scholarly journals The elementary unit of store-operated Ca2+ entry: local activation of CRAC channels by STIM1 at ER–plasma membrane junctions

2006 ◽  
Vol 174 (6) ◽  
pp. 815-825 ◽  
Author(s):  
Riina M. Luik ◽  
Minnie M. Wu ◽  
JoAnn Buchanan ◽  
Richard S. Lewis
Keyword(s):  
Plasma Membrane ◽  
Total Internal Reflection ◽  
Plasma Membranes ◽  
Patch Clamp Recording ◽  
Elementary Unit ◽  
Local Activation ◽  
Crac Channels ◽  
Store Depletion ◽  
Crac Channel ◽  
First Time

The activation of store-operated Ca2+ entry by Ca2+ store depletion has long been hypothesized to occur via local interactions of the endoplasmic reticulum (ER) and plasma membrane, but the structure involved has never been identified. Store depletion causes the ER Ca2+ sensor stromal interacting molecule 1 (STIM1) to form puncta by accumulating in junctional ER located 10–25 nm from the plasma membrane (see Wu et al. on p. 803 of this issue). We have combined total internal reflection fluorescence (TIRF) microscopy and patch-clamp recording to localize STIM1 and sites of Ca2+ influx through open Ca2+ release–activated Ca2+ (CRAC) channels in Jurkat T cells after store depletion. CRAC channels open only in the immediate vicinity of STIM1 puncta, restricting Ca2+ entry to discrete sites comprising a small fraction of the cell surface. Orai1, an essential component of the CRAC channel, colocalizes with STIM1 after store depletion, providing a physical basis for the local activation of Ca2+ influx. These studies reveal for the first time that STIM1 and Orai1 move in a coordinated fashion to form closely apposed clusters in the ER and plasma membranes, thereby creating the elementary unit of store-operated Ca2+ entry.

scholarly journals Ca2+ store depletion causes STIM1 to accumulate in ER regions closely associated with the plasma membrane

2006 ◽  
Vol 174 (6) ◽  
pp. 803-813 ◽  
Author(s):  
Minnie M. Wu ◽  
JoAnn Buchanan ◽  
Riina M. Luik ◽  
Richard S. Lewis
Keyword(s):  
Plasma Membrane ◽  
Jurkat Cells ◽  
Causal Role ◽  
Cell Periphery ◽  
Patch Clamp Recording ◽  
Channel Activation ◽  
Crac Channels ◽  
Store Depletion ◽  
Electron Microscopy Analysis ◽  
Crac Channel

Stromal interacting molecule 1 (STIM1), reported to be an endoplasmic reticulum (ER) Ca2+ sensor controlling store-operated Ca2+ entry, redistributes from a diffuse ER localization into puncta at the cell periphery after store depletion. STIM1 redistribution is proposed to be necessary for Ca2+ release–activated Ca2+ (CRAC) channel activation, but it is unclear whether redistribution is rapid enough to play a causal role. Furthermore, the location of STIM1 puncta is uncertain, with recent reports supporting retention in the ER as well as insertion into the plasma membrane (PM). Using total internal reflection fluorescence (TIRF) microscopy and patch-clamp recording from single Jurkat cells, we show that STIM1 puncta form several seconds before CRAC channels open, supporting a causal role in channel activation. Fluorescence quenching and electron microscopy analysis reveal that puncta correspond to STIM1 accumulation in discrete subregions of junctional ER located 10–25 nm from the PM, without detectable insertion of STIM1 into the PM. Roughly one third of these ER–PM contacts form in response to store depletion. These studies identify an ER structure underlying store-operated Ca2+ entry, whose extreme proximity to the PM may enable STIM1 to interact with CRAC channels or associated proteins.

Enhanced exocytotic-like insertion of Orai1 into the plasma membrane upon intracellular Ca2+ store depletion

2008 ◽  
Vol 294 (6) ◽  
pp. C1323-C1331 ◽  
Author(s):  
Geoffrey E. Woodard ◽  
Ginés M. Salido ◽  
Juan A. Rosado
Keyword(s):  
Plasma Membrane ◽  
Botulinum Neurotoxin ◽  
Surface Expression ◽  
Membrane Expression ◽  
Botulinum Neurotoxin A ◽  
Crac Channels ◽  
Store Depletion ◽  
Protein Receptor ◽  
Intracellular Ca ◽  
Crac Channel

Ca+ release-activated Ca2+ (CRAC) channels are activated when free Ca2+ concentration in the intracellular stores is substantially reduced and mediate sustained Ca2+ entry. Recent studies have identified Orai1 as a CRAC channel subunit. Here we demonstrate that passive Ca2+ store depletion using the inhibitor of the sarcoendoplasmic reticulum Ca2+-ATPase, thapsigargin (TG), enhances the surface expression of Orai1, a process that depends on rises in cytosolic free Ca2+ concentration, as demonstrated in cells loaded with dimethyl BAPTA, an intracellular Ca2+ chelator that prevented TG-evoked cytosolic free Ca2+ concentration elevation. Similar results were observed with a low concentration of carbachol. Cleavage of the soluble N-ethylmaleimide-sensitive-factor attachment protein receptor, synaptosomal-assiciated protein-25 (SNAP-25), with botulinum neurotoxin A impaired TG-induced increase in the surface expression of Orai1. In addition, SNAP-25 cleaving by botulinum neurotoxin A reduces the maintenance but not the initial stages of store-operated Ca2+ entry. In aggregate, these findings demonstrate that store depletion enhances Orai1 plasma membrane expression in an exocytotic manner that involves SNAP-25, a process that contributes to store-dependent Ca2+ entry.

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.

Transbilayer movement of fluorescent and spin-labeled phospholipids in the plasma membrane of human fibroblasts: a quantitative approach

1996 ◽  
Vol 109 (3) ◽  
pp. 687-698 ◽  
Author(s):  
T. Pomorski ◽  
P. Muller ◽  
B. Zimmermann ◽  
K. Burger ◽  
P.F. Devaux ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Initial Velocity ◽  
Plasma Membranes ◽  
Atp Hydrolysis ◽  
Human Fibroblasts ◽  
Atp Depletion ◽  
Aminophospholipid Translocase ◽  
Order Of Magnitude ◽  
Complicating Factors ◽  
First Time

All phospholipids in the plasma membrane of eukaryotic cells are subject to a slow passive transbilayer movement. In addition, aminophospholipids are recognized by the so-called aminophospholipid translocase, and are rapidly moved from the exoplasmic to the cytoplasmic leaflet of the plasma membrane at the expense of ATP hydrolysis. Though these principal pathways of transbilayer movement of phospholipids probably apply to all eukaryotic plasma membranes, studies of the actual kinetics of phospholipid redistribution have been largely confined to non-nucleated cells (erythrocytes). Experiments on nucleated cells are complicated by endocytosis and metabolism of the lipid probes inserted into the plasma membrane. Taking these complicating factors into account, we performed a detailed kinetic study of the transbilayer movement of short-chain fluorescent (N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl); NBD) and, for the first time, spin-labeled analogues of phosphatidylcholine (PC), -ethanolamine (PE), -serine (PS), and sphingomyelin (SM) in the plasma membrane of cultured human gingival fibroblasts. At 20 degrees C, the passive transbilayer diffusion of NBD analogues was very slow, and the choline-containing NBD analogues were internalized predominantly by endocytosis. Spin-labeled analogues of PC and SM showed higher passive transbilayer diffusion rates, and probably entered the cell by both passive transbilayer movement and endocytosis. In contrast, the rapid uptake of NBD- and spin-labeled aminophospholipid analogues could be mainly ascribed to the action of the aminophospholipid translocase, since it was inhibited by ATP depletion and N-ethylmaleimide pretreatment. The initial velocity of NBD-aminophospholipid translocation was eight to ten times slower than that of the corresponding spin-labeled lipid, and the half-times of redistribution of NBD-PS and spin-labeled PS were 7.2 and 3.6 minutes, respectively. Our data indicate that in human fibroblasts the initial velocity of aminophospholipid translocation is at least one order of magnitude higher than that in human erythrocytes, which should be sufficient to maintain the phospholipid asymmetry in the plasma membrane.

scholarly journals Thrombocytopoiesis--analysis by membrane tracer and freeze-fracture studies on fresh human and cultured mouse megakaryocytes.

1984 ◽  
Vol 99 (2) ◽  
pp. 390-402 ◽  
Author(s):  
D Zucker-Franklin ◽  
S Petursson
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Freeze Fracture ◽  
Precursor Cell ◽  
Membrane Domains ◽  
Thin Sections ◽  
Energy Dependent ◽  
First Time ◽  
Later Phase ◽  
Peripheral Cytoplasm

The origin of platelets (Pt) from megakaryocytes (MK) is beyond question, but the mechanism whereby Pts are released from the precursor cell is still debated. A widely-held theory claims that the MK plasma membrane invaginates to form demarcation membranes (DMS), which delineate Pt territories. Accordingly, Pts would be derived mostly from the periphery of the MK, and the MK and Pt plasma membranes would have to be virtually identical. Since, on morphologic grounds, this theory is untenable, several aspects of thrombocytopoiesis were reexamined with the help of membrane tracer and freeze-fracture analyses of freshly-collected human and cultured mouse MK. To our surprise, freeze-cleavage of the MK plasma membrane revealed that the vast majority of intramembranous particles (IMP) remained associated with the protoplasmic leaflet (P face), whereas the partition coefficient of IMPs of the platelet membrane was the reverse. This is the first time that any difference between MK and Pt membranes has been determined. Replicas of freeze-fractured MK that were in the process of thrombocytopoiesis revealed an additional novel phenomenon, i.e., numerous areas of membrane discontinuity that appeared to be related to Pt discharge. When such areas were small, the IMP were lined up along the margin of the crevice. At a later phase, a labyrinth of fenestrations was observed. Thin sections of MK at various stages of differentiation showed that Pt territories were fully demarcated before connections of the DMS with the surface could be found. Therefore, the Pt envelope is probably not derived from invaginations of the MK plasma membrane. When living, MK were incubated with cationic ferritin or peroxidase at 37 degrees C, the tracers entered into the DMS but did not delineate all membranes with which the DMS was in continuity, suggesting the existence of distinctive membrane domains. Interiorization of tracer was not energy-dependent, but arrested at low temperatures. At 4 degrees C the DMS remained empty, unless there was evidence that Pts had been released. In such instances, the tracers outlined infoldings of peripheral cytoplasm that was devoid of organelles. Thus, the majority of Pts seem to originate from the interior of the MK, and the surface membranes of the two cells differ in origin and structure. The observations do not only throw new light on the process of thrombocytopoiesis, but also strengthen the possibility that MKs and Pts may be subject to different stimuli.

scholarly journals Rapid inactivation of depletion-activated calcium current (ICRAC) due to local calcium feedback.

1995 ◽  
Vol 105 (2) ◽  
pp. 209-226 ◽  
Author(s):  
A Zweifach ◽  
R S Lewis
Keyword(s):  
Time Course ◽  
Measurement Techniques ◽  
Fast Inactivation ◽  
Patch Clamp Recording ◽  
Crac Channels ◽  
Whole Cell Patch Clamp ◽  
Close Proximity ◽  
Rapid Inactivation ◽  
Voltage Dependent ◽  
Crac Channel

Rapid inactivation of Ca2+ release-activated Ca2+ (CRAC) channels was studied in Jurkat leukemic T lymphocytes using whole-cell patch clamp recording and [Ca2+]i measurement techniques. In the presence of 22 mM extracellular Ca2+, the Ca2+ current declined with a biexponential time course (time constants of 8-30 ms and 50-150 ms) during hyperpolarizing pulses to potentials more negative than -40 mV. Several lines of evidence suggest that the fast inactivation process is Ca2+ but not voltage dependent. First, the speed and extent of inactivation are enhanced by conditions that increase the rate of Ca2+ entry through open channels. Second, inactivation is substantially reduced when Ba2+ is present as the charge carrier. Third, inactivation is slowed by intracellular dialysis with BAPTA (12 mM), a rapid Ca2+ buffer, but not by raising the cytoplasmic concentration of EGTA, a slower chelator, from 1.2 to 12 mM. Recovery from fast inactivation is complete within 200 ms after repolarization to -12 mV. Rapid inactivation is unaffected by changes in the number of open CRAC channels or global [Ca2+]i. These results demonstrate that rapid inactivation of ICRAC results from the action of Ca2+ in close proximity to the intracellular mouths of individual channels, and that Ca2+ entry through one CRAC channel does not affect neighboring channels. A simple model for Ca2+ diffusion in the presence of a mobile buffer predicts multiple Ca2+ inactivation sites situated 3-4 nm from the intracellular mouth of the pore, consistent with a location on the CRAC channel itself.

scholarly journals Microdomains of High Calcium Are Not Required for Exocytosis in Rbl-2h3 Mucosal Mast Cells

2001 ◽  
Vol 153 (2) ◽  
pp. 339-350 ◽  
Author(s):  
Sahar F. Mahmoud ◽  
Clare Fewtrell
Keyword(s):  
Plasma Membrane ◽  
Mast Cells ◽  
Crac Channels ◽  
Store Depletion ◽  
Mucosal Mast Cells ◽  
High Calcium ◽  
Constant Potential

We have previously shown that store-associated microdomains of high Ca2+ are not essential for exocytosis in RBL-2H3 mucosal mast cells. We have now examined whether Ca2+ microdomains near the plasma membrane are required, by comparing the secretory responses seen when Ca2+ influx was elicited by two very different mechanisms. In the first, antigen was used to activate the Ca2+ release–activated Ca2+ (CRAC) current (ICRAC) through CRAC channels. In the second, a Ca2+ ionophore was used to transport Ca2+ randomly across the plasma membrane. Since store depletion by Ca2+ ionophore will also activate ICRAC, different means of inhibiting ICRAC before ionophore addition were used. Ca2+ responses and secretion in individual cells were compared using simultaneous indo-1 microfluorometry and constant potential amperometry. Secretion still takes place when the increase in intracellular Ca2+ occurs diffusely via the Ca2+ ionophore, and at an average intracellular Ca2+ concentration that is no greater than that observed when Ca2+ entry via CRAC channels triggers secretion. Our results suggest that microdomains of high Ca2+ near the plasma membrane, or associated with mitochondria or Ca2+ stores, are not required for secretion. Therefore, we conclude that modest global increases in intracellular Ca2+ are sufficient for exocytosis in these nonexcitable cells.

scholarly journals Bioelectric characteristics of Chara fragilis (Characeae) cells

2020 ◽  
Vol 77 (5) ◽  
pp. 398-404
Author(s):  
A.E. Hasanova ◽  
◽  
N.A. Musayev ◽  
Keyword(s):  
Plasma Membrane ◽  
Membrane Transport ◽  
Plasma Membranes ◽  
Stress Factors ◽  
K Channels ◽  
Wide Range ◽  
Exogenous Stress ◽  
Membrane Pumps ◽  
First Time ◽  
Characeae Cells

For the first time the data on the distribution of the potential (ϕm) and resistance (Rm) in the plasma membrane and cell wall (R0) of Chara fragilis cells were obtained using the Hogg method by the number of C. fragilis cells in standard conditions. The Hogg method allows simultaneous measurement of electrophysiological parameters, such as ϕm and Rm. The stationary values of the membrane potential and resistance varied in a rather wide range: –90...–300 mV and 1.0–32.6 Ohm·m2, respectively. The average values of ϕm were –183.0 ± 4.9 mV, Rm –9.0 ± 1.2 Ohm·m2. Using standard modifiers of membrane transport, the electrogenic activity of the cells was differentiated into two types: K+-channels and H+-membrane pumps. The activation ranges of the K+-inward rectifying channels and K+-outward rectifying channels are –130...–50 and –300...–162 mV, respectively. The cytosolic activity of K+-ions was 61.6 mmol/L. Since in our research, C. fragilis cells were studied for the first time, their electrogenic activity and ionic conductivity were tested using ammonium metavanadate (VO3ˉ) – a proton pump inhibitor and tetraethylammonium (TEA+) – a universal blocker of K+-channels of plasma membranes. Chara fragilis cells can be recommended as a test object for establishing the mechanisms of changes in plasma membrane transport under exogenous stress factors influence.

scholarly journals An essential and NSF independent role for α-SNAP in store-operated calcium entry

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Yong Miao ◽  
Cathrine Miner ◽  
Lei Zhang ◽  
Phyllis I Hanson ◽  
Adish Dani ◽  
...  
Keyword(s):  
Essential Role ◽  
Calcium Release ◽  
Calcium Entry ◽  
Snare Complex ◽  
Functional Coupling ◽  
Crac Channels ◽  
Store Depletion ◽  
Store Operated Calcium Entry ◽  
Crac Channel

Store-operated calcium entry (SOCE) by calcium release activated calcium (CRAC) channels constitutes a primary route of calcium entry in most cells. Orai1 forms the pore subunit of CRAC channels and Stim1 is the endoplasmic reticulum (ER) resident Ca2+ sensor. Upon store-depletion, Stim1 translocates to domains of ER adjacent to the plasma membrane where it interacts with and clusters Orai1 hexamers to form the CRAC channel complex. Molecular steps enabling activation of SOCE via CRAC channel clusters remain incompletely defined. Here we identify an essential role of α-SNAP in mediating functional coupling of Stim1 and Orai1 molecules to activate SOCE. This role for α-SNAP is direct and independent of its known activity in NSF dependent SNARE complex disassembly. Importantly, Stim1-Orai1 clustering still occurs in the absence of α-SNAP but its inability to support SOCE reveals that a previously unsuspected molecular re-arrangement within CRAC channel clusters is necessary for SOCE.

scholarly journals Relationship between phosphoinositide kinase activities and protein tyrosine phosphorylation in plasma membranes from A431 cells

1990 ◽  
Vol 272 (3) ◽  
pp. 665-670 ◽  
Author(s):  
B Payrastre ◽  
M Plantavid ◽  
M Breton ◽  
E Chambaz ◽  
H Chap
Keyword(s):  
Plasma Membrane ◽  
Tyrosine Phosphorylation ◽  
Plasma Membranes ◽  
Dependent Manner ◽  
Protein Tyrosine Phosphorylation ◽  
A431 Cells ◽  
Phosphoinositide Kinase ◽  
Epidermal Growth ◽  
First Time ◽  
Dose Dependent

Production of PtdIns(4)P and PtdIns(4,5)P2 by plasma-membrane preparations from A431 cells was selectively stimulated in a dose-dependent manner by epidermal growth factor (EGF) in the presence of Na3VO4. Na3VO4 itself mimicked this effect, which was overcome after treatment by a specific phosphotyrosyl phosphatase isolated from A431 cells. PtdIns and PtdIns(4)P kinase activities were present in phosphotyrosyl-proteins isolated from EGF- and/or Na3VO4-stimulated A431 cells by immunoaffinity using an anti-phosphotyrosine antibody. These data suggest for the first time an EGF-dependent regulation of PtdIns 4-kinase and PtdIns(4)P 5-kinase activities by a mechanism involving a tyrosine-phosphorylation process.

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