Na+ pump α2-subunit expression modulates Ca2+ signaling

2003 ◽  
Vol 284 (2) ◽  
pp. C475-C486 ◽  
Author(s):  
Vera A. Golovina ◽  
Hong Song ◽  
Paul F. James ◽  
Jerry B. Lingrel ◽  
Mordecai P. Blaustein
Keyword(s):  
Plasma Membrane ◽  
Selective Reduction ◽  
Cyclopiazonic Acid ◽  
Cell Types ◽  
Membrane Microdomains ◽  
Α Subunit ◽  
Published Evidence ◽  
Subunit Expression ◽  
Mouse Fetuses ◽  
Free Media

The role of the Na+ pump α2-subunit in Ca2+ signaling was examined in primary cultured astrocytes from wild-type (α2 +/+ = WT) mouse fetuses and those with a null mutation in one [α2 +/− = heterozygote (Het)] or both [α2 −/− = knockout (KO)] α2 genes. Na+ pump catalytic (α) subunit expression was measured by immunoblot; cytosol [Na+] ([Na+]cyt) and [Ca2+] ([Ca2+]cyt) were measured with sodium-binding benzofuran isophthalate and fura 2 by using digital imaging. Astrocytes express Na+ pumps with both α1- (≈80% of total α) and α2- (≈20% of total α) subunits. Het astrocytes express ≈50% of normal α2; those from KO express none. Expression of α1 is normal in both Het and KO cells. Resting [Na+]cyt = 6.5 mM in WT, 6.8 mM in Het ( P > 0.05 vs. WT), and 8.0 mM in KO cells ( P < 0.001); 500 nM ouabain (inhibits only α2) equalized [Na+]cyt at 8 mM in all three cell types. Resting [Ca2+]cyt = 132 nM in WT, 162 nM in Het, and 196 nM in KO cells (both P < 0.001 vs. WT). Cyclopiazonic acid (CPA), which inhibits endoplasmic reticulum (ER) Ca2+ pumps and unloads the ER, induces transient (in Ca2+-free media) or sustained (in Ca2+-replete media) elevation of [Ca2+]cyt. These Ca2+ responses to 10 μM CPA were augmented in Het as well as KO cells. When CPA was applied in Ca2+-free media, the reintroduction of Ca2+ induced significantly larger transient rises in [Ca2+]cyt (due to Ca2+ entry through store-operated channels) in Het and KO cells than in WT cells. These results correlate with published evidence that α2 Na+ pumps and Na+/Ca2+ exchangers are confined to plasma membrane microdomains that overlie the ER. The data suggest that selective reduction of α2 Na+ pump activity can elevate local [Na+] and, via Na+/Ca2+ exchange, [Ca2+] in the tiny volume of cytosol between the plasma membrane and ER. This, in turn, augments adjacent ER Ca2+ stores and thereby amplifies Ca2+ signaling without elevating bulk [Na+]cyt.

Membrane microdomains: lessons from microscopy

1995 ◽  
Vol 53 ◽  
pp. 776-777
Author(s):  
J.M. Robinson ◽  
J.M Oliver
Keyword(s):  
Spatial Distribution ◽  
Plasma Membrane ◽  
Epithelial Cells ◽  
Plasma Membranes ◽  
Cell Types ◽  
Imaging Modalities ◽  
Membrane Microdomains ◽  
Membrane Domains ◽  
Lateral Heterogeneity ◽  
Functional Importance

Specialized regions of plasma membranes displaying lateral heterogeneity are the focus of this Symposium. Specialized membrane domains are known for certain cell types such as differentiated epithelial cells where lateral heterogeneity in lipids and proteins exists between the apical and basolateral portions of the plasma membrane. Lateral heterogeneity and the presence of microdomains in membranes that are uniform in appearance have been more difficult to establish. Nonetheless a number of studies have provided evidence for membrane microdomains and indicated a functional importance for these structures.This symposium will focus on the use of various imaging modalities and related approaches to define membrane microdomains in a number of cell types. The importance of existing as well as emerging imaging technologies for use in the elucidation of membrane microdomains will be highlighted. The organization of membrane microdomains in terms of dimensions and spatial distribution is of considerable interest and will be addressed in this Symposium.

What Can Epitope Specific Antibodies Tell us About the Organization of Caveolin in Cells?

2001 ◽  
Vol 7 (S2) ◽  
pp. 1030-1031
Author(s):  
J.M. Robinson
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Biochemical Characterization ◽  
Cell Types ◽  
Membrane Microdomains ◽  
Truncated Form ◽  
Coated Pits ◽  
N Terminus ◽  
Plasma Membrane Microdomains

There are three members of the caveolin (CAV) gene family that give rise to four polypeptides. These polypeptides are CAV-1α, CAV-1β, CAV-2, and CAV-3. The CAV-1β isoform is a truncated form of CAV-1α that lacks 31 amino acids at the N-terminus of the molecule. The CAV- 1β molecule arises through an alternative splicing mechanism.Caveolae are specialized plasma membrane microdomains that are expressed at high levels in some cell types (e.g., endothelium, adipocytes, fibroblasts). These specialized regions of the plasma membrane have a characteristic omega-shaped appearance with diameters ranging from 40-90 run. They are distinct from clathrin-coated pits since they lack the characteristic coated appearance in electron microscopy. Caveolae were among the first structures to be discovered by biological electron microscopy. However, biochemical characterization of these structures did not begin in earnest until a marker protein was identified. The initial marker was the 22-kDa protein known as caveolin.

scholarly journals Disruption of ankyrin B and caveolin-1 interaction sites alters Na+,K+-ATPase lateral diffusion in HEK293 cell plasma membranes

2017 ◽  
Author(s):  
Cornelia Junghans ◽  
Vladana Vukojević ◽  
Neslihan N. Tavraz ◽  
Eugene G. Maksimov ◽  
Werner Zuschratter ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Matrix Proteins ◽  
Membrane Microdomains ◽  
Membrane Targeting ◽  
Α Subunit ◽  
Caveolin 1 ◽  
Interaction Sites ◽  
Ankyrin B ◽  
Cellular Matrix

AbstractThe Na+,K+-ATPase is a plasma membrane ion transporter of high physiological importance for ion homeostasis and cellular excitability in electrically active tissues. Mutations in the genes coding for Na+,K+-ATPase α-subunit isoforms lead to severe human pathologies including Familial Hemiplegic Migraine type 2 (FHM2), Alternating Hemiplegia of Childhood (AHC), Rapid Dystonia Parkinsonism (RDP) or epilepsy. Many of the reported mutations lead to change- or loss-of-function effects, whereas others do not alter the functional properties, but lead to e.g. reduced protein stability, reduced protein expression or defective plasma membrane targeting. Na+,K+-ATPase frequently assembles with other membrane transporters or cellular matrix proteins in specialized plasma membrane microdomains, but the effects of these interactions on targeting or protein mobility are elusive so far. Mutational disruption of established interaction motifs of the Na+,K+-ATPase with ankyrin B and caveolin-1 are expected to result in changes in plasma membrane targeting, changes of the localization pattern, and of the diffusion behavior of the enzyme. We studied the consequences of mutations in these binding sites by monitoring diffusion of eGFP-labeled Na+,K+-ATPase constructs in the plasma membrane of living HEK293T cells by fluorescence correlation spectroscopy (FCS) as well as fluorescence recovery after photobleaching (FRAP) or photoswitching (FRAS) and observed significant differences compared to the wild-type enzyme, with synergistic effects for combinations of interaction site mutations. These measurements expand the possibilities to study the consequences of Na+,K+-ATPase mutations and provide information about the interaction of Na+,K+-ATPase α2-isoform with cellular matrix proteins, the cytoskeleton or other membrane protein complexes.

Clearance of store-released Ca2+ by the Na+-Ca2+ exchanger is diminished in aortic smooth muscle from Na+-K+-ATPase α2-isoform gene-ablated mice

2008 ◽  
Vol 294 (3) ◽  
pp. H1407-H1416 ◽  
Author(s):  
Ronald M. Lynch ◽  
Craig S. Weber ◽  
Kevin D. Nullmeyer ◽  
Edwin D. W. Moore ◽  
Richard J. Paul
Keyword(s):  
Smooth Muscle ◽  
Plasma Membrane ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Cyclopiazonic Acid ◽  
Selective Inhibition ◽  
Wild Type ◽  
Aortic Smooth Muscle ◽  
Plasmic Reticulum ◽  
Free Media

Two α-isoforms of the Na+-K+-ATPase are expressed in vascular smooth muscle cells (VSMCs). The α1-isoform is proposed to serve a cytosolic housekeeping role, whereas the α2-isoform modulates Ca2+ storage via coupling to the Na+-Ca2+ exchanger (NCX) in a subsarcolemmal compartment. To evaluate the ramifications of this proposed interaction, Ca2+-store load and the contributions of the primary Ca2+ transporters to Ca2+ clearance were studied in aortic VSMCs from embryonic wild-type (WT) and Na+-K+-ATPase α2-isoform gene-ablated, homozygous null knockout (α2-KO) mice. Ca2+ stores were unloaded by inhibiting the sarco(endo)plasmic reticulum Ca2+-ATPase with cyclopiazonic acid (CPA) in Ca2+-free media to limit Ca2+ influx. Ca2+ clearance by the plasma membrane Ca2+-ATPase (PMCA), NCX, or mitochondria was selectively inhibited. In WT VSMCs, NCX accounted for 90% of the Ca2+ efflux. In α2-KO VSMCs, preferential clearance of store-released Ca2+ by NCX was lost, whereas PMCA activity was increased. Selective inhibition of the α2-isoform (0.5 μM ouabain for 20 min), before treatment with CPA enhanced the store load in VSMCs from WT, but not α2-KO mice. A subsequent analysis of capacitative Ca2+ entry (CCE) indicated that the magnitude of Ca2+ influx was significantly greater in α2-KO cells. Our findings support the concept of a subsarcolemmal space where the α2-isoform coupled with NCX modulates Ca2+-store function and, thereby, CCE.

scholarly journals Plasma membrane rafts and chaperones in cytokine/STAT signaling.

2003 ◽  
Vol 50 (3) ◽  
pp. 583-594 ◽  
Author(s):  
Pravin B Sehgal
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Heat Shock Protein 90 ◽  
Cell Types ◽  
Janus Kinase ◽  
Membrane Microdomains ◽  
Target Cells ◽  
Surface Receptors ◽  
Stat Signaling ◽  
And Function

We and others have recently obtained data suggesting that cytokine-STAT signaling in many different cell-types is a chaperoned pathway initiated at the level of specialized plasma membrane microdomains called "rafts" (the "raft-STAT signaling hypothesis"). These findings are of broad significance in that all cytokines and growth factors initiate signaling in target cells by interacting with respective cell-surface receptors. The new data suggest that raft microdomains represent the units of function at the cell-surface through which ligand-stimulated STAT signaling is initiated. Moreover, recent evidence shows the involvement of chaperone proteins in regulating the STAT signaling pathway. These chaperones include the human homolog of the tumorous imaginal disc 1 protein (hTid1) which associates with Janus kinase 2 (JAK2) at the level of the plasma membrane, heat shock protein 90 (HSP90) which associates with STAT3 and STAT1 proteins in caveolin-1-containing raft and cytoplasmic complexes, and glucose regulated protein 58 (GRP58/ER-60/ERp57), a thiol dependent protein-disulfide isomerase, found in association with STAT3 "statosome" complexes in the cytosol and in the raft fraction. We suggest a function of the HSP90 chaperone system in preserving IL-6/STAT3 signaling in liver cells in the context of fever. The identification and function of protein partners associated with specific STAT species in rafts and in cytosolic complexes, and in the efficient departure of cytokine-activated STATs from the cytosolic face of rafts towards the cell nucleus are now areas of active investigation.

scholarly journals Plasma Membrane Microdomains Act as Concentration Platforms to Facilitate Intoxication by Aerolysin

1999 ◽  
Vol 147 (1) ◽  
pp. 175-184 ◽  
Author(s):  
Laurence Abrami ◽  
F. Gisou van der Goot
Keyword(s):  
Plasma Membrane ◽  
Target Cell ◽  
Soluble Protein ◽  
Cell Types ◽  
Living Cells ◽  
Membrane Microdomains ◽  
Toxin Concentration ◽  
Glycosyl Phosphatidylinositol ◽  
Plasma Membrane Microdomains

It has been proposed that the plasma membrane of many cell types contains cholesterol-sphingolipid–rich microdomains. Here, we analyze the role of these microdomains in promoting oligomerization of the bacterial pore-forming toxin aerolysin. Aeroly-sin binds to cells, via glycosyl phosphatidylinositol- anchored receptors, as a hydrophilic soluble protein that must polymerize into an amphipathic ring-like complex to form a pore. We first show that oligomerization can occur at &gt;105-fold lower toxin concentration at the surface of living cells than in solution. Our observations indicate that it is not merely the number of receptors on the target cell that is important for toxin sensitivity, but their ability to associate transiently with detergent resistant microdomains. Oligomerization appears to be promoted by the fact that the toxin bound to its glycosyl phosphatidylinositol-anchored receptors, can be recruited into these microdomains, which act as concentration devices.

scholarly journals The exosome journey: from biogenesis to uptake and intracellular signalling

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Sonam Gurung ◽  
Dany Perocheau ◽  
Loukia Touramanidou ◽  
Julien Baruteau
Keyword(s):  
Clinical Trials ◽  
Plasma Membrane ◽  
Scientific Community ◽  
Cell Types ◽  
Clinical Applications ◽  
Clinical Development ◽  
Intracellular Signalling ◽  
Clinical Settings ◽  
Health And Disease ◽  
Adequate Definition

AbstractThe use of exosomes in clinical settings is progressively becoming a reality, as clinical trials testing exosomes for diagnostic and therapeutic applications are generating remarkable interest from the scientific community and investors. Exosomes are small extracellular vesicles secreted by all cell types playing intercellular communication roles in health and disease by transferring cellular cargoes such as functional proteins, metabolites and nucleic acids to recipient cells. An in-depth understanding of exosome biology is therefore essential to ensure clinical development of exosome based investigational therapeutic products. Here we summarise the most up-to-date knowkedge about the complex biological journey of exosomes from biogenesis and secretion, transport and uptake to their intracellular signalling. We delineate the major pathways and molecular players that influence each step of exosome physiology, highlighting the routes of interest, which will be of benefit to exosome manipulation and engineering. We highlight the main controversies in the field of exosome research: their adequate definition, characterisation and biogenesis at plasma membrane. We also delineate the most common identified pitfalls affecting exosome research and development. Unravelling exosome physiology is key to their ultimate progression towards clinical applications.

scholarly journals Plasma membrane integrity in health and disease: significance and therapeutic potential

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Catarina Dias ◽  
Jesper Nylandsted
Keyword(s):  
Plasma Membrane ◽  
Therapeutic Potential ◽  
Calcium Influx ◽  
Membrane Integrity ◽  
Cell Types ◽  
Physical Parameters ◽  
Membrane Repair ◽  
Plasma Membrane Integrity ◽  
Repair Mechanisms ◽  
And Function

AbstractMaintenance of plasma membrane integrity is essential for normal cell viability and function. Thus, robust membrane repair mechanisms have evolved to counteract the eminent threat of a torn plasma membrane. Different repair mechanisms and the bio-physical parameters required for efficient repair are now emerging from different research groups. However, less is known about when these mechanisms come into play. This review focuses on the existence of membrane disruptions and repair mechanisms in both physiological and pathological conditions, and across multiple cell types, albeit to different degrees. Fundamentally, irrespective of the source of membrane disruption, aberrant calcium influx is the common stimulus that activates the membrane repair response. Inadequate repair responses can tip the balance between physiology and pathology, highlighting the significance of plasma membrane integrity. For example, an over-activated repair response can promote cancer invasion, while the inability to efficiently repair membrane can drive neurodegeneration and muscular dystrophies. The interdisciplinary view explored here emphasises the widespread potential of targeting plasma membrane repair mechanisms for therapeutic purposes.

Effects of red grape juice polyphenols in NADPH oxidase subunit expression in human neutrophils and mononuclear blood cells

2009 ◽  
Vol 102 (8) ◽  
pp. 1125-1135 ◽  
Author(s):  
Alberto Dávalos ◽  
Gema de la Peña ◽  
Carolina C. Sánchez-Martín ◽  
M. Teresa Guerra ◽  
Begoña Bartolomé ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Red Wine ◽  
Grape Juice ◽  
Cell Types ◽  
Human Neutrophils ◽  
Superoxide Anion Production ◽  
Oxidase Subunit ◽  
Subunit Expression ◽  
Red Grape Juice ◽  
Red Grape

The NADPH oxidase enzyme system is the main source of superoxide anions in phagocytic and vascular cells. NADPH oxidase-dependent superoxide generation has been found to be abnormally enhanced in several chronic diseases. Evidence is accumulating that polyphenols may have the potential to improve cardiovascular health, although the mechanism is not fully established. Consumption of concentrated red grape juice, rich in polyphenols, has been recently shown to reduce NADPH oxidase activity in circulating neutrophils from human subjects. In the present work we studied whether red grape juice polyphenols affected NADPH oxidase subunit expression at the transcription level. For this, we used human neutrophils and mononuclear cells from peripheral blood, HL-60-derived neutrophils and the endothelial cell line EA.hy926.Superoxide production was measured with 2′7′-dichlorofluorescein diacetate or lucigenin, mRNA expression by real-time RT-PCR and protein expression by Western blot. Each experiment was performed at least three times. In all cell types tested, red grape juice, dealcoholised red wine and pure polyphenols decreased superoxide anion production. Red grape juice and dealcoholised red wine selectively reduced p47phox, p22phox and gp91phox expression at both mRNA and protein levels, without affecting the expression of p67phox. Pure polyphenols, particularly quercetin, also reduced NADPH oxidase subunit expression, especially p47phox, in all cell types tested. The present results showing that red grape juice polyphenols reduce superoxide anion production provide an alternative mechanism by which consumption of grape derivatives may account for a reduction of oxidative stress associated with cardiovascular and/or inflammatory diseases related to NADPH oxidase superoxide overproduction.

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