Affinity For IP3 Research Articles

Dissociation of inositol 1,4,5-trisphosphate from IP3 receptors contributes to termination of Ca2+ puffs

Ca2+ puffs are brief, localized Ca2+ signals evoked by physiological stimuli that arise from the coordinated opening of a few clustered inositol 1,4,5-trisphosphate receptors (IP3Rs). However, the mechanisms that control the amplitude and termination of Ca2+ puffs are unresolved. To address these issues, we expressed SNAP-tagged IP3R3 in HEK cells without endogenous IP3Rs and used total internal reflection fluorescence microscopy to visualize the subcellular distribution of IP3Rs and the Ca2+ puffs that they evoke. We first confirmed that SNAP-IP3R3 were reliably identified and that they evoked normal Ca2+ puffs after photolysis of a caged analog of IP3. We show that increased IP3R expression caused cells to assemble more IP3R clusters, each of which contained more IP3Rs, but the mean amplitude of Ca2+ puffs (indicative of the number of open IP3Rs) was unaltered. We thus suggest that functional interactions between IP3Rs constrain the number of active IP3Rs within a cluster. Furthermore, Ca2+ puffs evoked by IP3R with reduced affinity for IP3 had undiminished amplitude, but the puffs decayed more quickly. The selective effect of reducing IP3 affinity on the decay times of Ca2+ puffs was not mimicked by exposing normal IP3R to a lower concentration of IP3. We conclude that distinct mechanisms constrain recruitment of IP3Rs during the rising phase of a Ca2+ puff and closure of IP3Rs during the falling phase, and that only the latter is affected by the rate of IP3 dissociation.

Combining IP3 affinity chromatography and bioinformatics reveals a novel protein-IP3 binding site on Plasmodium falciparum MDR1 transporter

Intracellular Ca2+ mobilization induced by second messenger IP3 controls many cellular events in most of the eukaryotic groups. Despite the increasing evidence of IP3-induced Ca2+ in apicomplexan parasites like Plasmodium, responsible for malaria infection, no protein with potential function as an IP3-receptor has been identified. The use of bioinformatic analyses based on previously known sequences of IP3-receptor failed to identify potential IP3-receptor candidates in any Apicomplexa. In this work, we combine the biochemical approach of an IP3 affinity chromatography column with bioinformatic meta-analyses to identify potential vital membrane proteins that present binding with IP3 in Plasmodium falciparum. Our analyses reveal that PF3D7_0523000, a gene that codes a transport protein associated with multidrug resistance as a potential target for IP3. This work provides a new insight for probing potential candidates for IP3-receptor in Apicomplexa.

Luminal Calcium Control of Type-1 Inositol 1,4,5-Trisphosphate Receptor

Inositol 1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (RyR) are homologous endo/sarcoplasmic reticulum Ca2+ release channels. IP3R and RyR are tightly controlled by both cytosolic and luminal Ca2+. The negatively charged residue E4872 in RyR2 (cardiac isoform) electrostatically interacts with luminal Ca2+, explaining RyR2's luminal Ca2+ dependency (Chen et al., 2014). The corresponding residue in IP3R type 1 is D2594, but its role (if any) in IP3R function is unknown. We hypothesized the IP3R1-D2594 also electrostatically interacts with luminal Ca2+ to determine IP3R1 luminal Ca2+ sensitivity. To test this possibility, we created a mouse knock-in model harboring the D2594K mutation in which the negatively charged IP3R1-D2594 residue was replaced by lysine, a positively charged residue. IP3R1-WT and -D2594K cerebella microsomes were isolated and reconstituted into planar lipid bilayers. Single channel IP3R1 studies showed the conductance properties of IP3R1-WT and -D2594K channels are similar. The IP3R1-D2594K channels exhibited WT-like IP3 affinity, but had substantially greater IP3 efficacy. The IP3R1-D2594K also had altered luminal Ca2+ sensitivity. Unlike IP3R1-WT, D2594K channel activity did not decrease at low luminal Ca2+ levels. In IP3R1-WT channels, the decrease in activity at these low luminal Ca2+ levels (i.e. IP3R1 luminal Ca2+ sensitivity) depends on the membrane potential. Taken together, these results indicate the IP3R1-D2594 residue, as it does in RyR2, electrostatically interacts with luminal Ca2+, explaining at least in part, the IP3R's sensitivity to luminal Ca2+. This newly identified mechanism together with luminal Ca2+ feed-through may explain the luminal control of IP3R1.

IP3 R1-Mediated Local Ca2+ Release Events are Enhanced in the Gain-of-Function D2594K Mutant Channel

Quantal Ca2+ release through the inositol 1,4,5-trisphosphate receptor (IP3R) is a well-defined and ubiquitous phenomenon. However, its molecular origin remains poorly understood. Our general assumption is that this process results from variations of the channel's IP3 affinity as a function of the intra-pore [Mg2+]/[Ca2+] ratio. This ratio may vary during Ca2+ release and could be sensed by a residue (D2594) located in the inner portion of the channel's permeation pore. The hypothesis tested in this work is that the gain-of-function behavior shown by the IP3R when the D2594 residue is replaced by the basic residue K, is explained by increased IP3 affinity of the channel. If so, we predicted this mutation would modify the biophysical attributes of the resulting local Ca2+ release events (puffs) resulting in larger, brighter, and/or longer puffs. To test this, Ca2+ puffs were recorded with real-time, fast Ca2+-imaging, confocal microscopy after perfusing the purinergic ligand ATP (30 & 50 nM) and puff biophysical attributes analyzed. Experiments were conducted in intact HEK cells expressing either WT or D2594K mutant IP3R. Our results indicate that D2594K transfected intact HEK cells exhibited Ca2+ puffs with no significant differences in duration, but more than 6-fold larger amplitude (WT 0.88 ± 0.24 deltaF/F0; D2594 6.1 ± 0.24 deltaF/F0) than HEK cells expressing the WT channel. In addition, D2594K-expressing cells showed more than 1.5-fold higher Ca2+ puff frequency at every tested [ATP] (WT: 2.8 & 3.3 puffs/cell⋅min at 30 & 50 nM ATP, respectively. D2594: 4.8 & 5.3 puffs/cell⋅min at 30 & 50 nM ATP, respectively). These results are consistent with our hypothesis that quantal IP3 R-mediated Ca2+ release is controlled by the D2594 residue which allosterically affects the channel's affinity for IP3.

Inhibition of cAMP-Dependent PKA Activates β2-Adrenergic Receptor Stimulation of Cytosolic Phospholipase A2 via Raf-1/MEK/ERK and IP3-Dependent Ca2+ Signaling in Atrial Myocytes.

We previously reported in atrial myocytes that inhibition of cAMP-dependent protein kinase (PKA) by laminin (LMN)-integrin signaling activates β2-adrenergic receptor (β2-AR) stimulation of cytosolic phospholipase A2 (cPLA2). The present study sought to determine the signaling mechanisms by which inhibition of PKA activates β2-AR stimulation of cPLA2. We therefore determined the effects of zinterol (0.1 μM; zint-β2-AR) to stimulate ICa,L in atrial myocytes in the absence (+PKA) and presence (-PKA) of the PKA inhibitor (1 μM) KT5720 and compared these results with atrial myocytes attached to laminin (+LMN). Inhibition of Raf-1 (10 μM GW5074), phospholipase C (PLC; 0.5 μM edelfosine), PKC (4 μM chelerythrine) or IP3 receptor (IP3R) signaling (2 μM 2-APB) significantly inhibited zint-β2-AR stimulation of ICa,L in–PKA but not +PKA myocytes. Western blots showed that zint-β2-AR stimulation increased ERK1/2 phosphorylation in–PKA compared to +PKA myocytes. Adenoviral (Adv) expression of dominant negative (dn) -PKCα, dn-Raf-1 or an IP3 affinity trap, each inhibited zint-β2-AR stimulation of ICa,L in + LMN myocytes compared to control +LMN myocytes infected with Adv-βgal. In +LMN myocytes, zint-β2-AR stimulation of ICa,L was enhanced by adenoviral overexpression of wild-type cPLA2 and inhibited by double dn-cPLA2S505A/S515A mutant compared to control +LMN myocytes infected with Adv-βgal. In–PKA myocytes depletion of intracellular Ca2+ stores by 5 μM thapsigargin failed to inhibit zint-β2-AR stimulation of ICa,L via cPLA2. However, disruption of caveolae formation by 10 mM methyl-β-cyclodextrin inhibited zint-β2-AR stimulation of ICa,L in–PKA myocytes significantly more than in +PKA myocytes. We conclude that inhibition of PKA removes inhibition of Raf-1 and thereby allows β2-AR stimulation to act via PKCα/Raf-1/MEK/ERK1/2 and IP3-mediated Ca2+ signaling to stimulate cPLA2 signaling within caveolae. These findings may be relevant to the remodeling of β-AR signaling in failing and/or aging heart, both of which exhibit decreases in adenylate cyclase activity.

IP3 and Ca(2+) signals in the heart: boost them or bust them?

IP3 and Ca(2+) signals in the heart: boost them or bust them?

Inhibition of Camp-Dependent PKA Activates β2-Adrenergic Receptor Stimulation of Cytosolic Phospholipase A2 via Raf-1/Mek/Erk and Ip3-Dependent Ca2+ Signaling in Atrial Myocytes

We previously reported in atrial myocytes that inhibition of PKA by laminin (LMN)-integrin signaling activates β2-adrenergic receptor (β2-AR) stimulation of cytosolic phospholipase A2 (cPLA2). Here, we determined the effects of zinterol (0.1 μM; zint-β2-AR) to stimulate ICa,L in atrial myocytes in the absence (+PKA) and presence (-PKA) of the PKA inhibitor (1 µM) KT5720 and compared these results with atrial myocytes attached to laminin (+LMN). Inhibition of Raf-1 (10 µM GW5074), phospholipase C (PLC; 0.5 μM edelfosine), PKC (4 μM chelerythrine) or IP3 receptor (IP3R) signaling (2 µM 2-APB) significantly inhibited zint-β2-AR stimulation of ICa,L in -PKA but not +PKA myocytes. Western blots showed that zint-β2-AR stimulation increased ERK1/2 phosphorylation in -PKA compared to +PKA myocytes. Adenoviral (Adv) expression of dominant negative (dn) -PKCα, dn-Raf-1 or an IP3 affinity trap, each inhibited zint-β2-AR stimulation of ICa,L in + LMN myocytes compared to control +LMN myocytes infected with Adv-βgal. In +LMN myocytes, zint-β2-AR stimulation of ICa,L was enhanced by adenoviral overexpression of wild-type cPLA2 and inhibited by double dn-cPLA2S505A/S515A mutant compared to control +LMN myocytes infected with Adv-βgal. In -PKA myocytes depletion of intracellular Ca2+ stores by 5 μM thapsigargin failed to inhibit zint-β2-AR stimulation of ICa,L via cPLA2. However, disruption of caveolae formation by 10 mM methyl-β-cyclodextrin inhibited zint-β2-AR stimulation of ICa,L in -PKA myocytes significantly more than in +PKA myocytes. We conclude that inhibition of PKA removes inhibition of Raf-1 and thereby allows β2-AR stimulation to act via PKCα/Raf-1/MEK/ERK1/2 and IP3-mediated Ca2+ signaling to stimulate cPLA2 signaling within caveolae. These findings may be relevant to the remodeling of β-AR signaling in failing and/or aging heart, both of which exhibit decreases in adenylate cyclase activity.

Functional Inositol 1,4,5-Trisphosphate Receptors Assembled from Concatenated Homo- and Heteromeric Subunits

Vertebrate genomes code for three subtypes of inositol 1,4,5-trisphosphate (IP3) receptors (IP3R1, -2, and -3). Individual IP3R monomers are assembled to form homo- and heterotetrameric channels that mediate Ca(2+) release from intracellular stores. IP3R subtypes are regulated differentially by IP3, Ca(2+), ATP, and various other cellular factors and events. IP3R subtypes are seldom expressed in isolation in individual cell types, and cells often express different complements of IP3R subtypes. When multiple subtypes of IP3R are co-expressed, the subunit composition of channels cannot be specifically defined. Thus, how the subunit composition of heterotetrameric IP3R channels contributes to shaping the spatio-temporal properties of IP3-mediated Ca(2+) signals has been difficult to evaluate. To address this question, we created concatenated IP3R linked by short flexible linkers. Dimeric constructs were expressed in DT40-3KO cells, an IP3R null cell line. The dimeric proteins were localized to membranes, ran as intact dimeric proteins on SDS-PAGE, and migrated as an ∼1100-kDa band on blue native gels exactly as wild type IP3R. Importantly, IP3R channels formed from concatenated dimers were fully functional as indicated by agonist-induced Ca(2+) release. Using single channel "on-nucleus" patch clamp, the channels assembled from homodimers were essentially indistinguishable from those formed by the wild type receptor. However, the activity of channels formed from concatenated IP3R1 and IP3R2 heterodimers was dominated by IP3R2 in terms of the characteristics of regulation by ATP. These studies provide the first insight into the regulation of heterotetrameric IP3R of defined composition. Importantly, the results indicate that the properties of these channels are not simply a blend of those of the constituent IP3R monomers.

Analysis of the phospholipase C-δ1 pleckstrin homology domain using native polyacrylamide gel electrophoresis

The phospholipase C (PLC)-δ1 pleckstrin homology (PH) domain has a characteristic short α-helix (α2) from residues 82 to 87. The contributions of the α2-helix toward the inositol 1,4,5-trisphosphate (IP3) binding activity and thermal stability of the PLC-δ1 PH domain were investigated using native polyacrylamide gel electrophoresis (PAGE). Native PAGE analyses of gel migration shift induced by IP3 binding and of protein aggregation induced by heating indicated that disruption of the α-helical conformation by replacement of Lys86 with proline resulted in reduced affinity for IP3 and in thermal destabilization of the IP3-binding state. Although the mutant protein with replacement of Lys86 with alanine showed a slight reduction in thermal stability, the IP3-binding affinity was similar to that of the wild-type protein. Replacement of Phe87 with alanine, but not with tyrosine, also resulted in reduced affinity for IP3 and in thermal instability. These results indicated that the helical conformation of the α2-helix and the phenyl ring of Phe87 play important roles in the IP3-binding activity and thermal stability of the PLC-δ1 PH domain. Based on these results, the biological role of the α2-helix of the PLC-δ1 PH domain is discussed in terms of membrane binding.

Common allosteric mechanisms between ryanodine and inositol-1,4,5-trisphosphate receptors

Ryanodine receptors (RyRs) are calcium release channels found in the membrane of the endoplasmic reticulum (ER). We recently described the crystal structure of the RyR1 N-terminal disease hot spot. It is built up by three domains that show clear structural homology with the inositol-1,4,5-triphosphate (IP3) binding core and suppressor domain of IP3 receptors (IP3Rs) . Here we analyze the structural features of the domains in both calcium release channels, and propose a model for the closed state of the IP3R N-terminal region. This model explains the effect of the suppressor domain on the affinity for IP3 and is supported by mutational studies performed previously. We propose a mechanism whereby opening of both RyR and IP3R is allosterically coupled to a displacement of the N-terminal domain from the following two domains. This displacement can be affected by disease mutations, glutathionylation of a highly reactive cysteine residue, or ligand binding.

β2-Adrenergic Receptor (β2-AR) Stimulation of Cytosolic Phospholipase A2 (cPLA2) Is Dependent on PKC and IP3-Mediated Ca2+ Signaling in Atrial Myocytes

We reported that inhibition of adenylate cyclase (AC)/cAMP by cell attachment to laminin (+LMN) or pharmacological (KT5720) inhibition of cAMP-dependent protein kinase (PKA) in cells not attached to LMN (-LMN-PKA), activates β2-AR stimulation of ICa,L via cPLA2 signaling. The present study determined the role of PKC and IP3-mediated Ca2+ release in β2-AR/cPLA2 signaling. As previously reported, 0.1 μM zinterol (β2-AR agonist) stimulation of L-type Ca2+ current (ICa,L) was unaffected by 10 μM AACOCF3 (cPLA2 inhibitor) in cells not attached to LMN (-LMN) but was significantly inhibited in +LMN and -LMN-PKA myocytes. Zinterol stimulation of ICa,L in -LMN-PKA myocytes was blocked by 5 μM U73122 (PLC inhibitor) and significantly inhibited by 4 μM chelerythrine (PKC inhibitor). Zinterol stimulation of ICa,L in -LMN myocytes was unaffected by inhibition of IP3-receptors (IP3Rs) by 2 μM 2-APB, but was significantly inhibited in +LMN and -LMN-PKA myocytes. Cells were cultured on LMN (24 hrs) with an adenovirus IP3 affinity trap to inhibit IP3-dependent Ca2+ signaling. Compared to control cells (β-gal), zinterol stimulation of ICa,L was significantly inhibited in cells infected with IP3 trap. Laser scanning confocal microscopy (fluo-4) revealed that zinterol stimulation of +LMN myocytes elicited local intracellular Ca2+ release events in 1 mM tetracaine (blocks RyR Ca2+ release), that were blocked by 2-APB. We conclude that inhibition of cAMP/PKA activates β2-AR stimulation of ICa,L via cPLA2 which is dependent on PKC and IP3-mediated Ca2+ signaling. These findings may be relevant to the remodeling of β-AR signaling in diseased (fibrotic) and/or aging atria, both of which exhibit decreases in AC activity.

Nuclear calcium signaling by inositol trisphosphate in GH3 pituitary cells

It has been proposed that nuclear and cytosolic Ca2+ ([Ca2+]N and [Ca2+]C) may be regulated independently. We address here the issue of whether inositol trisphosphate (IP3) can, bypassing changes of [Ca2+]C, produce direct release of Ca2+ into the nucleoplasm. We have used targeted aequorins to selectively measure and compare the changes in [Ca2+]C and [Ca2+]N induced by IP3 in GH3 pituitary cells. Heparin, an IP3 inhibitor that does not permeate the nuclear pores, abolished the [Ca2+]C peaks but inhibited only partly the [Ca2+]N peaks. The permeant inhibitor 2-aminoethoxy-diphenyl-borate (2-APB) blocked both responses. Removal of ATP also inhibited more strongly the [Ca2+]C than [Ca2+]N peak. The [Ca2+]N and [Ca2+]C responses differed also in their sensitivity to IP3, the nuclear response showing higher affinity. Among IP3 receptors, type 2 (IP3R2) has a higher affinity for IP3 and is not inactivated by ATP removal. We find that IP3R2 immunoreactivity is present inside the nucleus whereas the other IP3R subtypes are detected only in the cytoplasm. The nuclear envelope (NE) of GH3 cells showed deep invaginations into the nucleoplasm, with cytosol and cytoplasmic organella inside. These results indicate that GH3 pituitary cells possess mechanisms able to produce selective increases of [Ca2+]N.

Calcium regulation of inositol 1,4,5-trisphosphate receptors

Ca 2+ exerts both a stimulatory and inhibitory effect on type-I IP 3R channel activity. However, the structural determinants of Ca 2+ sensing in IP 3Rs are not fully understood. Previous studies by others have identified eight domains of the type-I IP 3R that bind 45Ca 2+ when expressed as GST-fusion proteins. We have mutated six highly conserved acidic residues within the second of these domains (aa378–450) in the full-length IP 3R and measured the Ca 2+regulation of IP 3-mediated Ca 2+ release in COS-7 cells. 45Ca 2+flux assays measured with a maximal [IP 3] (1 μM) indicate that one of the mutants retained a Ca 2+ sensitivity that was not significantly different from control (E411Q), three of the mutants show an enhanced Ca 2+ inhibition (D426N, E428Q and E439Q) and two of the mutants were relatively insensitive to Ca 2+ inhibition (D442N and D444N). IP 3 dose–response relationships indicated that the sensitivity to Ca 2+ inhibition and affinity for IP 3 were correlated for three of the constructs. Other mutants with enhanced IP 3 sensitivity (e.g. R441Q and a type-II/I IP 3R chimera) were also less sensitive to Ca 2+ inhibition. We conclude that the acidic residues within the aa378–450 segment are unlikely to represent a single functional Ca 2+ binding domain and do not contribute to Ca 2+ activation of the receptor. The different effects of the mutations may be related to their location within two clusters of acidic residues identified in the crystal structure of the ligand-binding domain [I. Bosanac, J.R. Alattia, T.K. Mal, et al., Structure of the inositol 1,4,5-trisphosphate receptor binding core in complex with its ligand, Nature 420 (2002) 696–700]. The data support the view that all IP 3R isoforms may display a range of Ca 2+ sensitivities that are determined by multiple sites within the protein and markedly influenced by the affinity of the receptor for IP 3.

Determinants of adenophostin A binding to inositol trisphosphate receptors.

Inositol 1,4,5-trisphosphate (IP(3)) receptors from cerebellum and recombinant type 1 IP(3) receptors expressed in Sf9 cells had indistinguishable affinities for IP(3) ( K (d)=6.40+/-0.48 nM) and adenophostin A ( K (d)=0.89+/-0.05 nM). In cytosol-like medium, each of the three mammalian IP(3) receptor subtypes when expressed in Sf9 cells bound adenophostin A with greater affinity than IP(3). It has been suggested that adenophostin A binds with high affinity only in the presence of ATP, but we found that adenophostin A similarly displaced [(3)H]IP(3) from type 1 IP(3) receptors whatever the ATP concentration. N-terminal fragments of the type 1 receptor were expressed with and without the S1 splice site; its removal had no effect on [(3)H]IP(3) binding to the 1-604 protein, but abolished binding to the 224-604 protein. The 1-604 fragment and full-length receptor bound adenophostin A with the same affinity, but the fragment had 3-fold greater affinity for IP(3), suggesting that C-terminal residues selectively inhibit IP(3) binding. The 224-604S1(+) fragment bound IP(3) and adenophostin A with increased affinity, but as with the 1-604 fragment it bound adenophostin A with only 2-fold greater affinity than IP(3). High-affinity binding of adenophostin A may be partially determined by its 2'-phosphate interacting more effectively than the 1-phosphate of IP(3) with residues within the IP(3)-binding core. This may account for the 2-fold greater affinity of adenophostin A relative to IP(3) for the minimal IP(3)-binding domain. In addition we suggest that C-terminal residues, which impede access of IP(3), may selectively interact with adenophostin A to allow it unhindered access to the IP(3)-binding domain.

A new fluorescent biosensor for inositol trisphosphate.

An intracellular second messenger d-myo-inositol-1,4,5-trisphosphate (IP3) is a key biological signaling molecule that controls the cellular Ca2+ concentration. We report the preparation and evaluation of a functionalized protein-based sensor for IP3 by exploring the selective IP3 binding properties of pleckstrin homology (PH) domain. Signal transduction is imparted to the protein by mutation of proximal residues to cysteine and then alkylation of the active site by various fluorophore derivatives. This creates functionalized proteins that show micromolar affinity for IP3, reasonably strong fluorescence emission, and wavelength changes in the fluorophore and selectivity higher than the original PH domain among different inositol phosphate derivatives.

Expression and distribution of the type 1 and type 3 inositol 1,4, 5-trisphosphate receptor in developing vascular smooth muscle.

The recent discoveries of inositol 1,4,5-trisphosphate (IP3) receptor subtypes with different affinities for IP3 and their potential involvement in development has important consequences for vascular smooth muscle. This study has examined the expression and distribution of the type 1 and type 3 IP3 receptor subtypes in developing rat vascular smooth muscles. Immunoblotting of portal vein and aorta from neonatal (2 to 4 days) and fully developed (6 weeks) rats revealed significantly higher levels of the type 3 IP3 receptor expression in neonatal, compared with developed, vascular smooth muscles. In contrast, expression of the type 1 IP3 receptor in neonates was lower compared with developed vascular smooth muscles. Immunolocalization of the type 3 IP3 receptors in neonatal tissues revealed that staining corresponded to the distribution of the sarcoplasmic reticulum (visualized by osmium ferricyanide staining of thin tissue sections), which suggested localization of the type 3 IP3 receptor throughout the sarcoplasmic reticulum network. We conclude that type 3 IP3 receptors are the predominant subtype in the development of vascular smooth muscle and are distributed throughout the sarcoplasmic reticulum in these cells. The switch in isoforms of the IP3 receptor during development from the type 3 with low affinity for IP3 to the higher-affinity type 1 receptor may play a role in calcium-mediated regulation of developing vascular smooth muscle.

Cooperative Formation of the Ligand-binding Site of the Inositol 1,4,5-Trisphosphate Receptor by Two Separable Domains

Limited trypsin digestion of mouse cerebellar membrane fractions leads to fragmentation of the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) into five major components (Yoshikawa, F., Iwasaki, H., Michikawa, T., Furuichi, T., and Mikoshiba, K. (1999) J. Biol. Chem. 274, 316-327). Here we report that trypsin-fragmented mouse IP3R1 (mIP3R1) retains significant inositol 1,4,5-trisphosphate (IP3) binding activity that is comparable to the intact receptor in affinity, capacity, and specificity. This is despite the fact that the IP3-binding core (residues 226-578), which is close to the minimum for high affinity binding, is completely split into two tryptic fragments at the Arg-343 and/or Arg-345, around the center of the core. Furthermore, we have examined whether binding activity could be complemented in vitro by mixing two distinct glutathione S-transferase (GST) fusion proteins, which were respectively composed of residues 1-343 and 341-604, almost corresponding to two split binding components, and separately expressed in Escherichia coli. The GST-fused residues 1-343 (GN) showed no binding affinity for IP3, whereas the GST-fused residues 341-604 (GC) displayed weak but definite activity with an affinity >100-fold lower than that of the native receptor. Upon mixing of both GN and GC, a high affinity site comparable to the native site appeared. We suggest that the IP3-binding pocket consists of two non-covalently but tightly associated structural domains each of which has a discrete function: the C-terminal domain alone has low affinity for IP3, whereas the N-terminal one alone is incapable of binding but is capable of potentiating binding affinity.

A novel role for calmodulin: Ca2+-independent inhibition of type-1 inositol trisphosphate receptors.

Calmodulin inhibits both inositol 1,4,5-trisphosphate (IP3) binding to, and IP3-evoked Ca2+ release by, cerebellar IP3 receptors [Patel, Morris, Adkins, O'Beirne and Taylor (1997) Proc. Natl. Acad. Sci. U. S.A. 94, 11627-11632]. In the present study, full-length rat type-1 and -3 IP3 receptors were expressed at high levels in insect Spodoptera frugiperda 9 cells and the effects of calmodulin were examined. In the absence of Ca2+, calmodulin caused a concentration-dependent and reversible inhibition of [3H]IP3 binding to type-1 IP3 receptors by decreasing their apparent affinity for IP3. The effect was not reproduced by high concentrations of troponin C, parvalbumin or S-100. Increasing the medium free [Ca2+] ([Ca2+]m) inhibited [3H]IP3 binding to type-1 receptors, but the further inhibition caused by a submaximal concentration of calmodulin was similar at each [Ca2+]m. In the absence of Ca2+, 125I-calmodulin bound to a single site on each type-1 receptor subunit and to an additional site in the presence of Ca2+. There was no detectable binding of 125I-calmodulin to type-3 receptors and binding of [3H]IP3 was insensitive to calmodulin at all [Ca2+]m. Both peptide and conventional Ca2+-calmodulin antagonists affected neither [3H]IP3 binding directly nor the inhibitory effect of calmodulin in the absence of Ca2+, but each caused a [Ca2+]m-dependent reversal of the inhibition of [3H]IP3 binding caused by calmodulin. Camstatin, a peptide that binds to calmodulin equally well in the presence or absence of Ca2+, reversed the inhibitory effects of calmodulin on [3H]IP3 binding at all [Ca2+]m. We conclude that calmodulin specifically inhibits [3H]IP3 binding to type-1 IP3 receptors: the first example of a protein regulated by calmodulin in an entirely Ca2+-independent manner. Inhibition of type-1 IP3 receptors by calmodulin may dynamically regulate their sensitivity to IP3 in response to the changes in cytosolic free calmodulin concentration thought to accompany stimulation of neurones.

Differential regulation of types-1 and -3 inositol trisphosphate receptors by cytosolic Ca2+

Biphasic regulation of inositol trisphosphate (IP3)-stimulated Ca2+ mobilization by cytosolic Ca2+ is believed to contribute to regenerative intracellular Ca2+ signals. Since cells typically express several IP3 receptor isoforms and the effects of cytosolic Ca2+ are not mediated by a single mechanism, it is important to resolve the properties of each receptor subtype. Full-length rat types-1 and -3 IP3 receptors were expressed in insect Sf9 cells at levels 10-40-fold higher than the endogenous receptors. The expressed receptors were glycosylated and assembled into tetramers, and binding of [3H]IP3 to each subtype was regulated by cytosolic Ca2+. The effects of increased [Ca2+] on native cerebellar and type-1 receptors expressed in Sf9 cells were indistinguishable. A maximally effective increase in [Ca2+] reversibly inhibited [3H]IP3 binding by approx. 50% by decreasing the number of IP3-binding sites (Bmax) without affecting their affinity for IP3. The effects of Ca2+ on type-3 receptors were more complex: increasing [Ca2+] first stimulated [3H]IP3 binding by increasing Bmax, and then inhibited it by causing a substantial decrease in the affinity of the receptor for IP3. The different effects of Ca2+ on the receptor subtypes were not a consequence of limitations in the availability of accessory proteins or of artifactual effects of Ca2+ on membrane structure. We conclude that Ca2+ can inhibit IP3 binding to types-1 and -3 IP3 receptors although by different mechanisms, and that IP3 binding to type-3 receptors is stimulated at intermediate [Ca2+]. A consequence of these differences is that, at resting cytosolic [Ca2+], type-3 receptors are more sensitive than type-1 receptors to IP3, but the situation reverses at higher cytosolic [Ca2+]. Such differences may be important in generating the spatially and temporally complex changes in cytosolic [Ca2+] evoked by receptors linked to IP3 formation.

Polarized expression of Ca2+ pumps in pancreatic and salivary gland cells. Role in initiation and propagation of [Ca2+]i waves.

The present study was aimed at localization of plasma membrane (PMCA) and intracellular (SERCA) Ca2+ pumps and characterizing their role in initiation and propagation of Ca2+ waves. Specific and polarized expression of Ca2+ pumps was observed in all epithelial cells examined. Immunolocalization revealed expression of PMCA in both the basolateral and luminal membranes of all cell types. SERCA2a appeared to be expressed in the luminal pole, whereas SERCA2b was expressed in the basal pole and the nuclear envelope of pancreatic acini. Interestingly, SERCA2b was found in the luminal pole of submandibular salivary gland acinar and duct cells. These cells expressed SERCA3 in the basal pole. To examine the significance of the polarized expression of SERCA and perhaps PMCA pumps in secretory cells, we compared the effect of inhibition of SERCA pumps with thapsigargine and partial Ca2+ release with ionomycin on Ca2+ release evoked by agonists and Ca2+ uptake induced by antagonists. Despite their polarized expression, Ca2+ uptake by SERCA pumps and Ca2+ efflux by PMCA resulted in uniform reduction in [Ca2+]i. Surprisingly, inhibition of the SERCA pumps, but not Ca2+ release by ionomycin, eliminated the distinct initiation sites and propagated Ca2+ waves, leading to a uniform increase in [Ca2+]i. In addition, inhibition of SERCA pumps reduced the rate of Ca2+ release from internal stores. The implication of these findings to rates of Ca2+ diffusion in the cytosol, compartmentalization of Ca2+ signaling complexes, and mechanism of Ca2+ wave propagation are discussed.