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+.     

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.     

Quantal release, incremental detection, and long-period Ca2+ oscillations in a model based on regulatory Ca2+-binding sites along the permeation pathway.

Quantal release, incremental detection, and oscillations are three types of Ca2+ responses that can be obtained in different conditions, after stimulation of the intracellular Ca2+ stores by submaximum concentrations of inositol 1,4,5-triphosphate (InsP3). All three phenomena are thought to occur through the regulatory properties of the InsP3 receptor/Ca2+ channel. In the present study, we perform further analysis of the model (Swillens et al., 1994, Proc. Natl. Acad. Sci. USA. 91:10074-10078) previously proposed for transient InsP3-induced Ca2+ release, based on the bell-shaped dependence of the InsP3 receptor activity on the Ca2+ level and on the existence of an intermediate Ca2+ domain located around the mouth of the channel. We show that Ca2+ oscillations also arise in the latter model. Conditions for the occurrence of the various behaviors are investigated. Numerical simulations also show that the existence of an intermediate Ca2+ domain can markedly increase the period of oscillations. Periods on the order of 1 min can indeed be accounted for by the model when one assigns realistic values to the kinetic constants of the InsP3 receptor, which, in the absence of a domain, lead to oscillations with periods of a few seconds. Finally, theoretical support in favor of a positive cooperativity in the regulation of the InsP3 receptor by Ca2+ is presented.     

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.     

The InsP3 receptor: its role in neuronal physiology and neurodegeneration

The InsP3 receptor is a ligand-gated channel that releases Ca2+ from intracellular stores in a variety of cell types, including neurons. Genetic studies from vertebrate and invertebrate model systems suggest that coordinated rhythmic motor functions are most susceptible to changes in Ca2+ release from the InsP3 receptor. In many cases, the InsP3 receptor interacts with other signaling mechanisms that control levels of cytosolic Ca2+, suggesting that the maintenance of Ca2+ homeostasis in normal cells could be controlled by the activity of the InsP3R. In support of this idea, recent studies show that altered InsP3 receptor activity can be partially responsible for Ca2+ dyshomeostasis seen in many neurodegenerative conditions. These observations open new avenues for carrying out genetic and drug screens that target InsP3R function in neurodegenerative conditions.     

Regulation of type 1 inositol 1,4,5-trisphosphate-gated calcium channels by InsP3 and calcium: Simulation of single channel kinetics based on ligand binding and electrophysiological analysis.

Cytosolic calcium acts as both a coagonist and an inhibitor of the type 1 inositol 1,4,5-trisphosphate (InsP3)-gated Ca channel, resulting in a bell-shaped Ca dependence of channel activity (Bezprozvanny, I., J. Watras, and B.E. Ehrlich. 1991. Nature. 351:751-754; Finch, E.A., T.J. Turner, and S.M. Goldin. 1991. Science. 252: 443-446; Iino, M. 1990. J. Gen. Physiol. 95:1103-1122). The ability of Ca to inhibit channel activity, however, varies dramatically depending on InsP3 concentration (Combettes, L., Z. Hannaert-Merah, J.F. Coquil, C. Rousseau, M. Claret, S. Swillens, and P. Champeil. 1994. J. Biol. Chem. 269:17561-17571; Kaftan, E.J., B.E. Ehrlich, and J. Watras. 1997. J. Gen. Physiol. 110:529-538). In the present report, we have extended the characterization of the effect of cytosolic Ca on both InsP3 binding and InsP3-gated channel kinetics, and incorporated these data into a mathematical model capable of simulating channel kinetics. We found that cytosolic Ca increased the Kd of InsP3 binding approximately 3.5-fold, but did not influence the maximal number of binding sites. The ability of Ca to decrease InsP3 binding is consistent with the rightward shift in the bell-shaped Ca dependence of InsP3-gated Ca channel activity. High InsP3 concentrations are able to overcome the Ca-dependent inhibition of channel activity, apparently due to a low affinity InsP3 binding site (Kaftan, E.J., B.E. Ehrlich, and J. Watras. 1997. J. Gen. Physiol. 110:529-538). Constants from binding analyses and channel activity determinations were used to develop a mathematical model that fits the complex Ca-dependent regulation of the type 1 InsP3-gated Ca channel. This model accurately simulated both steady state data (channel open probability and InsP3 binding) and kinetic data (channel activity and open time distributions), and yielded testable predictions with regard to the regulation of this intracellular Ca channel. Information gained from these analyses, and our current molecular model of this Ca channel, will be important for understanding the basis and regulation of intracellular Ca waves and oscillations in intact cells.     

The inositol 1,4,5-trisphosphate receptor of cerebellum. Mn2+ permeability and regulation by cytosolic Mn2+

The inositol 1,4,5-trisphosphate receptor (InsP3R), an intracellular calcium release channel, is found in virtually all cells and is abundant in the cerebellum. We used Mn2+ as a tool to study two aspects of the cerebellar InsP3R. First, to investigate the structure of the ion pore, Mn2+ permeation through the channel was determined. We found that Mn2+ can pass through the InsP3R; the selectivity sequence for divalent cations is Ba2+ > Sr2+ > Ca2+ > Mg2+ > Mn2+. Second, to begin characterization of the cytosolic regulatory sites responsible for the Ca(2+)-dependent modulation of InsP3R function, the ability of Mn2+ to replace Ca2+ was investigated. We show that Mn2+, as Ca2+, modulates InsP3R activity with a bell-shaped dependence where the affinity of the activation site of the InsP3R is similar for both ions, but higher concentrations of Mn2+ were necessary to inhibit the channel. These results suggest that the two regulatory sites are structurally distinct. Our findings are also important for the understanding of cellular responses when Mn2+ is used to quench the intracellular fluorescence of Ca2+ indicator dyes.     

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.     

Signal-induced Ca2+ oscillations: properties of a model based on Ca(2+)-induced Ca2+ release.

We consider a simple, minimal model for signal-induced Ca2+ oscillations based on Ca(2+)-induced Ca2+ release. The model takes into account the existence of two pools of intracellular Ca2+, namely, one sensitive to inositol 1,4,5 trisphosphate (InsP3) whose synthesis is elicited by the stimulus, and one insensitive to InsP3. The discharge of the latter pool into the cytosol is activated by cytosolic Ca2+. Oscillations in cytosolic Ca2+ arise in this model either spontaneously or in an appropriate range of external stimulation; these oscillations do not require the concomitant, periodic variation of InsP3. The following properties of the model are reviewed and compared with experimental observations: (a) Control of the frequency of Ca2+ oscillations by the external stimulus or extracellular Ca2+; (b) correlation of latency with period of Ca2+ oscillations obtained at different levels of stimulation; (c) effect of a transient increase in InsP3; (d) phase shift and transient suppression of Ca2+ oscillations by Ca2+ pulses, and (e) propagation of Ca2+ waves. It is shown that on all these counts the model provides a simple, unified explanation for a number of experimental observations in a variety of cell types. The model based on Ca(2+)-induced Ca2+ release can be extended to incorporate variations in the level of InsP3 as well as desensitization of the InsP3 receptor; besides accounting for the phenomena described by the minimal model, the extended model might also account for the occurrence of complex Ca2+ oscillations.     

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+].     

The role of inositol 1,4,5-trisphosphate receptors in the regulation of bile secretion in health and disease

Ca2+ signaling via the inositol 1,4,5-trisphosphate receptor (InsP3R) is a ubiquitous mechanism for regulation of cell function, yet very little is known about the role of the InsP3R in specific disease states. Converging lines of evidence suggest that the liver may provide a model for the role of the InsP3R in health and disease. Ca2+ signaling is mediated entirely by the InsP3R in hepatocytes and cholangiocytes, the two types of epithelia in the liver. Here we review the role of specific InsP3R isoforms and the physiological effects of InsP3R-mediated Ca2+ signals in both of these types of epithelia. In addition, we review evidence that the InsP3R is lost from cholangiocytes in cholestatic forms of liver disease, and discuss this as a possible final common pathway for cholestasis.     

Single-channel properties in endoplasmic reticulum membrane of recombinant type 3 inositol trisphosphate receptor

The inositol 1,4,5-trisphosphate receptor (InsP(3)R) is an intracellular Ca(2+)-release channel localized in endoplasmic reticulum (ER) with a central role in complex Ca(2+) signaling in most cell types. A family of InsP(3)Rs encoded by several genes has been identified with different primary sequences, subcellular locations, variable ratios of expression, and heteromultimer formation. This diversity suggests that cells require distinct InsP(3)Rs, but the functional correlates of this diversity are largely unknown. Lacking are single-channel recordings of the recombinant type 3 receptor (InsP(3)R-3), a widely expressed isoform also implicated in plasma membrane Ca(2+) influx and apoptosis. Here, we describe functional expression and single-channel recording of recombinant rat InsP(3)R-3 in its native membrane environment. The approach we describe suggests a novel strategy for expression and recording of recombinant ER-localized ion channels in the ER membrane. Ion permeation and channel gating properties of the rat InsP(3)R-3 are strikingly similar to those of Xenopus type 1 InsP(3)R in the same membrane. Using two different two-electrode voltage clamp protocols to examine calcium store-operated calcium influx, no difference in the magnitude of calcium influx was observed in oocytes injected with rat InsP(3)R-3 cRNA compared with control oocytes. Our results suggest that if cellular expression of multiple InsP(3)R isoforms is a mechanism to modify the temporal and spatial features of [Ca(2+)](i) signals, then it must be achieved by isoform-specific regulation or localization of various types of InsP(3)Rs that have relatively similar Ca(2+) permeation properties.     

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.     

Antibody to the inositol trisphosphate receptor blocks thimerosal-enhanced Ca(2+)-induced Ca2+ release and Ca2+ oscillations in hamster eggs.

The sulfhydryl reagent thimerosal enhanced the sensitivity of hamster eggs to injected inositol 1,4,5-trisphosphate (InsP3) or Ca2+ to generate regenerative Ca2+ release from intracellular pools. A monoclonal antibody (mAb) to the InsP3 receptor blocked both the InsP3-induced Ca2+ release (IICR) and Ca(2+)-induced Ca2+ release (CICR). The mAb also blocked Ca2+ oscillations induced by thimerosal. The results indicate that thimerosal enhances IICR sensitized by cytosolic Ca2+, but not CICR from InsP3-insensitive pools, and causes repetitive Ca2+ releases from InsP3-sensitive pools.     

Kinetics of cytosolic Ca2+ concentration after photolytic release of 1-D-myo-inositol 1,4-bisphosphate 5-phosphorothioate from a caged derivative in guinea pig hepatocytes.

The influence of 1-D-myo-inositol 1,4,5-trisphosphate (InsP3) breakdown by InsP3 5-phosphatase in determining the time course of Ca2+ release from intracellular stores was investigated with flash photolytic release of a stable InsP3 derivative, 5-thio-InsP3, from a photolabile caged precursor. The potency and Ca(2+)-releasing properties of the biologically active D isomers of 5-thio-InsP3 and InsP3 itself were compared by photolytic release in guinea pig hepatocytes. After a light flash, cytosolic free calcium concentration ([Ca2+]i) showed an initial delay before rising quickly to a peak and declining more slowly to resting levels, with time course and amplitude generally similar to those seen with photolytic release of InsP3. Differences were a three- to eightfold lower potency of 5-thio-InsP3 in producing Ca2+ release, much longer delays between photolytic release and Ca2+ efflux with low concentrations of 5-thio-InsP3 than with InsP3, and persistent reactivation of Ca2+ release, producing periodic fluctuations of cytosolic [Ca2+]i with high concentrations of 5-thio-InsP3 but not InsP3 itself. The lower potency of 5-thio-InsP3 may be a result of a lower affinity for closed receptor/channels or a lower open probability of liganded receptor/channels. The longer delays with 5-thio-InsP3 at low concentration suggest that metabolism of InsP3 by 5-phosphatase may reduce the concentration sufficiently to prevent receptor activation and may have a similar effect on InsP3 concentration during hormonal activation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fast kinetics of calcium liberation induced in Xenopus oocytes by photoreleased inositol trisphosphate.

Inositol 1,4,5-trisphosphate (InsP3) acts on intracellular receptors to cause liberation of Ca2+ ions into the cytosol as repetitive spikes and propagating waves. We studied the processes underlying this regenerative release of Ca2+ by monitoring with high resolution the kinetics of Ca2+ flux evoked in Xenopus oocytes by flash photolysis of caged InsP3. Confocal microfluorimetry was used to monitor intracellular free [Ca2+] from femtoliter volumes within the cell, and the underlying Ca2+ flux was then derived from the rate of increase of the fluorescence signals. A threshold amount of InsP3 had to be photoreleased to evoke any appreciable Ca2+ signal, and the amount of liberated Ca2+ then increased only approximately fourfold with maximal stimulation, whereas the peak rate of increase of Ca2+ varied over a range of nearly 20-fold, reaching a maximum of approximately 150 microMs-1. Ca2+ flux increased as a first-order function of [InsP3]. Indicating a lack of cooperativity in channel opening, and was half-maximal with stimuli approximately 10 times threshold. After a brief photolysis flash, Ca2+ efflux began after a quiescent latent period that shortened from several hundred milliseconds with near-threshold stimuli to 25 ms with maximal flashes. This delay could not be explained by an initial "foot" of Ca2+ increasing toward a threshold at which regenerative release was triggered, and the onset of release seemed too abrupt to be accounted for by multiple sequential steps involved in channel opening. Ca2+ efflux increased to a maximum after the latent period in a time that reduced from > 100 ms to approximately 8 ms with increasing [InsP3] and subsequently declined along a two-exponential time course: a rapid fall with a time constant shortening from > 100 ms to approximately 25 ms with increasing [InsP3], followed by a much smaller fail persisting for several seconds. The results are discussed in terms of a model in which InsP3 receptors must undergo a slow transition after binding InsP3 before they can be activated by cytosolic Ca2+ acting as a co-agonist. Positive feedback by liberated Ca2+ ions then leads to a rapid increase in efflux to a maximal rate set by the proportion of receptors binding InsP3. Subsequently, Ca2+ efflux terminates because of a slower inhibitory action of cytosolic Ca2+ on gating of InsP3 receptor-channels.     

The store-operated calcium entry pathways in human carcinoma A431 cells: functional properties and activation mechanisms

Activation of phospholipase C (PLC)-mediated signaling pathways in nonexcitable cells causes the release of Ca2+ from intracellular Ca2+ stores and activation of Ca2+ influx across the plasma membrane. Two types of Ca2+ channels, highly Ca2+-selective ICRAC and moderately Ca2+-selective ISOC, support store-operated Ca2+ entry process. In previous patch-clamp experiments with a human carcinoma A431 cell line we described store-operated Imin/ICRACL plasma membrane Ca2+ influx channels. In the present paper we use whole-cell and single-channel recordings to further characterize store-operated Ca2+ influx pathways in A431 cells. We discovered that (a) ICRAC and ISOC are present in A431 cells; (b) ICRAC currents are highly selective for divalent cations and fully activate within 150 s after initiation of Ca2+ store depletion; (c) ISOC currents are moderately selective for divalent cations (PBa/PCs = 14.5) and require at least 300 s for full activation; (d) ICRAC and ISOC currents are activated by PLC-coupled receptor agonists; (e) ISOC currents are supported by Imin/ICRACL channels that display 8.5-10 pS conductance for sodium; (f) ICRAC single channel conductance for sodium is estimated at 0.9 pS by the noise analysis; (g) Imin/ICRACL channels are activated in excised patches by an amino-terminal fragment of InsP3R1 (InsP3R1N); and (h) InsP3 binding to InsP3R1N is necessary for activation of Imin/ICRACL channels. Our findings provide novel information about store-operated Ca2+ influx pathways in A431 cells.     

Functional and biochemical analysis of the type 1 inositol (1,4,5)-trisphosphate receptor calcium sensor

Modulation of the type 1 inositol (1,4,5)-trisphosphate receptors (InsP(3)R1) by cytosolic calcium (Ca(2+)) plays an essential role in their signaling function, but structural determinants and mechanisms responsible for the InsP(3)R1 regulation by Ca(2+) are poorly understood. Using DT40 cell expression system and Ca(2+) imaging assay, in our previous study we identified a critical role of E2100 residue in the InsP(3)R1 modulation by Ca(2+). By using intrinsic tryptophan fluorescence measurements in the present study we determined that the putative InsP(3)R1 Ca(2+)-sensor region (E1932-R2270) binds Ca(2+) with 0.16 micro M affinity. We further established that E2100D and E2100Q mutations decrease Ca(2+)-binding affinity of the putative InsP(3)R1 Ca(2+)-sensor region to 1 micro M. In planar lipid bilayer experiments with recombinant InsP(3)R1 expressed in Spodoptera frugiperda cells we discovered that E2100D and E2100Q mutations shifted the peak of the InsP(3)R1 bell-shaped Ca(2+) dependence from 0.2 micro M to 1.5 micro M Ca(2+). In agreement with the biochemical data, we found that the apparent affinities of Ca(2+) activating and inhibitory sites of the InsP(3)R1 were 0.2 micro M for the wild-type channels and 1-2 micro M Ca(2+) for the E2100D and E2100Q mutants. The results obtained in our study support the hypothesis that E2100 residue forms a part of the InsP(3)R1 Ca(2+) sensor.