A model of the dynamics of intracellular calcium based on refined kinetics of inhibiting the inositol-1,4,5-trisphoshate-sensitive calcium channel

Several new models of intracellular calcium dynamics based on refined inositol-1,4,5-triphosphate-sensitive calcium channel kinetics were studied. The refined kinetic schemes take into account that a cytosolic calcium cannot inhibit inositol-1,4,5-triphosphate receptors when they are bound to inositol-1,4,5-triphosphate. The mathematical analysis of intracellular calcium dynamics based on one of these schemes allowed us to show how different types of Ca response to extracellular stimuli, such as excitability, oscillations, sustained elevation of Ca and frequency encoding can arise with a reasonably good fit to experimental data.     

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.     

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.     

The calcium-mobilizing effect of inositol-1,4,5-triphosphate in rod outer segments and disks]

The effect of inositol-1,4,5-trisphosphate (IP3) on the release of calcium ions from retinal rod discs was studied. It was shown that the release of Ca2+ from discs is an electroneutral process. The intradiscal calcium concentration during the release of the ion from the organelle decreases by 1 mM. It was found that the IP3-dependent release of Ca2+ ions from discs is activated by guanosine triphosphate and beta gamma-transducin. The increase in calcium concentration in the medium also activates the IP3-dependent release of Ca2+ ions from discs, which probably is due to the stimulation of phospholipase C. It is suggested that the functional role of the release of ions in related not to phototransduction but to slow regulatory and adaptation processes in the photoreceptor cell.     

Native structure and arrangement of inositol-1,4,5-trisphosphate receptor molecules in bovine cerebellar Purkinje cells as studied by quick-freeze deep-etch electron microscopy.

We used quick-freeze deep-etch replica electron microscopy to visualize the native structure of inositol-1,4,5-trisphosphate receptor (IP3R) in the cell. In the dendrites of Purkinje neurons of bovine cerebellum there were many vesicular organelles whose surfaces were covered with a two-dimensional crystalline array of molecules. Detailed examination of the cytoplasmic true surface of such vesicles in replica revealed that the structural unit, identified as IP3R by immunocytochemistry and subsequent Fourier analysis, is a square-shaped assembly and is aligned so that the side of the square is inclined by approximately 20 degrees from the row-line of the lattice. Comparison with the ryanodine receptor (RyaR), another intracellular Ca2+ channel on the endoplasmic reticulum, suggested that IP3R, unlike RyaR, has a very compact structure, potentially reflecting the crucial difference in the function of the cytoplasmic portion of the molecule.     

Three-dimensional rearrangements within inositol 1,4,5-trisphosphate receptor by calcium

Allosteric binding of calcium ion (Ca2+) to inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) controls channel gating within IP3R. Here, we present biochemical and electron microscopic evidence of Ca2+-sensitive structural changes in the three-dimensional structure of type 1 IP3R (IP3R1). Low concentrations of Ca2+ and high concentrations of Sr2+ and Ba2+ were shown to be effective for the limited proteolysis of IP3R1, but Mg2+ had no effect on the proteolysis. The electron microscopy and the limited proteolysis consistently demonstrated that the effective concentration of Ca2+ for conformational changes in IP3R1 was <10(-7) m and that the IP3 scarcely affected the conformational states. The structure of IP3R1 without Ca2+, as reconstructed by three-dimensional electron microscopy, had a "mushroom-like" appearance consisting of a large square-shaped head and a small channel domain linked by four thin bridges. The projection image of the "head-to-head" assembly comprising two particles confirmed the mushroom-like side view. The "windmill-like" form of IP3R1 with Ca2+ also contains the four bridges connecting from the IP3-binding domain toward the channel domain. These data suggest that the Ca2+-specific conformational change structurally regulates the IP3-triggered channel opening within IP3R1.     

Cyclic AMP-dependent phosphorylation of the inositol-1,4,5-trisphosphate receptor inhibits Ca2+ release from platelet membranes.

Purified internal platelet membranes were treated with the catalytic subunit of protein kinase A to determine its effect on inositol-1,4,5-trisphosphate (IP3)-mediated Ca2+ release. Release kinetics were monitored using rhod-2, a Ca(2+)-specific fluorophore. Protein kinase A maximally inhibited the rate of IP3-mediated Ca2+ release by approximately 30% at saturating IP3 (10 microM). This inhibition was eliminated by pretreatment with a specific kinase inhibitor peptide. Partial purification of the platelet IP3 receptor showed that both endogenous kinases and added A kinase directly phosphorylate the receptor. Since the IP3 receptor is phosphorylated in the absence of added kinase, the observed inhibition (30%) by protein kinase A does not represent the maximal effect of phosphorylation.     

The ligand binding site and transduction mechanism in the inositol-1,4,5-triphosphate receptor.

The inositol-1,4,5-triphosphate (InsP3) receptor consists of a homotetramer of highly conserved 313 kd subunits that contain multiple transmembrane regions in the C-terminal part of the protein. The receptor was expressed in COS cells and its domain structure was studied by mutagenesis. Deletion of the transmembrane regions from the receptor results in the synthesis of a soluble receptor protein that efficiently binds InsP3 but which instead of associating into homotetramers remains monomeric. This result suggests a role for the transmembrane regions in the association of the receptor subunits into tetramers but not in ligand binding. To localize the ligand binding site, further cDNAs encoding truncated receptor proteins were constructed. Assays of InsP3 binding to these truncated InsP3 receptors revealed that sequences in the N-terminal fourth of the InsP3 receptor are sufficient for ligand binding. Accordingly, each subunit of the InsP3 receptor homotetramer contains an independent ligand binding site that is located on the N-terminal ends of each subunit and is separated from the putative channel-forming transmembrane regions by greater than 1400 amino acids. Gel filtration experiments demonstrate a large conformational change of the receptor as a function of ligand binding, suggesting a mechanism by which ligand binding might cause channel opening.     

Two-state conformational changes in inositol 1,4,5-trisphosphate receptor regulated by calcium

Inositol 1,4,5-trisphosphate receptor (IP3R) is a highly controlled calcium (Ca2+) channel gated by inositol 1,4,5-trisphosphate (IP3). Multiple regulators modulate IP3-triggered pore opening by binding to discrete allosteric sites within IP3R. Accordingly we have postulated that these regulators structurally control ligand gating behavior; however, no structural evidence has been available. Here we show that Ca2+, the most pivotal regulator, induced marked structural changes in the tetrameric IP3R purified from mouse cerebella. Electron microscopy of the IP3R particles revealed two distinct structures with 4-fold symmetry: a windmill structure and a square structure. Ca2+ reversibly promoted a transition from the square to the windmill with relocations of four peripheral IP3-binding domains, assigned by binding to heparin-gold. Ca2+-dependent susceptibilities to limited digestion strongly support the notion that these alterations exist. Thus, Ca2+ appeared to regulate IP3 gating activity through the rearrangement of functional domains.     

Release of Ca2+ from plant hypocotyl microsomes by inositol-1,4,5-trisphosphate

The effect of inositol-1,4,5-trisphosphate on Ca2+ release from microsomes isolated from dark-grown zucchini (Cucurbita pepo L.) hypocotyls was studied. Up to 30% of the Ca2+ taken up by the microsomes in the presence of 2mM ATP, was released by mumolar concentrations of inositol-1,4, 5-trisphosphate. This release was very rapid (less than 0.5 min) and was followed by a slower re-uptake of Ca2+. The microsomal levels of Ca2+ previously attained were not re-established within 5 min. External concentration of free Ca2+ was maintained in the 10(-8)M region during the release. This is the first time that inositol-1,4,5-trisphosphate has been shown to have a regulatory effect on Ca2+ in plant membrane fractions. Phosphoinositides may be important in signal transduction in plant cells, by altering the cytoplasmic Ca2+ activity, a function already known in animal cells.     

Functional importance of inositol-1,4,5-triphosphate-induced intracellular Ca2+ mobilization in galanin-induced microglial migration

Galanin (GAL) is a neuropeptide which is up-regulated following neuronal axotomy or inflammation. One subtype of GAL receptor (GalR2) is reported to be expressed in the brain's immune cell population, microglia. In the present study, we investigated the effect of GAL on microglial migration and compared the mechanism with that of bradykinin (BK). GAL significantly increased the migration of rat cultured microglia at 0.1 pM. The GAL-induced signal cascade was partly similar to that induced by BK. It was not dependent on G(i/o) protein but involved activation of protein kinase C, phosphoinositide 3-kinase and Ca(2+)-dependent K(+) channels. However, reverse-mode activation of the Na(+) /Ca(2+) -exchanger 1 was not involved in GAL-induced microglial migration, unlike BK-induced migration. Likewise, nominally-free extracellular Ca(2+) inhibited BK-induced migration but not GAL-induced migration. An inositol-1,4,5-triphosphate receptor antagonist significantly inhibited GAL-induced migration. GAL-induced Ca(2+) signaling did not induce nitric oxide synthase expression, but up-regulated class II major histocompatibility complex expression. These results indicate that activation of inositol-1,4,5-triphosphate receptor and increase in intracellular Ca(2+) are important for GAL-induced migration and immunoreactivity in microglia. The differences in down-stream signal transduction induced by GAL and BK suggest that GAL and BK may control distinct microglial functions under pathological conditions.     

Stimulations of arachidonate release and inositol-1,4,5-triphosphate formation are mediated by distinct G-proteins in human platelets

Addition of fluoroaluminate to human platelet suspension stimulated thromboxane synthesis and inositol-1,4,5-triphosphate formation in a time and dose dependent manner. Neomycin inhibited markedly fluoroaluminate induced inositol-1,4,5-triphosphate formation without significantly affecting thromboxane synthesis. Preincubation of platelets with PGE1, also inhibited significantly inositol-1,4,5-triphosphate formation with modest reduction of thromboxane synthesis. On the contrary, pretreatment of platelets with pertussis toxin inhibited fluoroaluminate stimulated thromboxane synthesis without affecting inositol-1,4,5-triphosphate formation. Similarly, preincubation of platelets with phorbol ester, PMA, inhibited markedly thromboxane synthesis with modest reduction of inositol-1,4,5-triphosphate formation. These results indicate that inositol-1,4,5-triphosphate formation and arachidonate release and thromboxane synthesis are controlled separately and are mediated by different G-proteins which are coupled to phospholipase C and phospholipase A2 respectively in platelets.     

GTP requirement for inositol-1,4,5-trisphosphate-induced Ca2+ release from sarcoplasmic reticulum in smooth muscle

We have examined inositol-1,4,5-trisphosphate (IP3)-induced Ca2+ release from the sarcoplasmic reticulum (SR) in the skinned vascular smooth muscle. The amount of Ca2+ in the SR was estimated indirectly by caffeine-induced contraction of the skinned preparation. The Ca2+ release from the SR by IP3 required GTP. A non-hydrolyzable analogue of GTP, guanosine 5'-(beta gamma-imido) triphosphate (GppNHp) could substitute for GTP in the IP3-induced Ca2+ release. These results suggest an involvement of GTP-binding protein in the mechanism of Ca2+ release from the SR by IP3 in smooth muscle.     

Ca2+- and inositol-1,4,5-triphosphate induced release of Ca2+ in the microsomal fraction of the rat brain

Using the fluorescent probes, Quin 2 and chlortetracycline, a comparative study of the Ca2+ and inositol-1.4.5-triphosphate (IP3)-induced Ca2+ release from rabbit skeletal muscle sarcoplasmic reticulum (SR) terminal cisterns and rat brain microsomal vesicles was carried out. It was shown that Ca2+ release from rat brain microsomal vesicles is induced both by IP3 and Ca2+, whereas that in SR terminal cisterns is induced only by Ca2+. Data from chlorotetracycline fluorescence analysis revealed that CaCl2 (50 microM) causes the release of 15-20% and 40-50% of the total Ca2+ pool accumulated in rat brain microsomal vesicles and rabbit SR terminal cisterns, respectively. Using Quin 2, it was found that IP3 used at the optimal concentration (1.5 mM) caused the release of 0.4-0.6 nmol of Ca2+ per mg microsomal protein, which makes up to 10-15% of the total Ca2+ pool. IP3 does not induce Ca2+ release in SR. Preliminary release of Ca2+ from brain microsomes induced by IP3 diminishes the liberation of this cation induced by Ca2+. It is suggested that brain microsomes contain a Ca2+ pool which is exhausted under the action of the both effectors, Ca2+ and IP3.     

Cytosolic inositol 1,4,5-trisphosphate dynamics during intracellular calcium oscillations in living cells

We developed genetically encoded fluorescent inositol 1,4,5-trisphosphate (IP3) sensors that do not severely interfere with intracellular Ca2+ dynamics and used them to monitor the spatiotemporal dynamics of both cytosolic IP3 and Ca2+ in single HeLa cells after stimulation of exogenously expressed metabotropic glutamate receptor 5a or endogenous histamine receptors. IP3 started to increase at a relatively constant rate before the pacemaker Ca2+ rise, and the subsequent abrupt Ca2+ rise was not accompanied by any acceleration in the rate of increase in IP3. Cytosolic [IP3] did not return to its basal level during the intervals between Ca2+ spikes, and IP3 gradually accumulated in the cytosol with a little or no fluctuations during cytosolic Ca2+ oscillations. These results indicate that the Ca2+ -induced regenerative IP3 production is not a driving force of the upstroke of Ca2+ spikes and that the apparent IP3 sensitivity for Ca2+ spike generation progressively decreases during Ca2+ oscillations.     

Inositol-1,4,5-triphosphate receptors mediate activity-induced synaptic Ca2+ signals in muscle fibers and Ca2+ overload in slow-channel syndrome

Strict control of calcium entry through excitatory synaptic receptors is important for shaping synaptic responses, gene expression, and cell survival. Disruption of this control may lead to pathological accumulation of Ca2+. The slow-channel congenital myasthenic syndrome (SCS), due to mutations in muscle acetylcholine receptor (AChR), perturbs the kinetics of synaptic currents, leading to post-synaptic Ca2+ accumulation. To understand the regulation of calcium signaling at the neuromuscular junction (NMJ) and the etiology of Ca2+ overload in SCS we studied the role of sarcoplasmic Ca2+ stores in SCS. Using fura-2 loaded dissociated fibers activated with acetylcholine puffs, we confirmed that Ca2+ accumulates around wild type NMJ and discovered that Ca2+ accumulates significantly faster around the NMJ of SCS transgenic dissociated muscle fibers. Additionally, we determined that this process is dependant on the activation, altered kinetics, and movement of Ca2+ ions through the AChR, although, surprisingly, depletion of intracellular stores also prevents the accumulation of this cation around the NMJ. Finally, we concluded that the sarcoplasmic reticulum is the main source of Ca2+ and that inositol-1,4,5-triphosphate receptors (IP3R), and to a lesser degree L-type voltage gated Ca2+ channels, are responsible for the efflux of this cation from intracellular stores. These results suggest that a signaling system mediated by the activation of AChR, Ca2+, and IP3R is responsible for localized Ca2+ signals observed in muscle fibers and the Ca2+ overload observed in SCS.     

The Pro335 --> Leu polymorphism of type 3 inositol 1,4,5-trisphosphate receptor found in mouse inbred lines results in functional change

Inositol 1,4,5-trisphosphate receptor (IP3R) is an intracellular Ca2+ channel involved in various cellular signaling. Type 3 IP3R (IP3R3) retains ligand-gated Ca2+ channel properties differing from other subtypes in terms of IP3-binding affinity and regulation of its channel activity by effector molecules. In this study, we found the natural Pro335 --> Leu polymorphism of mouse IP3R3 between BALB/c and C57BL/6J. We investigated the functional differences between Pro335IP3R3 and Leu335IP3R3 with purified receptors reconstituted into proteoliposomes as well as with soluble ligand binding domains. Pro335IP3R3 exhibited significantly higher IP3-binding affinity and IP3-induced Ca2+ release than those of Leu335IP3R3 in both forms of the receptor. Moreover, the polymorphic change caused differences in the effect of external Ca2+ on IP3-induced Ca2+ release. The Pro335 --> Leu substitution alters the conformation of soluble ligand binding domain as revealed by intrinsic fluorescence and circular dichroism spectra with or without Ca2+. The results indicate that the polymorphism of IP3R3 causes changes in receptor function, presumably affecting intracellular Ca2+ signaling.     

Effects of inositol-1,4,5-trisphosphate injections into salamander rods

Solitary rods were isolated by trituration of salamander (Ambystoma tigrinum) retinas. One barrel of an intracellular, double-barreled micropipette was used to record membrane voltage; the other barrel was used to pressure-inject inositol-1,4,5-trisphosphate. The injection of inositol-1,4,5 -trisphosphate induced a reversible hyperpolarization of the rod membrane. Injections of inositol-1,4,5-trisphosphate decreased the size of receptor potentials induced by dim lights. Conversely, light decreased the responses of the rod to injections of inositol-1,4,5-trisphosphate. These results suggest that inositol-1,4,5-trisphosphate might be involved in the modulation of rod membrane voltage during phototransduction.     

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

Ca2+ exerts both a stimulatory and inhibitory effect on type-I IP3R channel activity. However, the structural determinants of Ca2+ sensing in IP3Rs are not fully understood. Previous studies by others have identified eight domains of the type-I IP3R that bind 45Ca2+ 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 IP3R and measured the Ca2+ regulation of IP3-mediated Ca2+ release in COS-7 cells. 45Ca2+ flux assays measured with a maximal [IP3] (1 microM) indicate that one of the mutants retained a Ca2+ sensitivity that was not significantly different from control (E411Q), three of the mutants show an enhanced Ca2+ inhibition (D426N, E428Q and E439Q) and two of the mutants were relatively insensitive to Ca2+ inhibition (D442N and D444N). IP3 dose-response relationships indicated that the sensitivity to Ca2+ inhibition and affinity for IP3 were correlated for three of the constructs. Other mutants with enhanced IP3 sensitivity (e.g. R441Q and a type-II/I IP3R chimera) were also less sensitive to Ca2+ inhibition. We conclude that the acidic residues within the aa378-450 segment are unlikely to represent a single functional Ca2+ binding domain and do not contribute to Ca2+ 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 IP3R isoforms may display a range of Ca2+ sensitivities that are determined by multiple sites within the protein and markedly influenced by the affinity of the receptor for IP3.     

Bridging the gaps in 3D structure of the inositol 1,4,5-trisphosphate receptor-binding core

Calcium concentration is strictly regulated in all cells. The inositol 1,4,5-trisphosphate receptor (IP(3)R), which forms a homotetrameric Ca2+ release channel in the endoplasmic reticulum, is one of the key molecules responsible for this regulation. The opening of this channel requires binding of two intracellular messengers, which are inositol 1,4,5-trisphosphate (IP(3)) and Ca2+. To promote the Ca2+-channel gating and release from the endoplasmic reticulum, IP(3) binds to the amino-terminal region of IP(3)R. Recently, the crystal structure of IP(3)R-binding core in complex with its ligand was presented [I. Bosanac, J.R. Alattia, T.K. Mai, J. Chan, S. Talarico, F.K. Tong, K.I. Tong, F. Yoshikawa, T. Furuichi, M. Iwai, T. Michikawa, K. Mikoshiba, M. Ikura, Structure of the inositol 1,4,5-trisphosphate receptor binding core in complex with its ligand, Nature 420 (2002) 696-700; I. Bosanac, H. Yamazaki, T. Matsu-ura, T. Michikawa, K. Mikoshiba, M. Ikura, Crystal structure of the ligand-binding suppressor domain of type 1 inositol 1,4,5-trisphosphate receptor, Mol. Cell 17 (2005) 193-203]. The space positions of residues 289-301 (segment A), 320-350 (segment B), 373-386 (segment C), and 529-545 (segment D) were not determined by the X-ray crystallography. To bridge these gaps, the computer modeling of physiologically meaningful low-energy 3D structures of the segments A-D of the inositol 1,4,5-trisphosphate receptor has been carried out by using a hierarchical conformational search algorithm combining two approaches: knowledge-based homology modeling and ab initio conformational search strategy. The structure analysis suggests a Ca2+-binding site of high affinity formed by residues 296-335, several low-energy regular secondary structure units within the segment B, and a number of hinge regions within the segments A-D, important for the receptor functioning.