InsP3 Receptor Isoforms Research Articles

Novel Luminal Regulation of Inositol 1,4,5-Trisphosphate Receptor Channel Gating

The ubiquitous, mostly endoplasmic reticulum (ER)-localized inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) channel modulates cytoplasmic free [Ca2+] ([Ca2+]i) to generate complex Ca2+ signals by releasing Ca2+ from the ER lumen store. Whereas cytoplasmic InsP3 activation and biphasic [Ca2+]i regulation of InsP3R channel have been well studied, the effects of ER luminal free [Ca2+] ([Ca2+]ER) on InsP3R channel activity remains poorly understood and controversial. Using single-channel nuclear patch-clamp electrophysiology, we characterized a feed-through effect of [Ca2+]ER on InsP3R channel, whereby Ca2+ ion driven through an open InsP3R channel by the [Ca2+] gradient across the ER membrane can sufficiently raise the local [Ca2+]i around the channel to alter the its activity through its cytoplasmic activating and inhibitory Ca2+-binding sites. Furthermore, even in strong [Ca2+]i-buffering condition where InsP3R channel activity is not affected by Ca2+ flux across the channel, we still observed that [Ca2+]ER exerts a novel flux-independent inhibitory effect. This effect is profound, reducing InsP3R channel open probability in both saturating and sub-saturating [InsP3] by 80-90% in physiological [Ca2+]ER (∼300 μM) compared to non-physiologically low [Ca2+]ER (70 nM). The effect is observed in on-nucleus patch-clamp experiments using nuclei isolated from multiple cell types with different expression levels of various InsP3R isoforms. This effect was irreversibly abrogated when the nuclear membrane patch was excised into the luminal-side-out configuration, and the luminal side of the isolated membrane and channel was perfused with low (70 nM) [Ca2+] solution. Our observations suggest that the [Ca2+]ER inhibitory effect is likely mediated by some ER luminal peripheral accessory protein(s), which is lost during the perfusion.

Modeling of Molecular and Cellular Mechanisms Involved in Ca<sup>2+</sup> Signal Encoding in Airway Myocytes

In airway myocytes signal transduction via cytosolic calcium plays an important role. In relation with experimental results we review models of basic molecular and cellular mechanisms involved in the signal transduction from the myocyte stimulation to the activation of the contractile apparatus. We concentrate on mechanisms for encoding of input signals into Ca2+ signals and the mechanisms for their decoding. The mechanisms are arranged into a general scheme of cellular signaling, the so-called bow-tie architecture of signaling, in which calcium plays the role of a common media for cellular signals and links the encoding and decoding part. The encoding of calcium signals in airway myocytes is better known and is presented in more detail. In particular, we focus on three recent models taking into account the intracellular calcium handling and ion fluxes through the plasma membrane. The model of membrane conductances was originally proposed for predicting membrane depolarization and voltage-dependent Ca2+ influx triggered by initial cytosolic Ca2+ increase as observed on cholinergic stimulation. Cellular models of intracellular Ca2+ handling were developed to investigate the role of a mixed population of InsP3 receptor isoforms and the cellular environment in the occurrence of Ca2+ oscillations, and the respective role of the sarcoplasmic reticulum, mitochondria, and cytosolic Ca2+-binding proteins in cytosolic Ca2+ clearance. Modeling the mechanisms responsible for the decoding of calcium signals is developed in a lesser extent; however, the most recent theoretical studies are briefly presented in relation with the known experimental results.

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+] ∼400 nM were inhibited by 290 μM [Ca2+]i, exposure of the isolated nuclei to a bath solution with ultra-low [Ca2+] (<5 nM, for ∼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.

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 P o (∼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 P o 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+].

Regulation by Ca2+ and inositol 1,4,5-trisphosphate (InsP3) of single recombinant type 3 InsP3 receptor channels. Ca2+ activation uniquely distinguishes types 1 and 3 insp3 receptors.

The inositol 1,4,5-trisphosphate (InsP(3)) receptor (InsP3R) is an endoplasmic reticulum-localized Ca2+ -release channel that controls complex cytoplasmic Ca(2+) signaling in many cell types. At least three InsP3Rs encoded by different genes have been identified in mammalian cells, with different primary sequences, subcellular locations, variable ratios of expression, and heteromultimer formation. To examine regulation of channel gating of the type 3 isoform, recombinant rat type 3 InsP3R (r-InsP3R-3) was expressed in Xenopus oocytes, and single-channel recordings were obtained by patch-clamp electrophysiology of the outer nuclear membrane. Gating of the r-InsP3R-3 exhibited a biphasic dependence on cytoplasmic free Ca2+ concentration ([Ca2+]i). In the presence of 0.5 mM cytoplasmic free ATP, r-InsP3R-3 gating was inhibited by high [Ca2+]i with features similar to those of the endogenous Xenopus type 1 Ins3R (X-InsP3R-1). Ca2+ inhibition of channel gating had an inhibitory Hill coefficient of approximately 3 and half-maximal inhibiting [Ca2+]i (Kinh) = 39 microM under saturating (10 microM) cytoplasmic InsP3 concentrations ([InsP3]). At [InsP3] < 100 nM, the r-InsP3R-3 became more sensitive to Ca2+ inhibition, with the InsP(3) concentration dependence of Kinh described by a half-maximal [InsP3] of 55 nM and a Hill coefficient of approximately 4. InsP(3) activated the type 3 channel by tuning the efficacy of Ca2+ to inhibit it, by a mechanism similar to that observed for the type 1 isoform. In contrast, the r-InsP3R-3 channel was uniquely distinguished from the X-InsP3R-1 channel by its enhanced Ca2+ sensitivity of activation (half-maximal activating [Ca2+]i of 77 nM instead of 190 nM) and lack of cooperativity between Ca2+ activation sites (activating Hill coefficient of 1 instead of 2). These differences endow the InsP3R-3 with high gain InsP3-induced Ca2+ release and low gain Ca2+ -induced Ca2+ release properties complementary to those of InsP3R-1. Thus, distinct Ca2+ signals may be conferred by complementary Ca2+ activation properties of different InsP3R isoforms.

Regulation of Ca2+ signating in bile duct epithelia by InsP3 receptor isoforms

Regulation of Ca2+ signating in bile duct epithelia by InsP3 receptor isoforms

Regulation of Ca2+ signating in bile duct epithelia by InsP3 receptor isoforms

Regulation of Ca2+ signating in bile duct epithelia by InsP3 receptor isoforms

Subtype identification and functional properties of inositol 1,4,5-trisphosphate receptors in heart and aorta

One of the major mechanisms by which hormones elevate intracellular Ca2+levels is by generating the second messenger inositol 1,4,5-trisphosphate (InsP3), which activates a Ca2+channel (InsP3receptor) located in the endoplasmic reticulum (ER). This study undertakes to identify the InsP3receptor subtypes (isoforms) in heart and aorta and to characterize their functional properties. The InsP3receptor isoforms were identified from rat heart and aorta tissues using both reverse-transcriptase polymerase chain reaction (RT-PCR) to assess the presence of mRNA for the different isoforms and immunochemistry using InsP3receptor isoform-specific antibodies. Functional studies included ligand binding experiments using [3H]InsP3and InsP3-induced Ca2+release studies using Fluo-3 as the Ca2+sensing dye. All three isoforms of the InsP3receptor were identified using RT-PCR and immunochemical analyses. [3H]InsP3binding studies using microsomes derived from these tissues showed that heart had a 3-fold lower abundance of InsP3receptors than aorta, while both have considerably lower abundance than the well characterized cerebellar microsomes. The affinity of the InsP3binding to the receptor was also different in the three tissues. In cerebellum the Kdwas 60 nM, while aorta had a much higher Kdof 220 nM. Heart microsomes, appeared to show two classes of binding affinity with Kds of 150 nM and 60 nM. Furthermore, the effects of free [Ca2+] on [3H]InsP3binding levels were also different for the three tissues. InsP3binding to both cerebellar and aorta microsomes decreased by 90% and 60%, respectively, above 30 nM free [Ca2+], while InsP3binding to heart was relatively insensitive to changes in [Ca2+]. At maximal InsP3concentrations, aorta microsomes were able to release about 5% of the accumulated Ca2+, compared to 25% by cerebellar microsomes. Heart microsomes, however, showed only very little InsP3-induced Ca2+release ( <0.5%). The EC50concentration for InsP3-induced Ca2+release was 1.2 μ M for aorta while that for cerebellum was 0.3μ M. Known agonists of the cerebellar InsP3receptor such as 3-deoxy InsP3and adenophostin A were also able to mobilize Ca2+from aorta microsomes. In addition, the competitive antagonist heparin and the non-competitive antagonists of the cerebellar InsP3receptor, tetracaine and tetrahexylammonium chloride, were also able to block InsP3-induced Ca2+release from aorta microsomes.

ATP-induced Ca2+ signals in bronchial epithelial cells.

Ca2+-dependent Cl- secretion in the respiratory tract occurs physiologically or under pathophysiological conditions when inflammatory mediators are released. The mechanism of intracellular Ca2+ release was investigated in the immortalized bronchial epithelial cell line 16HBE14o-. Experiments on both intact and permeabilized cells revealed that only inositol 1,4,5-trisphosphate (InsP3) receptors and not ryanodine receptors are involved in intracellular Ca2+ release. The expression pattern of the three InsP3 receptor isoforms was assessed both at the mRNA and at the protein level. The level of expression at the mRNA level was type 3 (92.5%) >> type 2 (5.4%) > type 1 (2.1%) and this rank order was also observed at the protein level. The ATP-induced Ca2+ signals in the intact cell, consisting of abortive Ca2+ spikes or fully developed [Ca2+] rises and intracellular Ca2+ waves, were indicative of positive feedback of Ca2+ on the InsP3 receptors. Low Ca2+ concentrations stimulated and high Ca2+ concentrations inhibited InsP3-induced Ca2+ release in permeabilized 16HBE14o- cells. We localized a cytosolic Ca2+-binding site between amino acid residues 2077 and 2101 in the type-2 InsP3 receptor and between amino acids 2030 and 2050 in the type-3 InsP3 receptor by expressing the respective parts of these receptors as glutathione S-transferase fusion proteins in bacteria. We conclude that the InsP3 receptor isoforms expressed in 16HBE14o- cells (mainly type-3 and type-2) are stimulated by Ca2+ and that this phenomenon contributes to the ATP-induced Ca2+ signals in intact 16HBE14o- cells.

Distribution of inositol 1,4,5-trisphosphate receptor isoforms, SERCA isoforms and Ca2+ binding proteins in RBLm2H3 rat basophilic leukemia cells

RBL-2H3 rat basophilic leukemia cells were homogenized and fractionated. A fraction F3 obtained by differential centrifugation was 6-fold enriched in [3H]-inositol 1,4,5-trisphosphate (InsP3) binding activity, while the NADH-cytochrome c oxidoreductase and sulphatase-C activities were only 3.8- and 2.9-fold enriched, respectively. Furthermore, the three InsP3 receptor (InsP3R) isoforms, two sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA) isoforms (2b and 3) as well as four Ca 2+ binding proteins (calreticulin, calnexin, protein disulfide isomerase (PDI) and BiP), were present in this fraction. Fraction F3 was, therefore, further purified on a discontinuous sucrose density gradient, and the 3 resulting fractions were analyzed. The InsP3 binding sites were distributed over the gradient and did not co-migrate with the RNA. We examined the relative content of the three InsP3R isoforms, of both SERCA2b and 3, as well as that of the four Ca 2+ binding proteins in fraction F3 and the sucrose density gradient fractions. Ins P3R-1 and InsP3R-2 showed a similar distribution, with the highest level in the light and intermediate density fractions. InsP3R-3 distributed differently, with the highest level in the intermediate density fraction. Both SERCA isoforms distributed similarly to InsP3R-1 and InsP3R-2. SERCA3 was present at a very low level in the high density fraction. Calreticulin and BiP showed a pattern similar to that of InsP3R-1 and InsP3R-2 and the SERCs. PDI was clearly enriched in the light density fraction while calnexin was broadly distributed. These results indicate a heterogeneous distribution of the three InsP3R isoforms, the two SERCA isoforms and the four Ca2+ binding proteins investigated. This heterogeneity may underlie specialization of the Ca2+ stores and the subsequent initiation of intracellular Ca2+ signals.

Isoprenylated human brain type I inositol 1,4,5-trisphosphate 5-phosphatase controls Ca2+ oscillations induced by ATP in Chinese hamster ovary cells.

D-myo-Inositol 1,4,5-trisphosphate (InsP3) 5-phosphatase and 3-kinase are thought to be critical regulatory enzymes in the control of InsP3 and Ca2+ signaling. In brain and many other cells, type I InsP3 5-phosphatase is the major phosphatase that dephosphorylates InsP3 and D-myo-inositol 1,3,4,5-tetrakisphosphate. The type I 5-phosphatase appears to be associated with the particulate fraction of cell homogenates. Molecular cloning of the human brain enzyme identifies a C-terminal farnesylation site CVVQ. Post-translational modification of this enzyme promotes membrane interactions and changes in specific activity. We have now compared the cytosolic Ca2+ ([Ca2+]i) responses induced by ATP, thapsigargin, and ionomycin in Chinese hamster ovary (CHO-K1) cells transfected with the intact InsP3 5-phosphatase and with a mutant in which the C-terminal cysteine cannot be farnesylated. [Ca2+]i was also measured in cells transfected with an InsP3 3-kinase construct encoding the A isoform. The Ca2+ oscillations detected in the presence of 1 microM ATP in control cells were totally lost in 87.5% of intact (farnesylated) InsP3 5-phosphatase-transfected cells, while such a loss occurred in only 1.1% of the mutant InsP3 5-phosphatase-transfected cells. All cells overexpressing the InsP3 3-kinase also responded with an oscillatory pattern. However, in contrast to control cells, the [Ca2+]i returned to base-line levels in between a couple of oscillations. The [Ca2+]i responses to thapsigargin and ionomycin were identical for all cells. The four cell clones compared in this study also behaved similarly with respect to capacitative Ca2+ entry. In permeabilized cells, no differences in extent of InsP3-induced Ca2+ release nor in the threshold for InsP3 action were observed among the four clones and no differences in the expression levels of the various InsP3 receptor isoforms could be shown between the clones. Our data support the contention that the ATP-induced increase in InsP3 concentration in transfected CHO-K1 cells is essentially restricted to the site of its production near the plasma membrane, where it can be metabolized by the type I InsP3 5-phosphatase. This enzyme directly controls the [Ca2+]i response and the Ca2+ oscillations in intact cells.

Multiple Inositol 1,4,5-Trisphosphate Receptor Isoforms Are Present in Platelets

Platelets are activated by an increase in cytosolic Ca2+, and a portion of this increase is derived from inositol 1,4,5-trisphosphate (InsP3)-mediated Ca2+release from internal stores via the InsP3receptor. There is some uncertainty concerning the localization of the InsP3receptor within platelets, and experiments were designed to help resolve this question. [3H]InsP3binding to unphosphorylated and phosphorylated platelet internal membranes revealed both low and high affinity InsP3binding sites, indicating the presence of more than one isoform of InsP3receptor within the internal membranes. Phosphorylation did not significantly affect InsP3binding. In contrast, a single class of high affinity sites was observed in plasma membranes indicating only one type of InsP3receptor. Western blotting of platelet internal and plasma membranes with antibodies against the three major InsP3receptor isoforms revealed that the internal membranes contain both type 1 and type 2 InsP3receptors while the plasma membrane contains only InsP3receptor type 2.

Receptors linked to changes in intracellular calcium concentration[Ca 2+] i in a human erythroleukemia cell line and in non-transformed human erythroid precursor cells

One of the major mechanisms by which hormones elevate intracellular Ca2+levels is by generating the second messenger inositol 1,4,5-trisphosphate (InsP3), which activates a Ca2+channel (InsP3receptor) located in the endoplasmic reticulum (ER). This study undertakes to identify the InsP3receptor subtypes (isoforms) in heart and aorta and to characterize their functional properties. The InsP3receptor isoforms were identified from rat heart and aorta tissues using both reverse-transcriptase polymerase chain reaction (RT-PCR) to assess the presence of mRNA for the different isoforms and immunochemistry using InsP3receptor isoform-specific antibodies. Functional studies included ligand binding experiments using [3H]InsP3and InsP3-induced Ca2+release studies using Fluo-3 as the Ca2+sensing dye. All three isoforms of the InsP3receptor were identified using RT-PCR and immunochemical analyses. [3H]InsP3binding studies using microsomes derived from these tissues showed that heart had a 3-fold lower abundance of InsP3receptors than aorta, while both have considerably lower abundance than the well characterized cerebellar microsomes. The affinity of the InsP3binding to the receptor was also different in the three tissues. In cerebellum the Kdwas 60 nM, while aorta had a much higher Kdof 220 nM. Heart microsomes, appeared to show two classes of binding affinity with Kds of 150 nM and 60 nM. Furthermore, the effects of free [Ca2+] on [3H]InsP3binding levels were also different for the three tissues. InsP3binding to both cerebellar and aorta microsomes decreased by 90% and 60%, respectively, above 30 nM free [Ca2+], while InsP3binding to heart was relatively insensitive to changes in [Ca2+]. At maximal InsP3concentrations, aorta microsomes were able to release about 5% of the accumulated Ca2+, compared to 25% by cerebellar microsomes. Heart microsomes, however, showed only very little InsP3-induced Ca2+release ( <0.5%). The EC50concentration for InsP3-induced Ca2+release was 1.2 μ M for aorta while that for cerebellum was 0.3μ M. Known agonists of the cerebellar InsP3receptor such as 3-deoxy InsP3and adenophostin A were also able to mobilize Ca2+from aorta microsomes. In addition, the competitive antagonist heparin and the non-competitive antagonists of the cerebellar InsP3receptor, tetracaine and tetrahexylammonium chloride, were also able to block InsP3-induced Ca2+release from aorta microsomes.