic stimulation were investigated in the organ of Corti, the auditory sensory epithelium. Adenosine 59-triphosphate (ATP, 50-100 microM) applied focally by pressure increased the intracellular free Ca2+ concentration ([Ca2+]i). At the same time ATP evoked an early inward current that was followed by an outward component, reflecting a sustained Ca2+-dependent reduction of the pre-stimulus offset current. These responses were maintained when Ca2+ was removed from the extracellular medium (0 [Ca2+]o), indicating a contribution to Ca2+ signalling from P2Y metabotropic receptors. UV photolysis of caged inositol 1,4,5-triphosphate (InsP3, 16 microM) produced Ca2+ responses similar to those evoked by exogenous ATP, accompanied by reduction of the offset current. In Deiters' cells uncoupled by octanol (1mM), ATP activated only the early inward current, suggesting that functional gap junctions are required in the late phase of the current responses. Following the delivery of UV flashes to pairs of Deiters' cells loaded with caged InsP3, the electrical coupling ratio (CR), monitored by double patch-clamp recordings, was strongly attenuated. These data support the idea that, by promoting inflow of cations and by controlling gap-junction conductance in a Ca2+-and InsP3-dependent way, ATP might serve a protective role in the cochlea.     

Stimulus-responsive and rapid formation of inositol pentakisphosphate in cultured adrenal chromaffin cells

When [3H]inositol-prelabeled cultured bovine adrenal chromaffin cells were stimulated with high K+ (56 mM) and nicotine (10 microM), a large and transient increase in [3H]inositol 1,3,4,5,6-pentakisphosphate (InsP5) accumulation was observed. The accumulation reached the maximum level at 15 s and then declined to the basal level at 2 min. The time course of accumulation of InsP5 was parallel to that of [3H]inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). Angiotensin II (Ang II) (10 microM) rapidly accumulated InsP5, but the level was sustained for 2 min. With a slower time course and a lesser amount than InsP5, high K+, nicotine, and Ang II caused an accumulation of [3H]inositol 1,3,4,5-tetrakisphosphate and [3H]inositol hexakisphosphate. Veratridine (100 microM), maitotoxin (10 ng/ml), ATP (30 microM), platelet-derived growth factor (10 ng/ml), and endothelin (10 ng/ml) also induced the InsP5 accumulation. High K+, nicotine, veratridine, and maitotoxin induced an increase in 45Ca2+ uptake, whereas Ang II, ATP, platelet-derived growth factor, and endothelin did not cause 45Ca2+ uptake. Nifedipine, a calcium channel antagonist, inhibited the high K(+)-induced InsP5 accumulation but failed to affect the Ang II-induced InsP5 accumulation. In an EGTA-containing and Ca2(+)-depleted medium, the high K(+)-induced InsP5 accumulation was completely inhibited, whereas the InsP5 accumulation induced by Ang II was not significantly inhibited. 12-O-tetradecanoylphorbol-13-acetate inhibited partially the Ang II-induced InsP5 accumulation but failed to inhibit the high K(+)-induced accumulation. In those experiments, the changes of InsP5 accumulation were closely correlated to those of Ins(1,4,5)P3. In the chromaffin cell homogenate, [3H] Ins(1,4,5)P3 was converted eventually to [3H]InsP5 through [3H]inositol 1,3,4,6-tetrakisphosphate. Taken together, the above results suggest that InsP5 is rapidly formed by a variety of stimulants and that the formation of InsP5 may occur through two mechanisms, i.e. Ca2+ uptake-dependent and Ca2+ uptake-independent ones in cultured adrenal chromaffin cells.     

Regulation of Ca²⁺ release through inositol 1,4,5-trisphosphate receptors by adenine nucleotides in parotid acinar cells

Secretagogue-stimulated intracellular Ca(2+) signals are fundamentally important for initiating the secretion of the fluid and ion component of saliva from parotid acinar cells. The Ca(2+) signals have characteristic spatial and temporal characteristics, which are defined by the specific properties of Ca(2+) release mediated by inositol 1,4,5-trisphosphate receptors (InsP(3)R). In this study we have investigated the role of adenine nucleotides in modulating Ca(2+) release in mouse parotid acinar cells. In permeabilized cells, the Ca(2+) release rate induced by submaximal [InsP(3)] was increased by 5 mM ATP. Enhanced Ca(2+) release was not observed at saturating [InsP(3)]. The EC(50) for the augmented Ca(2+) release was ～8 μM ATP. The effect was mimicked by nonhydrolysable ATP analogs. ADP and AMP also potentiated Ca(2+) release but were less potent than ATP. In acini isolated from InsP(3)R-2-null transgenic animals, the rate of Ca(2+) release was decreased under all conditions but now enhanced by ATP at all [InsP(3)]. In addition the EC(50) for ATP potentiation increased to ～500 μM. These characteristics are consistent with the properties of the InsP(3)R-2 dominating the overall features of InsP(3)R-induced Ca(2+) release despite the expression of all isoforms. Finally, Ca(2+) signals were measured in intact parotid lobules by multiphoton microscopy. Consistent with the release data, carbachol-stimulated Ca(2+) signals were reduced in lobules exposed to experimental hypoxia compared with control lobules only at submaximal concentrations. Adenine nucleotide modulation of InsP(3)R in parotid acinar cells likely contributes to the properties of Ca(2+) signals in physiological and pathological conditions.     

Spatial and temporal aspects of Ca2+ signaling mediated by P2Y receptors in cultured rat hippocampal astrocytes

ATP produces a variety of Ca2+ responses in astrocytes. To address the complex spatio-temporal Ca2+ signals, we analyzed the ATP-evoked increase in intracellular Ca2+ concentration ([Ca2+]i) in cultured rat hippocampal astrocytes using fura-2 or fluo-3 based Ca2+ imaging techniques. ATP at less than 10 nM produced elementary Ca2+ release event "puffs" in a manner independent of extracellular Ca2+. Stimulation with higher ATP concentrations (3 or 10 micro M) resulted in global Ca2+ responses such as intercellular Ca2+ wave. These Ca2+ responses were mainly mediated by metabotropic P2Y receptors. ATP acting on both P2Y1 and P2Y2 receptors produced a transient Ca2+ release by inositol 1,4,5-trisphosphate (InsP3). When cells were stimulated with ATP much longer, the transient [Ca2+]i elevation was followed by sustained Ca2+ entry from the extracellular space. This sustained rise in [Ca2+]i was inhibited by Zn2+ (<10 micro M), an inhibitor of capacitative Ca2+ entry (CCE). CCE induced by cyclopiazonic acid or thapsigargin and Ca2+ entry evoked by ATP share the same pharmacological profile in astrocytes. Taken together, the hierarchical Ca2+ responses to ATP were observed in hippocampal astrocytes, i.e., puffs, global Ca2+ release by InsP3, and CCE in response to depletion of InsP3-sensitive Ca2+ stores. It should be noted that these Ca2+ signals and their modulation by Zn2+ could occur in the hippocampus in situ since both ATP and Zn2+ are rich in the hippocampus and could be released by excitatory stimulation.