Nicotinic acetylcholine receptor α7 regulates cAMP signal within lipid rafts

2003 ◽  
Vol 285 (3) ◽  
pp. C567-C574 ◽  
Author(s):  
Jin Oshikawa ◽  
Yoshiyuki Toya ◽  
Takayuki Fujita ◽  
Masato Egawa ◽  
Junichi Kawabe ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Lipid Rafts ◽  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Plasma Membranes ◽  
Gradient Centrifugation ◽  
Membrane Microdomains ◽  
Cholesterol Depletion ◽  
Nicotinic Acetylcholine ◽  
Density Fractions

Neuronal nicotinic acetylcholine receptors (nAChRs) are made of multiple subunits with diversified functions. The nAChR α7-subunit has a property of high Ca2+ permeability and may have specific functions and localization within the plasma membrane as a signal transduction molecule. In PC-12 cells, fractionation by sucrose gradient centrifugation revealed that nAChRα7 existed in low-density, cholesterol-enriched plasma membrane microdomains known as lipid rafts where flotillin also exists. In contrast, nAChR α5- and β2-subunits were located in high-density fractions, out of the lipid rafts. Type 6 adenylyl cyclase (AC6), a calcium-inhibitable isoform, was also found in lipid rafts and was coimmunoprecipitated with nAChRα7. Cholesterol depletion from plasma membranes with methyl-β-cyclodextrin redistributed nAChRα7 and AC6 diffusely within plasma membranes. Nicotine stimulation reduced forskolin-stimulated AC activity by 35%, and this inhibition was negated by either treatment with α-bungarotoxin, a specific antagonist of nAChRα7, or cholesterol depletion from plasma membranes. The effect of cholesterol depletion was negated by the addition of cholesterol. These data suggest that nAChRα7 has a specific membrane localization relative to other nAChR subunits and that lipid rafts are necessary to localize nAChRα7 with AC within plasma membranes. In addition, nAChRα7 may regulate the AC activity via Ca2+ within lipid rafts.

scholarly journals Pseudotypes of Vesicular Stomatitis Virus with CD4 Formed by Clustering of Membrane Microdomains during Budding

2005 ◽  
Vol 79 (11) ◽  
pp. 7077-7086 ◽  
Author(s):  
Erica L. Brown ◽  
Douglas S. Lyles
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Lipid Rafts ◽  
Immunoelectron Microscopy ◽  
Vesicular Stomatitis Virus ◽  
Plasma Membranes ◽  
Membrane Microdomains ◽  
Virus Budding ◽  
Vesicular Stomatitis ◽  
Membrane Components

ABSTRACT Many plasma membrane components are organized into detergent-resistant membrane microdomains referred to as lipid rafts. However, there is much less information about the organization of membrane components into microdomains outside of lipid rafts. Furthermore, there are few approaches to determine whether different membrane components are colocalized in microdomains as small as lipid rafts. We have previously described a new method of determining the extent of organization of proteins into membrane microdomains by analyzing the distribution of pairwise distances between immunogold particles in immunoelectron micrographs. We used this method to analyze the microdomains involved in the incorporation of the T-cell antigen CD4 into the envelope of vesicular stomatitis virus (VSV). In cells infected with a recombinant virus that expresses CD4 from the viral genome, both CD4 and the VSV envelope glycoprotein (G protein) were found in detergent-soluble (nonraft) membrane fractions. However, analysis of the distribution of CD4 and G protein in plasma membranes by immunoelectron microscopy showed that both were organized into membrane microdomains of similar sizes, approximately 100 to 150 nm. In regions of plasma membrane outside of virus budding sites, CD4 and G protein were present in separate membrane microdomains, as shown by double-label immunoelectron microscopy data. However, virus budding occurred from membrane microdomains that contained both G protein and CD4, and extended to approximately 300 nm, indicating that VSV pseudotype formation with CD4 occurs by clustering of G protein- and CD4-containing microdomains.

scholarly journals Spatiotemporal Analysis of Differential Akt Regulation in Plasma Membrane Microdomains

2008 ◽  
Vol 19 (10) ◽  
pp. 4366-4373 ◽  
Author(s):  
Xinxin Gao ◽  
Jin Zhang
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Lipid Rafts ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Spatiotemporal Analysis ◽  
Membrane Microdomains ◽  
Cholesterol Depletion ◽  
Growth Factor Signaling

As a central kinase in the phosphatidylinositol 3-kinase pathway, Akt has been the subject of extensive research; yet, spatiotemporal regulation of Akt in different membrane microdomains remains largely unknown. To examine dynamic Akt activity in membrane microdomains in living cells, we developed a specific and sensitive fluorescence resonance energy transfer-based Akt activity reporter, AktAR, through systematic testing of different substrates and fluorescent proteins. Targeted AktAR reported higher Akt activity with faster activation kinetics within lipid rafts compared with nonraft regions of plasma membrane. Disruption of rafts attenuated platelet-derived growth factor (PDGF)-stimulated Akt activity in rafts without affecting that in nonraft regions. However, in insulin-like growth factor-1 (IGF)-1 stimulation, Akt signaling in nonraft regions is dependent on that in raft regions. As a result, cholesterol depletion diminishes Akt activity in both regions. Thus, Akt activities are differentially regulated in different membrane microdomains, and the overall activity of this oncogenic pathway is dependent on raft function. Given the increased abundance of lipid rafts in some cancer cells, the distinct Akt-activating characteristics of PDGF and IGF-1, in terms of both effectiveness and raft dependence, demonstrate the capabilities of different growth factor signaling pathways to transduce differential oncogenic signals across plasma membrane.

scholarly journals Cholesterol-Enriched Membrane Microdomains Are Required for Inducing Host Cell Cytoskeleton Rearrangements in Response to Attaching-Effacing Escherichia coli

2005 ◽  
Vol 73 (11) ◽  
pp. 7113-7125 ◽  
Author(s):  
Jason D. Riff ◽  
John W. Callahan ◽  
Philip M. Sherman
Keyword(s):  
Escherichia Coli ◽  
Plasma Membrane ◽  
Lipid Rafts ◽  
Skin Fibroblasts ◽  
Membrane Microdomains ◽  
Membrane Cholesterol ◽  
Cholesterol Depletion ◽  
Host Membrane ◽  
Niemann Pick ◽  
Cytoskeletal Rearrangements

ABSTRACT The diarrheal pathogens enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain CL56 and enteropathogenic Escherichia coli (EPEC) O127:H6 strain E2348/69 adhere intimately to epithelial cells through attaching-effacing lesions, which are characterized by rearrangements of the host cytoskeleton, intimate adherence, and destruction of microvilli. These cytoskeletal responses require activation of host signal transduction pathways. Lipid rafts are signaling microdomains enriched in sphingolipid and cholesterol in the plasma membrane. The effect of perturbing plasma membrane cholesterol on bacterial intimate adherence was assessed. Infection of both HEp-2 cells and primary skin fibroblasts with strains CL56 and E2348/69 caused characteristic rearrangements of the cytoskeleton at sites of bacterial adhesion. CL56- and E2348/69-induced cytoskeletal rearrangements were inhibited following cholesterol depletion. Addition of exogenous cholesterol to depleted HEp-2 cells restored cholesterol levels and rescued bacterially induced α-actinin mobilization. Quantitative bacterial adherence assays showed that EPEC adherence to HEp-2 cells was dramatically reduced following cholesterol depletion, whereas the adherence of EHEC remained high. Cytoskeletal rearrangements on skin fibroblasts obtained from children with Niemann-Pick type C disease were markedly reduced. These findings indicate that host membrane cholesterol contained in lipid rafts is necessary for the cytoskeletal rearrangements following infection with attaching-effacing Escherichia coli. Differences in initial adherence indicate divergent roles for host membrane cholesterol in the pathogenesis of EHEC and EPEC infections.

scholarly journals The Assembly of GM1 Glycolipid- and Cholesterol-Enriched Raft-Like Membrane Microdomains Is Important for Giardial Encystation

2015 ◽  
Vol 83 (5) ◽  
pp. 2030-2042 ◽  
Author(s):  
Atasi De Chatterjee ◽  
Tavis L. Mendez ◽  
Sukla Roychowdhury ◽  
Siddhartha Das
Keyword(s):  
Lipid Rafts ◽  
Plasma Membranes ◽  
Gradient Centrifugation ◽  
Cyst Formation ◽  
Neuraminidase Inhibitor ◽  
Major Constituent ◽  
Membrane Microdomains ◽  
Content Type ◽  
Cyst Production ◽  
Raft Domains

Although encystation (or cyst formation) is an important step of the life cycle ofGiardia, the cellular events that trigger encystation are poorly understood. Because membrane microdomains are involved in inducing growth and differentiation in many eukaryotes, we wondered if these raft-like domains are assembled by this parasite and participate in the encystation process. Since the GM1 ganglioside is a major constituent of mammalian lipid rafts (LRs) and known to react with cholera toxin B (CTXB), we used Alexa Fluor-conjugated CTXB and GM1 antibodies to detect giardial LRs. Raft-like structures in trophozoites are located in the plasma membranes and on the periphery of ventral discs. In cysts, however, they are localized in the membranes beneath the cyst wall. Nystatin and filipin III, two cholesterol-binding agents, and oseltamivir (Tamiflu), a viral neuraminidase inhibitor, disassembled the microdomains, as evidenced by reduced staining of trophozoites with CTXB and GM1 antibodies. GM1- and cholesterol-enriched LRs were isolated fromGiardiaby density gradient centrifugation and found to be sensitive to nystatin and oseltamivir. The involvement of LRs in encystation could be supported by the observation that raft inhibitors interrupted the biogenesis of encystation-specific vesicles and cyst production. Furthermore, culturing of trophozoites in dialyzed medium containing fetal bovine serum (which is low in cholesterol) reduced raft assembly and encystation, which could be rescued by adding cholesterol from the outside. Our results suggest thatGiardiais able to form GM1- and cholesterol-enriched lipid rafts and these raft domains are important for encystation.

scholarly journals Cytosolic [Ca2+] regulation of InsP3-evoked puffs

2012 ◽  
Vol 449 (1) ◽  
pp. 167-173 ◽  
Author(s):  
Michiko Yamasaki-Mann ◽  
Angelo Demuro ◽  
Ian Parker
Keyword(s):  
Plasma Membrane ◽  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Xenopus Oocytes ◽  
Single Channel ◽  
Building Blocks ◽  
Electrochemical Gradient ◽  
Intact Cells ◽  
Nicotinic Acetylcholine ◽  
Insp3 Receptors

InsP3-mediated puffs are fundamental building blocks of cellular Ca2+ signalling, and arise through the concerted opening of clustered InsP3Rs (InsP3 receptors) co-ordinated via Ca2+-induced Ca2+ release. Although the Ca2+ dependency of InsP3Rs has been extensively studied at the single channel level, little is known as to how changes in basal cytosolic [Ca2+] would alter the dynamics of InsP3-evoked Ca2+ signals in intact cells. To explore this question, we expressed Ca2+-permeable channels (nicotinic acetylcholine receptors) in the plasma membrane of voltage-clamped Xenopus oocytes to regulate cytosolic [Ca2+] by changing the electrochemical gradient for extracellular Ca2+ entry, and imaged Ca2+ liberation evoked by photolysis of caged InsP3. Elevation of basal cytosolic [Ca2+] strongly increased the amplitude and shortened the latency of global Ca2+ waves. In oocytes loaded with EGTA to localize Ca2+ signals, the number of sites at which puffs were observed and the frequency and latency of puffs were strongly dependent on cytosolic [Ca2+], whereas puff amplitudes were only weakly affected. The results of the present study indicate that basal cytosolic [Ca2+] strongly affects the triggering of puffs, but has less of an effect on puffs once they have been initiated.

scholarly journals Metabolic stability and antigenic modulation of nicotinic acetylcholine receptors on bovine adrenal chromaffin cells.

1988 ◽  
Vol 107 (3) ◽  
pp. 1147-1156 ◽  
Author(s):  
L S Higgins ◽  
D K Berg
Keyword(s):  
Plasma Membrane ◽  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Chromaffin Cells ◽  
Splanchnic Nerve ◽  
Temperature Sensitive ◽  
Adrenal Chromaffin Cells ◽  
Antigenic Modulation ◽  
Nicotinic Acetylcholine ◽  
Adrenal Chromaffin

Bovine adrenal chromaffin cells have nicotinic acetylcholine receptors (AChRs) that are activated by the splanchnic nerve, resulting in release of catecholamines from the cells. Examination of the AChRs can provide information about the regulation and turnover of synaptic components on neurons and endocrine cells. Previous studies have shown that mAb 35 recognizes the AChR on the cells. Here we show that mAb 35 can remove AChRs from the surface of the cells by antigenic modulation, and that the modulation can be used together with other methods to examine the stability and turnover of the receptors in the plasma membrane. Unexpectedly, the results indicate a disparity between the rate at which AChRs reappear on the cells and the rate at which the ACh response recovers after preexisting AChRs have been removed. Exposure of bovine adrenal chromaffin cultures to mAb 35 results in a parallel decrease in the magnitude of the nicotinic response and the number of AChRs on the cells. The decrease depends on the concentration and divalence of mAb 35, and on the time and temperature of the incubation. The antibody induces receptor aggregation in the plasma membrane under conditions where receptor loss subsequently occurs. After binding to receptor, mAb 35 appears to be internalized, degraded, and released from the cells through a temperature sensitive pathway that requires lysosomal function. These features are characteristic of antigenic modulation. Appearance of new AChRs on the cells either after antigenic modulation or after blockade of existing AChRs with monovalent antibody fragments occurs at a rate equivalent to 3% of the receptors present on control cells per hour. The rate of receptor loss from the cells was measured in the presence of either tunicamycin or puromycin to block appearance of new receptors. Both conditions indicated a receptor half-life of approximately 24 h and a rate of loss of approximately 3%/h. The finding that the rate of receptor loss equaled the rate of receptor appearance was consistent with the observation that the total number of AChRs on untreated cells did not increase with time. In the presence of tunicamycin, loss of receptor-mediated response to nicotine also occurred with a half-time of 24 h. Paradoxically, the rate of recovery of the nicotinic response, determined using two procedures, was more than twice as great as the rate at which new AChRs appeared on the cells.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Antibody-induced crosslinking and cholesterol-sensitive, anomalous diffusion of nicotinic acetylcholine receptors

2019 ◽  
Author(s):  
Alejo Mosqueira ◽  
Pablo A. Camino ◽  
Francisco J. Barrantes
Keyword(s):  
Single Molecule ◽  
Anomalous Diffusion ◽  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Dynamic Equilibrium ◽  
Switching Behavior ◽  
Live Cells ◽  
Cholesterol Depletion ◽  
Antigenic Modulation ◽  
Nicotinic Acetylcholine

AbstractSynaptic strength depends on the number of cell-surface neurotransmitter receptors in dynamic equilibrium with intracellular pools. Dysregulation of this homeostatic balance occurs e.g. in myasthenia gravis, an autoimmune disease characterized by a decrease in the number of postsynaptic nicotinic acetylcholine receptors (nAChRs). Monoclonal antibody mAb35 mimics this effect. Here we use STORM nanoscopy to characterize the individual and ensemble dynamics of mAb35-crosslinked receptors in the clonal cell line CHO-K1/A5, which robustly expresses adult muscle-type nAChRs. Antibody labeling of live cells results in 80% receptor immobilization. The remaining mobile fraction exhibits a heterogeneous combination of Brownian and anomalous diffusion. Single-molecule trajectories exhibit a two-state switching behavior between free Brownian walks and anticorrelated walks within confinement areas. The latter act as permeable fences (∼34 nm radius, ∼400 ms lifetime). Dynamic clustering, trapping and immobilization also occur in larger nanocluster zones (120-180 nm radius) with longer lifetimes (11 ± 1 s), in a strongly cholesterol-sensitive manner. Cholesterol depletion increases the size and average duration of the clustering phenomenon; cholesterol enrichment has the opposite effect. The disclosed high proportion of mAb35-crosslinked immobile receptors, together with their anomalous, cholesterol-sensitive diffusion and clustering, provides new insights into the antibody-enhanced antigenic modulation that leads to physiopathological internalization and degradation of receptors in myasthenia.A preliminary version of this work has appeared in the biorXiv repository: https://www.biorxiv.org/content/10.1101/744664v1. The study was not pre-registered.

scholarly journals Bcl-2 overexpression prevents calcium overload and subsequent apoptosis in dystrophic myotubes

2006 ◽  
Vol 395 (2) ◽  
pp. 267-276 ◽  
Author(s):  
Olivier Basset ◽  
François-Xavier Boittin ◽  
Christian Cognard ◽  
Bruno Constantin ◽  
Urs T. Ruegg
Keyword(s):  
Plasma Membrane ◽  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Calcium Overload ◽  
Mdx Mice ◽  
Mitochondrial Calcium ◽  
Protein Overexpression ◽  
Nicotinic Acetylcholine ◽  
Calcium Dependent ◽  
Inositol 1,4,5 Trisphosphate

Duchenne muscular dystrophy (DMD) is a lethal disease caused by the lack of the cytoskeletal protein dystrophin. Altered calcium homoeostasis and increased calcium concentrations in dystrophic fibres may be responsible for the degeneration of muscle occurring in DMD. In the present study, we used subsarcolemmal- and mitochondrial-targeted aequorin to study the effect of the antiapoptotic Bcl-2 protein overexpression on carbachol-induced near-plasma membrane and mitochondrial calcium responses in myotubes derived from control C57 and dystrophic (mdx) mice. We show that Bcl-2 overexpression decreases subsarcolemmal and mitochondrial calcium overload that occurs during activation of nicotinic acetylcholine receptors in dystrophic myotubes. Moreover, our results suggest that overexpressed Bcl-2 protein may prevent near-plasma membrane and mitochondrial calcium overload by inhibiting IP3Rs (inositol 1,4,5-trisphosphate receptors), which we have shown previously to be involved in abnormal calcium homoeostasis in dystrophic myotubes. Most likely as a consequence, the inhibition of IP3R function by Bcl-2 also inhibits calcium-dependent apoptosis in these cells.

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