scholarly journals Type 3 Inositol 1,4,5-Trisphosphate Receptor is a Crucial Regulator of Calcium Dynamics Mediated by Endoplasmic Reticulum in HEK Cells

Cells ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 275 ◽  
Author(s):  
Lili Yue ◽  
Liuqing Wang ◽  
Yangchun Du ◽  
Wei Zhang ◽  
Kozo Hamada ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Channel Activity ◽  
Double Knockout ◽  
Over Expression ◽  
Inositol 1 ◽  
Crac Channels ◽  
Crac Channel ◽  
Trisphosphate Receptor ◽  
Type 3

Being the largest the Ca2+ store in mammalian cells, endoplasmic reticulum (ER)-mediated Ca2+ signalling often involves both Ca2+ release via inositol 1, 4, 5-trisphosphate receptors (IP3R) and store operated Ca2+ entries (SOCE) through Ca2+ release activated Ca2+ (CRAC) channels on plasma membrane (PM). IP3Rs are functionally coupled with CRAC channels and other Ca2+ handling proteins. However, it still remains less well defined as to whether IP3Rs could regulate ER-mediated Ca2+ signals independent of their Ca2+ releasing ability. To address this, we generated IP3Rs triple and double knockout human embryonic kidney (HEK) cell lines (IP3Rs-TKO, IP3Rs-DKO), and systemically examined ER Ca2+ dynamics and CRAC channel activity in these cells. The results showed that the rate of ER Ca2+ leakage and refilling, as well as SOCE were all significantly reduced in IP3Rs-TKO cells. And these TKO effects could be rescued by over-expression of IP3R3. Further, results showed that the diminished SOCE was caused by NEDD4L-mediated ubiquitination of Orai1 protein. Together, our findings indicate that IP3R3 is one crucial player in coordinating ER-mediated Ca2+ signalling.

scholarly journals Regulation of Interorganellar Ca2+ Transfer and NFAT Activation by the Mitochondrial Ca2+ Uniporter

2021 ◽  
Author(s):  
Ryan E. Yoast ◽  
Scott M. Emrich ◽  
Xuexin Zhang ◽  
Ping Xin ◽  
Vikas Arige ◽  
...  
Keyword(s):  
Nuclear Translocation ◽  
Channel Activity ◽  
Crac Channels ◽  
Store Depletion ◽  
Agonist Stimulation ◽  
Dependent Inactivation ◽  
Nfat Activation ◽  
Crac Channel ◽  
Cellular Bioenergetics ◽  
Trisphosphate Receptor

Mitochondrial Ca2+ uptake is crucial for coupling receptor stimulation to cellular bioenergetics. Further, Ca2+ uptake by respiring mitochondria prevents Ca2+-dependent inactivation (CDI) of store-operated Ca2+ release-activated Ca2+ (CRAC) channels and inhibits Ca2+ extrusion to sustain cytosolic Ca2+ signaling. However, how Ca2+ uptake by the mitochondrial Ca2+ uniporter (MCU) shapes receptor-evoked interorganellar Ca2+ signaling is unknown. Here, we generated several cell lines with MCU-knockout (MCU-KO) as well as tissue-specific MCU-knockdown mice. We show that mitochondrial depolarization, but not MCU-KO, inhibits store-operated Ca2+ entry (SOCE). Paradoxically, despite enhancing Ca2+ extrusion and promoting CRAC channel CDI, MCU-KO increased cytosolic Ca2+ in response to store depletion. Further, physiological agonist stimulation in MCU-KO cells led to enhanced frequency of cytosolic Ca2+ oscillations, endoplasmic reticulum Ca2+ refilling, NFAT nuclear translocation and proliferation. However, MCU-KO did not affect inositol-1,4,5-trisphosphate receptor activity. Mathematical modeling supports that MCU-KO enhances cytosolic Ca2+, despite limiting CRAC channel activity.

Store-Operated Ca2+ Channels: Mechanism, Function, Pharmacology, and Therapeutic Targets

2021 ◽  
Vol 61 (1) ◽  
pp. 629-654
Author(s):  
Daniel Bakowski ◽  
Fraser Murray ◽  
Anant B. Parekh
Keyword(s):  
Channel Blockers ◽  
Channel Activity ◽  
Loss Of Function ◽  
Crac Channels ◽  
Crac Channel ◽  
Major Route ◽  
And Function ◽  
Therapeutic Perspective ◽  
Poor Efficacy ◽  
Ca2 Channels

Calcium (Ca2+) release–activated Ca2+ (CRAC) channels are a major route for Ca2+ entry in eukaryotic cells. These channels are store operated, opening when the endoplasmic reticulum (ER) is depleted of Ca2+, and are composed of the ER Ca2+ sensor protein STIM and the pore-forming plasma membrane subunit Orai. Recent years have heralded major strides in our understanding of the structure, gating, and function of the channels. Loss-of-function and gain-of-function mutants combined with RNAi knockdown strategies have revealed important roles for the channel in numerous human diseases, making the channel a clinically relevant target. Drugs targeting the channels generally lack specificity or exhibit poor efficacy in animal models. However, the landscape is changing, and CRAC channel blockers are now entering clinical trials. Here, we describe the key molecular and biological features of CRAC channels, consider various diseases associated with aberrant channel activity, and discuss targeting of the channels from a therapeutic perspective.

scholarly journals Permeant calcium ion feed-through regulation of single inositol 1,4,5-trisphosphate receptor channel gating

2012 ◽  
Vol 140 (6) ◽  
pp. 697-716 ◽  
Author(s):  
Horia Vais ◽  
J. Kevin Foskett ◽  
Ghanim Ullah ◽  
John E. Pearson ◽  
Don-On Daniel Mak
Keyword(s):  
Posttranslational Modifications ◽  
Imaging Techniques ◽  
Channel Gating ◽  
Channel Activity ◽  
Release Channel ◽  
Solution Exchange ◽  
Inositol 1,4,5 Trisphosphate ◽  
Direct Binding ◽  
Trisphosphate Receptor ◽  
Type 3

The ubiquitous inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) Ca2+ release channel plays a central role in the generation and modulation of intracellular Ca2+ signals, and is intricately regulated by multiple mechanisms including cytoplasmic ligand (InsP3, free Ca2+, free ATP4−) binding, posttranslational modifications, and interactions with cytoplasmic and endoplasmic reticulum (ER) luminal proteins. However, regulation of InsP3R channel activity by free Ca2+ in the ER lumen ([Ca2+]ER) remains poorly understood because of limitations of Ca2+ flux measurements and imaging techniques. Here, we used nuclear patch-clamp experiments in excised luminal-side-out configuration with perfusion solution exchange to study the effects of [Ca2+]ER on homotetrameric rat type 3 InsP3R channel activity. In optimal [Ca2+]i and subsaturating [InsP3], jumps of [Ca2+]ER from 70 nM to 300 µM reduced channel activity significantly. This inhibition was abrogated by saturating InsP3 but restored when [Ca2+]ER was raised to 1.1 mM. In suboptimal [Ca2+]i, jumps of [Ca2+]ER (70 nM to 300 µM) enhanced channel activity. Thus, [Ca2+]ER effects on channel activity exhibited a biphasic dependence on [Ca2+]i. In addition, the effect of high [Ca2+]ER was attenuated when a voltage was applied to oppose Ca2+ flux through the channel. These observations can be accounted for by Ca2+ flux driven through the open InsP3R channel by [Ca2+]ER, raising local [Ca2+]i around the channel to regulate its activity through its cytoplasmic regulatory Ca2+-binding sites. Importantly, [Ca2+]ER regulation of InsP3R channel activity depended on cytoplasmic Ca2+-buffering conditions: it was more pronounced when [Ca2+]i was weakly buffered but completely abolished in strong Ca2+-buffering conditions. With strong cytoplasmic buffering and Ca2+ flux sufficiently reduced by applied voltage, both activation and inhibition of InsP3R channel gating by physiological levels of [Ca2+]ER were completely abolished. Collectively, these results rule out Ca2+ regulation of channel activity by direct binding to the luminal aspect of the channel.

scholarly journals Single-Channel Properties in Endoplasmic Reticulum Membrane of Recombinant Type 3 Inositol Trisphosphate Receptor

2000 ◽  
Vol 115 (3) ◽  
pp. 241-256 ◽  
Author(s):  
Don-On Daniel Mak ◽  
Sean McBride ◽  
Viswanathan Raghuram ◽  
Yun Yue ◽  
Suresh K. Joseph ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Single Channel ◽  
Calcium Influx ◽  
Endoplasmic Reticulum Membrane ◽  
Release Channel ◽  
Cellular Expression ◽  
Recombinant Type ◽  
Specific Regulation ◽  
Trisphosphate Receptor ◽  
Type 3

The inositol 1,4,5-trisphosphate receptor (InsP3R) is an intracellular Ca2+-release channel localized in endoplasmic reticulum (ER) with a central role in complex Ca2+ signaling in most cell types. A family of InsP3Rs encoded by several genes has been identified with different primary sequences, subcellular locations, variable ratios of expression, and heteromultimer formation. This diversity suggests that cells require distinct InsP3Rs, but the functional correlates of this diversity are largely unknown. Lacking are single-channel recordings of the recombinant type 3 receptor (InsP3R-3), a widely expressed isoform also implicated in plasma membrane Ca2+ influx and apoptosis. Here, we describe functional expression and single-channel recording of recombinant rat InsP3R-3 in its native membrane environment. The approach we describe suggests a novel strategy for expression and recording of recombinant ER-localized ion channels in the ER membrane. Ion permeation and channel gating properties of the rat InsP3R-3 are strikingly similar to those of Xenopus type 1 InsP3R in the same membrane. Using two different two-electrode voltage clamp protocols to examine calcium store-operated calcium influx, no difference in the magnitude of calcium influx was observed in oocytes injected with rat InsP3R-3 cRNA compared with control oocytes. Our results suggest that if cellular expression of multiple InsP3R isoforms is a mechanism to modify the temporal and spatial features of [Ca2+]i signals, then it must be achieved by isoform-specific regulation or localization of various types of InsP3Rs that have relatively similar Ca2+ permeation properties.

Expression of the type 3 inositol 1, 4, 5-trisphosphate receptor according to the chronic liver disease etiology in hepatocellular carcinoma.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e16604-e16604
Author(s):  
Paulo Henrique Costa Diniz ◽  
Marcone Loiola dos Santos ◽  
Andressa França ◽  
Antônio Carlos Melo Lima Filho ◽  
Paula Vieira Teixeira Vidigal ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Liver Disease ◽  
Chronic Liver Disease ◽  
Mitotic Index ◽  
Chronic Liver ◽  
Receptor Expression ◽  
Cryptogenic Cirrhosis ◽  
Inositol 1 ◽  
Trisphosphate Receptor ◽  
Type 3

e16604 Background: The expression of type 3 isoform of the inositol 1, 4, 5-trisphosphate receptor (ITPR-3), an intracellular calcium (Ca2+) channel reported in liver cancer cells, is important in the Ca2+ signalling. Thus, it may be involved in the many events of hepatocarcinogenesis. Here, we investigated the ITPR-3 expression in hepatocellular carcinoma (HCC) and its association with clinicopathological parameters and long-term outcomes, according to the etiology of underlying chronic liver disease (CLD). Methods: Clinical and laboratory data from patients (n = 53) who underwent orthotopic liver transplantation for HCC treatment in a Brazilian referral center were retrospectively collected. After pathological reviewing of their explanted liver samples, ITPR-3 expression in both tumor and underlying cirrhosis were assessed by immunohistochemistry, and quantified using density histograms in the ImageJ software. Event (tumor recurrence or death from any cause) occurrence and event-free survival (EFS) were analysed. Results: Hepatitis C virus (HCV) (n = 31), alcohol abuse (n = 16) and cryptogenic cirrhosis (n = 6) were the underlying CLD etiology, and the groups were, in general, well balanced regarding clinicopathological indices. Median EFS was 78.9 months (range, 63.6-94.1). The ITPR-3 expression profile was cytoplasmatic, predominantly perinuclear, and was stronger in tumor than in adjacent cirrhosis, considering all etiologies together (intensity 9.1% higher in tumors, p < 0.001) However, analyzing each etiologic group, the cryptogenic was the only one in which there was no difference between tumor and underlying CLD. Comparing the ITPR-3 expression only in tumors, there was no difference regarding the etiology of CLD. The tumor ITPR-3 higher intensity was correlated with higher serum aspartate alanine-transferases (ALT) levels (p = 0.018) and lower mitotic index ( < 5 per 10 high power fields) (p = 0.009). There was no association between receptor expression and event occurrence or EFS. Conclusions: The ITPR-3 was expressed in HCC, regardless of the underlying CLD etiology. Its correlation with mitotic index, a cell proliferation marker, was demonstrated, but there were no associations with clinical outcomes. Apart from cryptogenic cirrhosis, ITPR-3 expression was more intense in tumors than in underlying cirrhosis. These findings suggest that ITPR-3 could have a role in carcinogenesis. However, the prognostic and therapeutic implications need to be investigated.

Demethylation increases the expression of type 3 inositol 1, 4, 5-trisphosphate receptor in hepatocytes

2018 ◽  
Vol 68 ◽  
pp. S129
Author(s):  
R.M. Florentino ◽  
A. França ◽  
A.C.M.L. Filho ◽  
M.C. Fonseca ◽  
A.G. Oliveira ◽  
...  
Keyword(s):  
Inositol 1 ◽  
Trisphosphate Receptor ◽  
Type 3

scholarly journals A Novel Modulator of STIM2-Dependent Store-Operated Ca2+ Channel Activity

Acta Naturae ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 140-146
Author(s):  
Anton Yu. Skopin ◽  
Andrey D. Grigoryev ◽  
Lyubov N. Glushankova ◽  
Alexey V. Shalygin ◽  
Guanghui Wang ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Small Molecules ◽  
Mammalian Cells ◽  
Calcium Influx ◽  
Channel Activity ◽  
Excitable Cells ◽  
Protein Activity ◽  
Physiological Conditions ◽  
The Moment

Store-operated Ca2+ entry is one of the main pathways of calcium influx into non-excitable cells, which entails the initiation of many intracellular processes. The endoplasmic reticulum Ca2+ sensors STIM1 and STIM2 are the key components of store-operated Ca2+ entry in mammalian cells. Under physiological conditions, STIM proteins are responsible for store-operated Ca2+ entry activation. The STIM1 and STIM2 proteins differ in their potency for activating different store-operated channels. At the moment, there are no selective modulators of the STIM protein activity. We screened a library of small molecules and found the 4-MPTC compound, which selectively inhibited STIM2-dependent store-operated Ca2+ entry (IC50 = 1 M) and had almost no effect on the STIM1-dependent activation of store-operated channels.

scholarly journals S-acylation of Orai1 regulates store-operated Ca2+ entry

2021 ◽  
Vol 135 (5) ◽  
Author(s):  
Savannah J. West ◽  
Goutham Kodakandla ◽  
Qioachu Wang ◽  
Ritika Tewari ◽  
Michael X. Zhu ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Cell Types ◽  
Central Component ◽  
Channel Activation ◽  
Crac Channels ◽  
Store Depletion ◽  
Crac Channel ◽  
Underlying Mechanisms ◽  
Different Cell Types

ABSTRACT Store-operated Ca2+ entry is a central component of intracellular Ca2+ signaling pathways. The Ca2+ release-activated channel (CRAC) mediates store-operated Ca2+ entry in many different cell types. The CRAC channel is composed of the plasma membrane (PM)-localized Orai1 channel and endoplasmic reticulum (ER)-localized STIM1 Ca2+ sensor. Upon ER Ca2+ store depletion, Orai1 and STIM1 form complexes at ER–PM junctions, leading to the formation of activated CRAC channels. Although the importance of CRAC channels is well described, the underlying mechanisms that regulate the recruitment of Orai1 to ER–PM junctions are not fully understood. Here, we describe the rapid and transient S-acylation of Orai1. Using biochemical approaches, we show that Orai1 is rapidly S-acylated at cysteine 143 upon ER Ca2+ store depletion. Importantly, S-acylation of cysteine 143 is required for Orai1-mediated Ca2+ entry and recruitment to STIM1 puncta. We conclude that store depletion-induced S-acylation of Orai1 is necessary for recruitment to ER–PM junctions, subsequent binding to STIM1 and channel activation.

scholarly journals STIM1, an essential and conserved component of store-operated Ca2+ channel function

2005 ◽  
Vol 169 (3) ◽  
pp. 435-445 ◽  
Author(s):  
Jack Roos ◽  
Paul J. DiGregorio ◽  
Andriy V. Yeromin ◽  
Kari Ohlsen ◽  
Maria Lioudyno ◽  
...  
Keyword(s):  
T Cells ◽  
Mammalian Cells ◽  
Hek293 Cells ◽  
Complete Suppression ◽  
Patch Clamp Recording ◽  
Common Component ◽  
Cellular Processes ◽  
Crac Channels ◽  
Crac Channel ◽  
Molecular Components

Store-operated Ca2+ (SOC) channels regulate many cellular processes, but the underlying molecular components are not well defined. Using an RNA interference (RNAi)-based screen to identify genes that alter thapsigargin (TG)-dependent Ca2+ entry, we discovered a required and conserved role of Stim in SOC influx. RNAi-mediated knockdown of Stim in Drosophila S2 cells significantly reduced TG-dependent Ca2+ entry. Patch-clamp recording revealed nearly complete suppression of the Drosophila Ca2+ release-activated Ca2+ (CRAC) current that has biophysical characteristics similar to CRAC current in human T cells. Similarly, knockdown of the human homologue STIM1 significantly reduced CRAC channel activity in Jurkat T cells. RNAi-mediated knockdown of STIM1 inhibited TG- or agonist-dependent Ca2+ entry in HEK293 or SH-SY5Y cells. Conversely, overexpression of STIM1 in HEK293 cells modestly enhanced TG-induced Ca2+ entry. We propose that STIM1, a ubiquitously expressed protein that is conserved from Drosophila to mammalian cells, plays an essential role in SOC influx and may be a common component of SOC and CRAC channels.

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