Interaction of chromogranin B and the near N-terminal region of chromogranin B with an intraluminal loop peptide of the inositol 1,4, 5-trisphosphate receptor

Given the interaction of the inositol 1,4,5-trisphosphate receptor (IP(3)R) with chromogranins A (CGA) and B (CGB), two major Ca(2+) storage proteins of secretory granules that have been shown to be IP(3)-sensitive intracellular Ca(2+) store of neuroendocrine cells, we have investigated the potential interaction of the intraluminal loop regions of the IP(3)R with both intact CGB and the conserved near N-terminal region of CGB. The interaction studies carried out with CGB and glutathione S-transferase fusion proteins of intraluminal loop regions of bovine type 1 IP(3)R showed that CGB interacts with intraluminal loop 3-2 (the second loop formed between transmembrane regions 5 and 6) of the IP(3)R at both pH 5.5 and 7.5. Analytical ultracentrifugation studies also indicated that CGB interacts with the same intraluminal loop region of the IP(3)R and the interaction was much stronger than that between CGA and the loop. Moreover, the conserved near N-terminal region of CGB also interacted with the intraluminal loop region of the IP(3)R. The CGB interaction with the IP(3)R intraluminal loop peptide at pH 7.5 showed a DeltaG(0) value of -8.1 kcal/mol at 37 degrees C for a 1:1 stoichiometry, indicating a K(d) of approximately 1.9 micrometer. These results give insight into the molecular organization of the IP(3)-sensitive Ca(2+) store.     

Localization of three types of the inositol 1,4,5-trisphosphate receptor/Ca(2+) channel in the secretory granules and coupling with the Ca(2+) storage proteins chromogranins A and B

Although the role of secretory granules as the inositol 1,4,5-trisphosphate (IP(3))-sensitive intracellular Ca(2+) store and the presence of the IP(3) receptor (IP(3)R)/Ca(2+) channel on the secretory granule membrane have been established, the identity of the IP(3)R types present in the secretory granules is not known. We have therefore investigated the presence of different types of IP(3)R in the secretory granules of bovine adrenal medullary chromaffin cells using immunogold electron microscopy and found the existence of all three types of IP(3)R in the secretory granules. To determine whether these IP(3)Rs interact with CGA and CGB, each IP(3)R isoform was co-transfected with CGA or CGB into NIH3T3 or COS-7 cells, and the expressed IP(3)R isoform and CGA or CGB were co-immunoprecipitated. From these studies it was shown that all three types of IP(3)R form complexes with CGA and CGB in the cells. To further confirm whether the IP(3)R isoforms and CGA and CGB form a complex in the secretory granules the potential interaction between all three isoforms of IP(3)R and CGA and CGB was tested by co-immunoprecipitation experiments of the mixture of secretory granule lysates and the granule membrane proteins. The three isoforms of IP(3)R were shown to form complexes with CGA and CGB, indicating the complex formation between the three isoforms of IP(3)R and CGA and CGB in the secretory granules. Moreover, the pH-dependent Ca(2+) binding property of CGB was also studied using purified recombinant CGB, and it was shown that CGB bound 93 mol of Ca(2+)/mol with a dissociation constant (K(d)) of 1.5 mm at pH 5.5 but virtually no Ca(2+) at pH 7.5. The high capacity, low affinity Ca(2+)-binding property of CGB at pH 5.5 is comparable with that of CGA and is in line with its role as a Ca(2+) storage protein in the secretory granules.     

Purification, pH-dependent conformational change, aggregation, and secretory granule membrane binding property of secretogranin II (chromogranin C).

Secretogranin II (SgII) is one of the three major proteins, the other two being chromogranins A (CGA) and B (CGB), of secretory granules of neuroendocrine cells. The Ca(2+) storage proteins CGA and CGB not only are coupled to the IP(3) receptor (IP(3)R)/Ca(2+) channels that exist on the secretory granule membrane but also are known to play key roles in secretory granule biogenesis. Unlike the better studied CGA and CGB, secretogranin II has never been completely purified in the native state and studied. We have therefore purified SgII in native form from bovine adrenal medulla and subjected it to biochemical characterization. Secretogranin II consisted of largely beta-sheet and random coil structures with a low level of alpha-helicity. Like CGA and CGB, it also underwent pH-dependent conformational changes, showing 9.5% alpha-helicity at pH 7.5 and 17.0% alpha-helicity at pH 5.5. Secretogranin II also underwent acidic pH- and Ca(2+)-dependent aggregation, and it was approximately 8-fold more sensitive than CGA to Ca(2+) in its pH-dependent aggregation but was 8-fold less sensitive than CGB. Further, similar to CGA and CGB that had interacted with the secretory granule membrane at the intragranular pH 5.5, SgII also interacted with the secretory granule membrane at pH 5.5 and dissociated from it at near-physiological pH 7.5, implying similar roles of SgII in the cell as those of CGA and CGB. Secretogranin II hence appeared to actively participate in secretory granule biogenesis as has been proposed for CGA and CGB.     

Subcellular distribution of chromogranins A and B in bovine adrenal chromaffin cells

The major secretory granule proteins chromogranins A (CGA) and B (CGB) have recently been shown to play critical roles in inositol 1,4,5-trisphosphate-dependent intracellular Ca(2+) mobilizations. We determined here the subcellular distribution of CGA and CGB based on 3D-images of chromaffin cells, and found that approximately 95% of cellular CGA was present in secretory granules while approximately 5% was in the endoplasmic reticulum (ER), whereas approximately 57% of cellular CGB was in secretory granules while approximately 24% and approximately 19% were in the ER and nucleus, respectively. These results suggest that chromogranins are at the center of intracellular Ca(2+) homeostasis in secretory cells.     

Activation of the inositol 1,4,5-trisphosphate receptor by the calcium storage protein chromogranin A

Secretory granules of neuroendocrine cells are inositol 1,4,5-trisphosphate (InsP(3))-sensitive Ca(2+) stores in which the Ca(2+) storage protein, chromogranin A (CGA), couples with InsP(3)-gated Ca(2+) channels (InsP(3)R) located in the granule membrane. The functional aspect of this coupling has been investigated via release studies and planar lipid bilayer experiments in the presence and absence of CGA. CGA drastically increased the release activity of the InsP(3)R by increasing the channel open probability by 9-fold and the mean open time by 12-fold. Our results show that CGA-coupled InsP(3)Rs are more sensitive to activation than uncoupled receptors. This modulation of InsP(3)R channel activity by CGA appears to be an essential component in the control of intracellular Ca(2+) concentration by secretory granules and may regulate the rate of vesicle fusion and exocytosis.     

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.     

Coupling of the inositol 1,4,5-trisphosphate receptor and chromogranins A and B in secretory granules

The secretory granules of neuroendocrine cells which contain large amounts of Ca(2+) and chromogranins have been demonstrated to release Ca(2+) in response to inositol 1,4,5-trisphosphate (IP(3)). Moreover, chromogranin A (CGA) has been shown to interact with several secretory granule membrane proteins, including the IP(3) receptor (IP(3)R). To determine whether the IP(3)Rs interact directly with chromogranins A and B (CGB), two major proteins of the secretory granules, we have used purified IP(3)R from bovine cerebellum in the interaction study with CGA and CGB, and have shown that chromogranins A and B directly interact with the IP(3)R at the intravesicular pH 5.5. Immunogold cytochemical study using the IP(3)R and CGA antibodies indicated that IP(3)R-labeled gold particles were localized in the periphery of the secretory granules, indicating the presence of the IP(3)Rs on the secretory granule membrane. To determine whether the IP(3)R and chromogranins A and B are physically linked in the cells, bovine type 1 IP(3)R (IP(3)R-1) and CGA or CGB are co-transfected into COS-7 cells and co-immunoprecipitation was carried out. Immunoprecipitation of the cell extracts demonstrated the presence of CGA-IP(3)R-1 and CGB-IP(3)R-1 complexes, respectively, indicating the complex formation between the IP(3)R and chromogranins A and B in native state.     

Presence of secretogranin II and high-capacity, low-affinity Ca2+ storage role in nucleoplasmic Ca2+ store vesicles

Chromogranins and secretogranins have traditionally been known as marker proteins of secretory granules that contain the highest concentrations of cellular calcium, reaching approximately 40 mM. In addition, chromogranin B was also shown to exist in the nucleus, localizing in the putative inositol 1,4,5-trisphosphate (IP3)-sensitive nucleoplasmic Ca2+ store vesicles. Chromogranins A (CGA) and B (CGB) are high-capacity, low-affinity Ca2+ binding proteins, binding 30-90 mol of Ca2+/mol with dissociation constants (Kd) of 1.5-4 mM. Yet the Ca2+-binding property of secretogranins has not been studied. Here, we show the localization of secretogranin II (SgII) in the nucleus, more specifically, in the IP3-sensitive nucleoplasmic Ca2+ store vesicles along with CGB and the IP3 receptors. We have also determined the Ca2+-binding property of SgII and found that SgII binds 61 mol of Ca2+/mol (910 nmol Ca2+/mg) with a Kd of 3.0 mM at the intragranular pH 5.5 and 30 mol of Ca2+/mol (440 nmol Ca2+/mg) with a Kd of 2.2 mM at a near-physiological pH 7.5. Chromogranin B also bound 50 mol of Ca2+/mol (670 nmol Ca2+/mg) with a Kd of 3.1 mM at pH 7.5. Given the high-capacity, low-affinity Ca2+-binding property of SgII and its presence in the IP3-sensitive nucleoplasmic Ca2+ store vesicles, these results suggest that SgII may function in the storage and control of Ca2+ in the nucleus through its interaction with CGB in the nucleoplasmic vesicles.     

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.     

Translational mobility of the type 3 inositol 1,4,5-trisphosphate receptor Ca2+ release channel in endoplasmic reticulum membrane

The inositol 1,4,5-trisphosphate receptor (InsP3R) is an integral membrane protein in the endoplasmic reticulum (ER) which functions as a ligand-gated Ca2+ release channel. InsP3-mediated Ca2+ release modulates the cytoplasmic free Ca2+ concentration ([Ca2+]i), providing a ubiquitous intracellular signal with high temporal and spatial specificity. Precise localization of the InsP3R is believed to be important for providing local [Ca2+] regulation and for ensuring efficient functional coupling between Ca2+ release sites by enabling graded recruitment of channels with increasing stimulus strength in the face of the intrinsically unstable regenerative process of Ca2+-induced Ca2+ release. Highly localized Ca2+ release has been attributed to the ability of the InsP3R channels to cluster and to be localized to discrete areas, suggesting that mechanisms may exist to restrict their movement. Here, we examined the lateral mobility of the type 3 isoform of the InsP3R (InsP3R3) in the ER membrane by performing confocal fluorescence recovery after photobleaching of an InsP3R3 with green fluorescent protein fused to its N terminus. In Chinese hamster ovary and COS-7 cells, the diffusion coefficient D was approximately 4 x 10(-10) cm2/s at room temperature, a value similar to that determined for other ER-localized integral membrane proteins, with a high fraction (approximately 75%) of channels mobile. D was modestly increased at 37 degrees C, and it as well as the mobile fraction were reversibly reduced by ATP depletion. Although disruption of the actin cytoskeleton (latrunculin) was without effect, disruption of microtubules (nocodazole) reduced D by half without affecting the mobile fraction. We conclude that the entire ER is continuous in these cells, with the large majority of InsP3R3 channels free to diffuse throughout it, at rates that are comparable with those measured for other polytopic ER integral membrane proteins. The observed InsP3R3 mobility may be higher than its intrinsic diffusional mobility because of additional ATP- and microtubule-facilitated motility of the channel.     

Functional coupling of chromogranin with the inositol 1,4,5-trisphosphate receptor shapes calcium signaling

Chromogranins A and B are high capacity, low affinity calcium (Ca(2+)) storage proteins that bind to the inositol 1,4,5-trisphosphate-gated receptor (InsP(3) R). Although most commonly associated with secretory granules of neuroendocrine cells, chromogranins have also been found in the lumen of the endoplasmic reticulum (ER) of many cell types. To investigate the functional consequences of the interaction between the InsP(3) R and the chromogranins, we disrupted the interaction between the two proteins by adding a chromogranin fragment, which competed with chromogranin for its binding site on the InsP(3)R. Responses were monitored at the single channel level and in intact cells. When using InsP(3) R type I incorporated into planar lipid bilayers and activated by cytoplasmic InsP(3) and luminal chromogranin, the addition of the fragment reversed the enhancing effect of chromogranin. Moreover, the expression of the fragment in the ER of neuronally differentiated PC12 cells attenuated agonist-induced intracellular Ca(2+) signaling. These results show that the InsP(3)R/chromogranin interaction amplifies Ca(2+) release from the ER and that chromogranin is an essential component of this intracellular channel complex.     

C-terminal domain of chromogranin B regulates intracellular calcium signaling

The versatility of intracellular calcium as a second messenger is seen in its ability to mediate opposing events such as neuronal cell growth and apoptosis. A leading hypothesis used to explain how calcium regulates such divergent signaling pathways is that molecules responsible for maintaining calcium homeostasis have multiple roles. For example, chromogranin B (CGB), a calcium binding protein found in secretory granules and in the lumen of the endoplasmic reticulum, buffers calcium and also binds to and amplifies the activity of the inositol 1,4,5-trisphosphate receptor (InsP(3)R). Previous studies have identified two conserved domains of CGB, an N-terminal domain (N-CGB) and a C-terminal domain (C-CGB). N-CGB binds to the third intraluminal loop of the InsP(3)R and inhibits binding of full-length CGB. This displacement of CGB decreases InsP(3)R-dependent calcium release and alters normal signaling patterns. In the present study, we further characterized the role of N-CGB and identified roles for C-CGB. The effect of N-CGB on calcium release depended upon endogenous levels of cellular CGB, whereas the regulatory effect of C-CGB was apparent regardless of endogenous levels of CGB. When either full-length CGB or C-CGB was expressed in cells, calcium transients were increased. Additionally, the calcium signal initiation site was altered upon C-CGB expression in neuronally differentiated PC12 and SHSY5Y cells. These results show that CGB has numerous regulatory roles and that CGB is a critical component in modulating InsP(3)R-dependent calcium signaling.     

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 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.     

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.     

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.     

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.     

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.