共查询到9条相似文献,搜索用时 4 毫秒
1.
Paula J. Bartlett Walson Metzger Lawrence D. Gaspers Andrew P. Thomas 《The Journal of biological chemistry》2015,290(30):18519-18533
How Ca2+ oscillations are generated and fine-tuned to yield versatile downstream responses remains to be elucidated. In hepatocytes, G protein-coupled receptor-linked Ca2+ oscillations report signal strength via frequency, whereas Ca2+ spike amplitude and wave velocity remain constant. IP3 uncaging also triggers oscillatory Ca2+ release, but, in contrast to hormones, Ca2+ spike amplitude, width, and wave velocity were dependent on [IP3] and were not perturbed by phospholipase C (PLC) inhibition. These data indicate that oscillations elicited by IP3 uncaging are driven by the biphasic regulation of the IP3 receptor by Ca2+, and, unlike hormone-dependent responses, do not require PLC. Removal of extracellular Ca2+ did not perturb Ca2+ oscillations elicited by IP3 uncaging, indicating that reloading of endoplasmic reticulum stores via plasma membrane Ca2+ influx does not entrain the signal. Activation and inhibition of PKC attenuated hormone-induced Ca2+ oscillations but had no effect on Ca2+ increases induced by uncaging IP3. Importantly, PKC activation and inhibition differentially affected Ca2+ spike frequencies and kinetics. PKC activation amplifies negative feedback loops at the level of G protein-coupled receptor PLC activity and/or IP3 metabolism to attenuate IP3 levels and suppress the generation of Ca2+ oscillations. Inhibition of PKC relieves negative feedback regulation of IP3 accumulation and, thereby, shifts Ca2+ oscillations toward sustained responses or dramatically prolonged spikes. PKC down-regulation attenuates phenylephrine-induced Ca2+ wave velocity, whereas responses to IP3 uncaging are enhanced. The ability to assess Ca2+ responses in the absence of PLC activity indicates that IP3 receptor modulation by PKC regulates Ca2+ release and wave velocity. 相似文献
2.
Rahul Chandrasekhar Kamil J. Alzayady Larry E. Wagner II David I. Yule 《The Journal of biological chemistry》2016,291(10):4846-4860
The ability of inositol 1,4,5-trisphosphate receptors (IP3R) to precisely initiate and generate a diverse variety of intracellular Ca2+ signals is in part mediated by the differential regulation of the three subtypes (R1, R2, and R3) by key functional modulators (IP3, Ca2+, and ATP). However, the contribution of IP3R heterotetramerization to Ca2+ signal diversity has largely been unexplored. In this report, we provide the first definitive biochemical evidence of endogenous heterotetramer formation. Additionally, we examine the contribution of individual subtypes within defined concatenated heterotetramers to the shaping of Ca2+ signals. Under conditions where key regulators of IP3R function are optimal for Ca2+ release, we demonstrate that individual monomers within heteromeric IP3Rs contributed equally toward generating a distinct ''blended'' sensitivity to IP3 that is likely dictated by the unique IP3 binding affinity of the heteromers. However, under suboptimal conditions where [ATP] were varied, we found that one subtype dictated the ATP regulatory properties of heteromers. We show that R2 monomers within a heterotetramer were both necessary and sufficient to dictate the ATP regulatory properties. Finally, the ATP-binding site B in R2 critical for ATP regulation was mutated and rendered non-functional to address questions relating to the stoichiometry of IP3R regulation. Two intact R2 monomers were sufficient to maintain ATP regulation in R2 homotetramers. In summary, we demonstrate that heterotetrameric IP3R do not necessarily behave as the sum of the constituent subunits, and these properties likely extend the versatility of IP3-induced Ca2+ signaling in cells expressing multiple IP3R isoforms. 相似文献
3.
Chaloux B Caron AZ Guillemette G 《Biology of the cell / under the auspices of the European Cell Biology Organization》2007,99(7):379-388
Background information. In endocrine cells, IP3R (inositol 1,4,5‐trisphosphate receptor), a ligand‐gated Ca2+ channel, plays an important role in the control of intracellular Ca2+ concentration. There are three subtypes of IP3R that are distributed differentially among cell types. RINm5F cells express almost exclusively the IP3R‐3 subtype. The purpose of the present study was to investigate the effect of PKA (protein kinase A) on the activity of IP3R‐3 in RINm5F cells. Results. We show that immunoprecipitated IP3R‐3 is a good substrate for PKA. Using a back‐phosphorylation approach, we show that endogenous PKA phosphorylates IP3R‐3 in intact RINm5F cells. [3H]IP3 (inositol 1,4,5‐trisphosphate) binding affinity and IP3‐induced Ca2+ release activity were enhanced in permeabilized cells that were pre‐treated with forskolin or PKA. The PKA‐induced enhancement of IP3R‐3 activity was also observed in intact RINm5F cells stimulated with carbachol and epidermal growth factor, two agonists that use different receptor types to activate phospholipase C. Conclusion. The results of the present study reveal a converging step where the cAMP and the Ca2+ signalling systems act co‐operatively in endocrine cell responses to external stimuli. 相似文献
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Wenting Guo Bo Sun Zhichao Xiao Yingjie Liu Yundi Wang Lin Zhang Ruiwu Wang S. R. Wayne Chen 《The Journal of biological chemistry》2016,291(5):2150-2160
Activation of the cardiac ryanodine receptor (RyR2) by elevating cytosolic Ca2+ is a central step in the process of Ca2+-induced Ca2+ release, but the molecular basis of RyR2 activation by cytosolic Ca2+ is poorly defined. It has been proposed recently that the putative Ca2+ binding domain encompassing a pair of EF-hand motifs (EF1 and EF2) in the skeletal muscle ryanodine receptor (RyR1) functions as a Ca2+ sensor that regulates the gating of RyR1. Although the role of the EF-hand domain in RyR1 function has been studied extensively, little is known about the functional significance of the corresponding EF-hand domain in RyR2. Here we investigate the effect of mutations in the EF-hand motifs on the Ca2+ activation of RyR2. We found that mutations in the EF-hand motifs or deletion of the entire EF-hand domain did not affect the Ca2+-dependent activation of [3H]ryanodine binding or the cytosolic Ca2+ activation of RyR2. On the other hand, deletion of the EF-hand domain markedly suppressed the luminal Ca2+ activation of RyR2 and spontaneous Ca2+ release in HEK293 cells during store Ca2+ overload or store overload-induced Ca2+ release (SOICR). Furthermore, mutations in the EF2 motif, but not EF1 motif, of RyR2 raised the threshold for SOICR termination, whereas deletion of the EF-hand domain of RyR2 increased both the activation and termination thresholds for SOICR. These results indicate that, although the EF-hand domain is not required for RyR2 activation by cytosolic Ca2+, it plays an important role in luminal Ca2+ activation and SOICR. 相似文献
5.
Szlufcik K Missiaen L Parys JB Callewaert G De Smedt H 《Biology of the cell / under the auspices of the European Cell Biology Organization》2006,98(1):1-14
Ca(2+) release via intracellular release channels, IP(3)Rs (inositol 1,4,5-trisphosphate receptors) and RyRs (ryanodine receptors), is perhaps the most ubiquitous and versatile cellular signalling mechanism, and is involved in a vast number of cellular processes. In addition to this classical release pathway there is limited, but yet persistent, information about less well-defined Ca(2+)-leak pathways that may play an important role in the control of the Ca(2+) load of the endo(sarco)plasmic reticulum. The mechanisms responsible for this 'basal' leak are not known, but recent data suggest that both IP(3)Rs and RyRs may also operate as Ca(2+)-leak channels, particularly in pathological conditions. Proteolytic cleavage or biochemical modification (such as hyperphosphorylation or nitrosylation), for example, occurring during conditions of cell stress or apoptosis, can functionally uncouple the cytoplasmic control domains from the channel domain of the receptor. Highly significant information has been obtained from studies of malfunctioning channels in various disorders; for example, RyRs in cardiac malfunction or genetic muscle diseases and IP(3)Rs in neurodegenerative diseases. In this review we aim to summarize the existing information about functionally uncoupled IP(3)R and RyR channels, and to discuss the concept that those channels can participate in Ca(2+)-leak pathways. 相似文献
6.
Zhou H Iwasaki H Nakamura T Nakamura K Maruyama T Hamano S Ozaki S Mizutani A Mikoshiba K 《Biochemical and biophysical research communications》2007,352(2):277-282
Capacitative calcium entry (CCE), the mechanism that replenishes the internal Ca2+ stores with Ca2+ from the extracellular milieu in response to depletion of the store, is mediated by Ca2+ channels in the plasma membrane generally referred to as store-operated channels (SOCs). However, the roles of SOCs in the more physiological context have been fully elucidated. 2-Aminoethyl diphenylborinate (2-APB) strongly inhibits SOCs, as well as inositol-1,4,5 trisphosphate (IP3) receptors. In the present study, we screened a library of 166 2-APB analogues for effects on CCE and IP3-induced Ca2+ release in order to discover specific SOC inhibitors, and found that some blocked both store-operated and receptor-operated Ca2+ influx more strongly and selectively than 2-APB. Indeed, these new compounds ceased the prolonged intracellular Ca2+ oscillations induced by a low concentration of ATP in CHO-K1 cells. These novel SOC inhibitors will be valuable pharmacological and biochemical tools for elucidating the physiological roles. 相似文献
7.
2-Aminoethoxydiphenyl borate (2-APB) is used as a pharmacological tool because it antagonizes inositol 1,4,5-trisphosphate receptors and store-operated Ca2+ (SOC) channels, and activates some TRP channels. Recently, we reported that 2-APB enhanced the increase in cytotoxic [Ca2+]i, resulting in cell death under external acidic conditions in rat pheochromocytoma cell line PC12. However, the molecular mechanism and functional role of the 2-APB-induced Ca2+ influx in PC12 have not been clarified. In this study, to identify the possible target for the action of 2-APB we examined the pharmacological and molecular properties of [Ca2+]i and secretory responses to 2-APB under extracellular low pH conditions. 2-APB dose-dependently induced a [Ca2+]i increase and dopamine release, which were greatly enhanced by the external acidification (pH 6.5). [Ca2+]i and secretory responses to 2-APB at pH 6.5 were inhibited by the removal of extracellular Ca2+ and SOC channel blockers such as SK&F96365, La3+ and Gd3+. PC12 expressed all SOC channel molecules, Orai 1, Orai 2 and Orai 3. When we used an siRNA system, downregulation of Orai 3, but not Orai 1 and Orai 2, attenuated both [Ca2+]i and secretory responses to 2-APB. These results suggest that 2-APB evokes external acid-dependent increases of [Ca2+]i and dopamine release in PC12 through the activation of Orai 3. The present results indicate that 2-APB may be a useful pharmacological tool for Orai channel-related signaling. 相似文献
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