IP3 receptors and store-operated Ca2+ entry: a license to fill.

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are widely expressed intracellular Ca2+ channels that evoke large local increases in cytosolic Ca2+ concentration. By depleting the ER of Ca2+, IP3Rs also activate store-operated Ca2+ entry (SOCE). Immobile IP3Rs close to the plasma membrane (PM) are the only IP3Rs that respond to physiological stimuli. The association of these 'licensed' IP3Rs with the ER-PM junctions where STIM interacts with Orai PM Ca2+ channels may define the autonomous functional unit for SOCE. Ca2+ entering cells through SOCE can be delivered directly to specific effectors, or it may reach them only after the Ca2+ has been sequestered by the ER and then released through IP3Rs. This 'tunnelling' of Ca2+ through the ER to IP3Rs selectively delivers Ca2+ to different effectors.Wellcome Trust BBSR

Inositol 1,4,5-trisphosphate receptors and their protein partners as signalling hubs.

Inositol 1,4,5-trisphosphate receptors (IP3 Rs) are expressed in nearly all animal cells, where they mediate the release of Ca(2+) from intracellular stores. The complex spatial and temporal organization of the ensuing intracellular Ca(2+) signals allows selective regulation of diverse physiological responses. Interactions of IP3 Rs with other proteins contribute to the specificity and speed of Ca(2+) signalling pathways, and to their capacity to integrate information from other signalling pathways. In this review, we provide a comprehensive survey of the proteins proposed to interact with IP3 Rs and the functional effects that these interactions produce. Interacting proteins can determine the activity of IP3 Rs, facilitate their regulation by multiple signalling pathways and direct the Ca(2+) that they release to specific targets. We suggest that IP3 Rs function as signalling hubs through which diverse inputs are processed and then emerge as cytosolic Ca(2+) signals.This work was supported by the Biotechnology and Biological Sciences Research Council (L0000075) and the Wellcome Trust (101844).This is the final version of the article. It first appeared from Wiley via http://dx.doi.org/10.1113/JP27113

Cooperative activation of IP3 receptors by sequential binding of IP3 and Ca2+ safeguards against spontaneous activity

AbstractBackground: Ca2+ waves allow effective delivery of intracellular Ca2+ signals to cytosolic targets. Propagation of these regenerative Ca2+ signals probably results from the activation of intracellular Ca2+ channels by the increase in cytosolic [Ca2+] that follows the opening of these channels. Such positive feedback is potentially explosive. Mechanisms that limit the spontaneous opening of intracellular Ca2+ channels are therefore likely to have evolved in parallel with the mechanism of Ca2+-induced Ca2+ release.Results: Maximal rates of 45Ca2+ efflux from permeabilised hepatocytes superfused with medium in which the [Ca2+] was clamped were cooperatively stimulated by inositol 1,4,5-trisphosphate (IP3). A minimal interval of ∼400 msec between IP3 addition and the peak rate of Ca2+ mobilisation indicate that channel opening does not immediately follow binding of IP3. Although the absolute latency of Ca2+ release was unaffected by further increasing the IP3 concentration, it was reduced by increased [Ca2+].Conclusions: We propose that the closed conformation of the IP3 receptor is very stable and therefore minimally susceptible to spontaneous activation; at least three (probably four) IP3 molecules may be required to provide enough binding energy to drive the receptor into a stable open conformation. We suggest that a further defence from noise is provided by an extreme form of coincidence detection. Binding of IP3 to each of its four receptor subunits unmasks a site to which Ca2+ must bind before the channel can open. As IP3 binding may also initiate receptor inactivation, there may be only a narrow temporal window during which each receptor subunit must bind both of its agonists if the channel is to open rather than inactivate

A genetically encoded toolkit of functionalized nanobodies against fluorescent proteins for visualizing and manipulating intracellular signalling.

BACKGROUND: Intrabodies enable targeting of proteins in live cells, but generating specific intrabodies against the thousands of proteins in a proteome poses a challenge. We leverage the widespread availability of fluorescently labelled proteins to visualize and manipulate intracellular signalling pathways in live cells by using nanobodies targeting fluorescent protein tags. RESULTS: We generated a toolkit of plasmids encoding nanobodies against red and green fluorescent proteins (RFP and GFP variants), fused to functional modules. These include fluorescent sensors for visualization of Ca2+, H+ and ATP/ADP dynamics; oligomerising or heterodimerising modules that allow recruitment or sequestration of proteins and identification of membrane contact sites between organelles; SNAP tags that allow labelling with fluorescent dyes and targeted chromophore-assisted light inactivation; and nanobodies targeted to lumenal sub-compartments of the secretory pathway. We also developed two methods for crosslinking tagged proteins: a dimeric nanobody, and RFP-targeting and GFP-targeting nanobodies fused to complementary hetero-dimerizing domains. We show various applications of the toolkit and demonstrate, for example, that IP3 receptors deliver Ca2+ to the outer membrane of only a subset of mitochondria and that only one or two sites on a mitochondrion form membrane contacts with the plasma membrane. CONCLUSIONS: This toolkit greatly expands the utility of intrabodies and will enable a range of approaches for studying and manipulating cell signalling in live cells

What about nitrogen? Using nitrogen as a carrier gas during the analysis of petroleum biomarkers by gas chromatography mass spectrometry

Open access via the Elsevier AgreementPeer reviewedPublisher PD

2,5-Di-(tert-butyl)-1,4-benzohydroquinone mobilizes inositol 1,4,5-trisphosphate-sensitive and -insensitive Ca2+ stores

AbstractIn permcabilized rat hepatocytcs a maximal concentration (25 μM) of 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ) mobilized 70% of sequestered Ca2+ and a half-maximal effect was produced by 1.7 μM tBuBHQ. Inositol 1,4,5-trisphosphate (Ins(1,4,5) P3) stimulated release of about 40% of the intracellular Ca2+ stores. Combined applications of a range of tBuBHQ concentrations with a maximal concentration of Ins(1,4,5)P3 demonstrated that tBuBHQ has slight selectivity for the Ca2+ transport process of the Ins(1,4,5) P3-sensitive stores. We conclude that the Ins(1,4,5) P3-sensitive stores arc a subset of those sensitive to tBuBHQ and that the latter is therefore unlikely to prove useful as a tool to discriminate Ins(1,4,5) P3-sensitive and -insensitive Ca2+ stores though it may provide opportunities to design more selective agents

A Comparison of Adaptations via Either a Linear Periodization or an Undulating Periodization Model of Weight Training

BACKGROUND: Resistance training has been proven to have a positive impact on parameters such as muscular strength, hypertrophy and endurance. Periodization is a progressive mode of training that has been shown to illicit greater results than programs that stay consistent throughout. The two prime modes of periodization are linear (LP), which progresses from high volume/low intensity to low volume/high intensity, and undulating (UP) which follows an oscillating volume/intensity design. The purpose of this study was to compare the effects of a 12 week linear program to those from an undulating program on strength and body composition when both programs reflect the entire NSCA repetition continuum. METHODS: 10 resistance trained men were matched according to weight-relative strength and randomly assigned to either a linear (N=4, 21 ± 2.5 yrs, 69.08 ± 0.69in, 1178.13 ± 19.5 lbs, 16.9 ± 5.2 %BF) or undulating (N=6, 20 ± 1.7 yrs, 70.27 ± 2.13 in, 180.17 ± 23.98 lbs, 13.67 ± 2.94 %BF) periodization program. Subjects participated in 3 days per week of supervised total body resistance training with repetitions and intensities reflecting the entire NSCA repetition continuum. At 0, 4, 8 and 12-weeks, subjects were tested on body composition via dual energy x-ray absorptiometry, 1RM strength, muscular endurance, vertical jump and anaerobic capacity. Statistical analyses utilized a two-way ANOVA with repeated measures for all criterion variables (p ≤ 0.05). Data are presented as mean ± SD changes from baseline values. RESULTS: Significant main effects for time (p \u3c 0.05) were observed on bench press (LP: 15.03 ± 8.02 lb; UP: 26.10 ± 5.05 lb), leg press (LP:161.07 ± 14.10 lb ; UP: 164.03 ± 55.20 lb), and Wingate peak power (LP: 148.21 ± 78.05 W; UP: 143.22 ± 137.04 W). However, no significant interactions were observed between groups on any of these parameters. Also, no significant group or time effects for time or differences between groups were observed in the measures of percent body fat, lean muscle mass, or vertical jump. CONCLUSION: Both undulating and linear periodization models of resistance training that reflect the entire repetition continuum can bring forth highly significant changes in strength and peak anaerobic power. However, despite the fact that the undulating method resulted in a 43% greater increase in upper body strength, there is no significant difference in the magnitude of the training adaptations that stem from the two methods over 12 weeks

Sustained signalling by PTH modulates IP3 accumulation and IP3 receptors through cyclic AMP junctions.

Parathyroid hormone (PTH) stimulates adenylyl cyclase through type 1 PTH receptors (PTH1R) and potentiates the Ca(2+) signals evoked by carbachol, which stimulates formation of inositol 1,4,5-trisphosphate (IP3). We confirmed that in HEK cells expressing PTH1R, acute stimulation with PTH(1-34) potentiated carbachol-evoked Ca(2+) release. This was mediated by locally delivered cyclic AMP (cAMP), but unaffected by inhibition of protein kinase A (PKA), exchange proteins activated by cAMP, cAMP phosphodiesterases (PDEs) or substantial inhibition of adenylyl cyclase. Sustained stimulation with PTH(1-34) causes internalization of PTH1R-adenylyl cyclase signalling complexes, but the consequences for delivery of cAMP to IP3R within cAMP signalling junctions are unknown. Here, we show that sustained stimulation with PTH(1-34) or with PTH analogues that do not evoke receptor internalization reduced the potentiated Ca(2+) signals and attenuated carbachol-evoked increases in cytosolic IP3. Similar results were obtained after sustained stimulation with NKH477 to directly activate adenylyl cyclase, or with the membrane-permeant analogue of cAMP, 8-Br-cAMP. These responses were independent of PKA and unaffected by substantial inhibition of adenylyl cyclase. During prolonged stimulation with PTH(1-34), hyperactive cAMP signalling junctions, within which cAMP is delivered directly and at saturating concentrations to its targets, mediate sensitization of IP3R and a more slowly developing inhibition of IP3 accumulation.Supported by the Wellcome Trust (101844) and the Biotechnology and Biological Sciences Research Council (L000075). AM was supported in part by the Central Institute of Medicinal and Aromatic Plants (CSIR), Lucknow, India.This is the final version of the article. It first appeared from the Company of Biologists via //http://jcs.biologists.org/content/128/2/408

Ca2+ Release by IP3 Receptors Is Required to Orient the Mitotic Spindle.

The mitotic spindle distributes chromosomes evenly to daughter cells during mitosis. The orientation of the spindle, guided by internal and external cues, determines the axis of cell division and thereby contributes to tissue morphogenesis. Progression through mitosis requires local Ca2+ signals at critical steps, and because store-operated Ca2+ entry is inhibited during mitosis, those signals probably require Ca2+ release through inositol 1,4,5-trisphosphate receptors (IP3Rs). In cells without IP3Rs, astral microtubules around the daughter centrosome are shorter than those at the mother centrosome, and the mitotic spindle fails to align with the substratum during metaphase. The misalignment is due to the spindle ineffectively detecting internal cues rather than a failure of cells to recognize the substratum. Expression of type 3 IP3R is sufficient to rescue spindle alignment, but only if the IP3R has a functional pore. We conclude that Ca2+ signals evoked by IP3Rs are required to orient the mitotic spindle

Effective Glucose Uptake by Human Astrocytes Requires Its Sequestration in the Endoplasmic Reticulum by Glucose-6-Phosphatase-β.

After its uptake into the cytosol, intracellular glucose is phosphorylated to glucose-6-phosphate (G6P), trapping it within the cell and preparing it for metabolism. In glucose-exporting tissues, like liver, G6P is transported into the ER, where it is dephosphorylated by G6Pase-α. The glucose is then returned to the cytosol for export [1, 2]. Defects in these pathways cause glycogen storage diseases [1]. G6Pase-β, an isozyme of G6Pase-α, is widely expressed [3, 4]. Its role in cells that do not export glucose is unclear, although mutations in G6Pase-β cause severe and widespread abnormalities [5-7]. Astrocytes, the most abundant cells in the brain, provide metabolic support to neurons, facilitated by astrocytic endfeet that contact blood capillaries or neurons [8-12]. Perivascular endfeet are the main site of glucose uptake by astrocytes [13], but in human brain they may be several millimeters away from the perineuronal processes [14]. We show that cultured human fetal astrocytes express G6Pase-β, but not G6Pase-α. ER-targeted glucose sensors [15, 16] reveal that G6Pase-β allows the ER of human astrocytes to accumulate glucose by importing G6P from the cytosol. Glucose uptake by astrocytes, ATP production, and Ca2+ accumulation by the ER are attenuated after knockdown of G6Pase-β using lentivirus-delivered shRNA and substantially rescued by expression of G6Pase-α. We suggest that G6Pase-β activity allows effective uptake of glucose by astrocytes, and we speculate that it allows the ER to function as an intracellular "highway" delivering glucose from perivascular endfeet to the perisynaptic processes