Dataset Information

Differential distribution, clustering, and lateral diffusion of subtypes of the inositol 1,4,5-trisphosphate receptor.

ABSTRACT: Regulation of inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)R) by IP(3) and Ca(2+) allows them to initiate and regeneratively propagate intracellular Ca(2+) signals. The distribution and mobility of IP(3)R determines the spatial organization of these Ca(2+) signals. Until now, there has been no systematic comparison of the distribution and mobility of the three mammalian IP(3)R subtypes in a uniform background. We used confocal microscopy and fluorescence recovery after photobleaching to define these properties for each IP(3)R subtype expressed heterologously in COS-7 cells. IP(3)R1 and IP(3)R3 were uniformly distributed within the membranes of the endoplasmic reticulum (ER), but the distribution of IP(3)R2 was punctate. The mobile fractions (M(f) = 84 ± 2 and 80 ± 2%) and diffusion coefficients (D = 0.018 ± 0.001 and 0.016 ± 0.002 ?m(2)/s) of IP(3)R1 and IP(3)R3 were similar. Other ER membrane proteins (ryanodine receptor type 1 and sarco/endoplasmic reticulum Ca(2+)-ATPase type 1) and a luminal protein (enhanced GFP with a KDEL retrieval sequence) had similar mobile fractions, suggesting that IP(3)R1 and IP(3)R3 move freely within an ER that is largely, although not entirely, continuous. IP(3)R2 was less mobile, but IP(3)R2 mobility differed between perinuclear (M(f) = 47 ± 4% and D = 0.004 ± 0.001 ?m(2)/s) and near-plasma membrane (M(f) = 64 ± 6% and D = 0.013 ± 0.004 ?m(2)/s) regions, whereas IP(3)R3 behaved similarly in both regions. We conclude that IP(3)R1 and IP(3)R3 diffuse freely within a largely continuous ER, but IP(3)R2 is more heterogeneously distributed and less mobile, and its mobility differs between regions of the cell.

SUBMITTER: Pantazaka E

PROVIDER: S-EPMC3123102 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Differential distribution, clustering, and lateral diffusion of subtypes of the inositol 1,4,5-trisphosphate receptor.

Pantazaka Evangelia E Taylor Colin W CW

The Journal of biological chemistry 20110505 26

Regulation of inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)R) by IP(3) and Ca(2+) allows them to initiate and regeneratively propagate intracellular Ca(2+) signals. The distribution and mobility of IP(3)R determines the spatial organization of these Ca(2+) signals. Until now, there has been no systematic comparison of the distribution and mobility of the three mammalian IP(3)R subtypes in a uniform background. We used confocal microscopy and fluorescence recovery after photobleaching to ...[more]

PMID: 21550988

Similar Datasets

Project description:The Ins(1,4,5)P3 regioisomers, Ins(1,4,6)P3 and Ins(1,3,6)P3, which can mimic the 1,4,5-arrangement on the inositol ring of Ins(1,4,5)P3, were examined for Ca2+ release by using four types of saponin-permeabilized cell possessing various abundances of receptor subtypes, with special reference to the relation of potency to receptor subtype. Ins(1,4,6)P3 and Ins(1,3,6)P3 were weak agonists in rat basophilic leukaemic cells (RBL cells), which possess predominantly subtype II receptors, with respective potencies of 1/200 and less than 1/500 that of Ins(1,4,5)P3 [the EC50 values were 0.2, 45 and more than 100 microM for Ins(1,4,5)P3, Ins(1,4,6)P3 and Ins(1,3,6)P3 respectively]. Similar rank order potencies were also evaluated for the displacement of [3H]Ins(1,4,5)P3 bound to RBL cell membranes by these regioisomers. However, they caused Ca2+ release from GH3 rat pituitary cells possessing predominantly subtype I receptors more potently; Ins(1,4,6)P3 and Ins(1,3,6)P3 evoked release at respective concentrations of only one-third and one-twentieth that of Ins(1,4,5)P3 (the EC50 values were 0.4, 1.2 and 8 microM for Ins(1,4,5)P3, Ins(1,4,6)P3 and Ins(1,3,6)P3 respectively). In COS-1 African green-monkey kidney cells, with the relative abundances of 37% of the subtype II and of 62% of the subtype III receptor, potencies of 1/40 and approx. 1/200 for Ins(1, 4,6)P3 and Ins(1,3,6)P3 respectively were exhibited relative to Ins(1,4,5)P3 (the EC50 values were 0.4, 15 and approx. 80 microM for Ins(1,4,5)P3, Ins(1,4,6)P3 and Ins(1,3,6)P3 respectively). In HL-60 human leukaemic cells, in spite of the dominant presence of subtype I receptors (71%), similar respective potencies to those seen with COS-1 cells were exhibited (the EC50 values were 0.3, 15 and approx. 100 microM for Ins(1,4,5)P3, Ins(1,4,6)P3 and Ins(1,3,6)P3 respectively). These results indicate that these regioisomers are the first ligands that distinguish between receptor subtypes; the present observations are of significance for the future design of molecules with enhanced selectivity.

Project description:We have compared the properties of the [3H]Ins(1,4,5)P3-binding sites from a number of tissues in an attempt to determine if heterogeneity exists within the Ins(1,4,5)P3-receptor family. The binding of Ins(1,4,5)P3 was characterized in detail by using membranes prepared from human uterine smooth muscle and bovine adrenal cortex. Ins(1,4,5)P3 exhibited an approx. 5 times greater affinity for the binding site in adrenal cortex (KD = 9.81 +/- 1.92 nM) compared with uterine smooth muscle (KD = 37.1 +/- 1.8 nM). The binding was dependent on pH in both tissues, with a maximum at pH 8.3; at this pH various inositol phosphates and nucleotides competed for the binding sites with similar potencies on both tissues. However, the binding of Ins(1,4,5)P3 to the uterine smooth-muscle membranes was Ca2(+)-sensitive, whereas that to the bovine adrenal cortex was not; furthermore, heparin displaced the binding of Ins(1,4,5)P3 in the uterus with an IC50 value (concn. of displacer giving 50% inhibition of specific binding) of 3.9 micrograms/ml (2.5, 6.4; lower, upper range), compared with a value of 22 (13, 30) micrograms/ml in adrenal cortex. In view of the ability of Ins(1,4,5)P3 and heparin to distinguish between these binding sites, their effect on other tissues was examined. Ins(1,4,5)P3 showed a similar affinity for receptors located in the bovine cerebellum to those in the bovine adrenal cortex, but heparin displaced Ins(1,4,5)P3 binding with a 5-fold greater affinity from the cerebellum. Ins(1,4,5)P3 had a 2-fold greater affinity for its receptor with human platelets, as compared with human uterus, but heparin was unable to distinguish between these sites. In guinea-pig ileum, Ins(1,4,5)P3 displayed a similar affinity for the receptors in the longitudinal muscle compared with the circular muscle, but heparin could distinguish between these sites. These data show that small differences exist between tissues, but no clear picture is apparent. It is possible that these results reflect tissue-dependent factors such as phosphorylation, the presence of calmedin etc., rather than the presence of receptor subtypes or species difference.

Dataset Information

Differential distribution, clustering, and lateral diffusion of subtypes of the inositol 1,4,5-trisphosphate receptor.

Publications

Differential distribution, clustering, and lateral diffusion of subtypes of the inositol 1,4,5-trisphosphate receptor.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets