[21] Selective labeling of neurotransmitter transporters at the cell surface

Daniels, Gwynn M.; Amara, Susan G.

doi:10.1016/s0076-6879(98)96023-2

Cited by 54 publications

(36 citation statements)

References 7 publications

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“…Cell Surface Biotinylation-Biotinylation of MLO-Y4 cells was performed based on a modification of previously published procedures (25,32 , and glycine and lysed in lysis buffer (133 mM NaCl, 5 mM KCl, 1% dextrose, and 20 mM HEPES) plus 1% sodium deoxycholate, 1% Triton X-100, 0.1% SDS, and protease inhibitors. Cell lysates were mixed with monomeric avidin beads and incubated for 1 h at room temperature.…”

Section: Methodsmentioning

confidence: 99%

Adaptation of Connexin 43-Hemichannel Prostaglandin Release to Mechanical Loading

Siller-Jackson

Burra

et al. 2008

Journal of Biological Chemistry

158

179

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Bone tissues respond to mechanical loading/unloading regimens to accommodate (re)modeling requirements; however, the underlying molecular mechanism responsible for these responses is largely unknown. Previously, we reported that connexin (Cx) 43 hemichannels in mechanosensing osteocytes mediate the release of prostaglandin, PGE 2 , a crucial factor for bone formation in response to anabolic loading. We show here that the opening of hemichannels and release of PGE 2 by shear stress were significantly inhibited by a potent antibody we developed that specifically blocks Cx43-hemichannels, but not gap junctions or other channels. The opening of hemichannels and release of PGE 2 are magnitude-dependent on the level of shear stress. Insertion of a rest period between stress enhances this response. Hemichannels gradually close after 24 h of continuous shear stress corresponding with reduced Cx43 expression on the cell surface, thereby reducing any potential negative effects of channels staying open for extended periods. These data suggest that Cx43-hemichannel activity associated with PGE 2 release is adaptively regulated by mechanical loading to provide an effective means of regulating levels of extracellular signaling molecules responsible for initiation of bone (re)modeling.The skeleton regulates its architecture and mass to meet structural and metabolic needs. To fulfill its structural functions, this complex tissue must adapt to loading and unloading while simultaneously regulating the metabolic demands of the skeleton. Numerous in vivo animal studies show the essential role of mechanical loading for bone formation and remodeling; however, the underlying molecular mechanisms, in particular how bone cells adapt to mechanical stimulation, remain largely uncharacterized.When mechanical forces are applied to bone, several potential stimuli occur including changes in hydrostatic pressure, direct cell strain, fluid flow, and electric potentials. These changes lead to fluid movement through the bone (1-3). Shear stress induced by mechanical loading facilitates the exchange of nutrients and bone modulators, and elicits biochemical responses. Osteocytes are well positioned in the bone to sense the magnitude of mechanical strain and are essential for the skeleton adaptive response to load. Experimental studies have shown that osteocytes are sensitive to stress applied to both intact bone tissue and in cell culture (4 -6). Encased within mineralized tissue, their dendritic morphology allows them to connect through small tunnels called canaliculi to form a threedimensional network not only with adjacent osteocytes but also to connect to cells on the bone surface and bone marrow.Connexins (Cx), 2 gap junction-forming proteins, belong to a multigene family expressing four transmembrane domains. The regions corresponding to transmembrane and extracellular domains are highly conserved. Cx43 has been identified in most types of bone cells (7-13) and is the major connexin expressed in osteocyte-like MLO-Y4 cells and primary osteocy...

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Section: Methodsmentioning

confidence: 99%

Adaptation of Connexin 43-Hemichannel Prostaglandin Release to Mechanical Loading

Siller-Jackson

Burra

et al. 2008

Journal of Biological Chemistry

158

179

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“…30,31 MDCK cells, grown either nonconfluently on cell culture plates (Figure 4) or confluently on filters ( Figure 5), were transfected with ClC-Kb alone or together with barttin fluorescence protein-fusion proteins. One day after transfection, the cells were incubated with 1.5 mg of biotin (sulfo-NHS-LC-biotin or sulfo-NHS-SS-biotin; Pierce, Rockford, IL) in PBS or triethanolamine buffer for 1 to 2 h. In experiments using filters, the side not incubated with biotin was incubated with biotinylation buffer only.…”

Section: Electrophysiologymentioning

confidence: 99%

Disease-Causing Dysfunctions of Barttin in Bartter Syndrome Type IV

Janssen

Scholl²,

Domeyer³

et al. 2009

Journal of the American Society of Nephrology

116

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Bartter syndrome type IV is an inherited human condition characterized by severe renal salt wasting and sensorineural deafness. The causal gene, BSND, encodes barttin, an accessory subunit of chloride channels located in the kidney and inner ear. Barttin modulates the stability, cell surface localization, and function of ClC-K channels; distinct mutations cause phenotypes of varying severity. For definition of the molecular basis of this diversity, the functional consequences of six disease-causing mutations (R8L, R8W, G10S, Q32X, G47R, and E88X) on ClC-K channel properties were studied by heterologous expression in renal cell lines, electrophysiology, confocal imaging, and biochemical analysis. Three missense mutations (R8L, R8W, and G10S) eliminated the function of ClC-K/barttin channels but did not prevent the insertion of the channels into the surface membrane. Another mutant that produces a mild renal phenotype (G47R) was capable of performing all functions of wild-type barttin but bound to ClC-K channels less effectively. The nonsense mutation E88X affected epithelial sorting, leading to equal amounts of barttin inserting into the basolateral and apical membranes, contrasting with the preferential apical insertion of wild-type barttin. Last, the nonsense mutation Q32X allowed barttin to associate with ClC-K channels but prevented surface membrane insertion and channel activation. These results demonstrate that Bartter syndrome type IV can be caused by various derangements in the function of barttin, likely contributing to the diversity of observed phenotypes.

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“…Biotinylation of the surface proteins from Chinese hamster ovary (CHO)-K1-transfected cells (3,24), followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (20) and Western blotting (2,18), was performed to investigate the localization of the wildtype and gB variants and also to estimate their surface expres-sion level compared to the wild-type protein. This approach was established (3) and successfully used for detection of surface-expressed EBV gH/gL complexes (24).…”

mentioning

confidence: 99%

“…This approach was established (3) and successfully used for detection of surface-expressed EBV gH/gL complexes (24). The membraneimpermeable biotinylation agent, sulfosuccinimidyl-6-(biotinamido) hexanoate (Pierce), was used to ensure labeling of only surface proteins (3). Actin served as a negative control for labeling of intracellular proteins.…”

mentioning

confidence: 99%

Hydrophobic Residues That Form Putative Fusion Loops of Epstein-Barr Virus Glycoprotein B Are Critical for Fusion Activity

Backović

Jardetzky

Longnecker

2007

J Virol

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To test the importance of the hydrophobic residues within the putative Epstein-Barr virus (EBV) glycoprotein B (gB) fusion loops in membrane fusion, WY 112-113 and WLIW [193][194][195][196] were mutated into alanine, glutamic acid, or the analogous residues from herpes simplex virus type 1 (HSV-1) gB (HR and RVEA). All gB variants exhibited cell surface expression, demonstrating that the substitutions did not perturb gB trafficking. None of six gB variants was, however, capable of mediating fusion with either epithelial or B cells. These data demonstrate that the bulky and hydrophobic EBV loop residues, which differ from the more hydrophilic HSV-1 residues and appear more compatible with membrane insertion, are essential for EBV gB-dependent fusion.Envelope glycoprotein B (gB) and glycoproteins H and L (gH/gL) form the core fusion machinery of all herpesviruses (32). The mechanism by which the three glycoproteins function to orchestrate membrane fusion is not fully understood. In varicella-zoster virus, cytomegalovirus, and human herpesvirus 8 (HHV-8), the action of gB or the gH/gL complex alone can result in fusion, although at a lower level than when all three glycoproteins are present (6,17,25). A truncated variant of Epstein-Barr virus (EBV) gB mediates fusion with epithelial cells at levels up to 60% of what is observed when gB, gH, and gL are transfected together (23,25). In the case of herpes simplex virus type 1 (HSV-1), the gH/gL complex seems to be responsible for the formation of a hemifusion intermediate, whereas gB is required to resolve the intermediate and complete fusion (33). The involvement of multiple proteins distinguishes herpesviruses from most other viruses where membrane merger is typically mediated by one fusion protein (16).Glycoprotein B is highly conserved throughout the herpesvirus family. HSV-1 gB exhibits 86% sequence identity with HSV-2 gB and 29% with EBV gB, while EBV and HHV-8 gB share 40% sequence identity. Although HSV-1 gB does not share any similarity with the fusion protein (G) of vesicular stomatitis virus (VSV) at the protein sequence level, the structural homology between the two proteins is notable (Fig. 1A) (11, 29). The only available structure of HSV-1 gB (11) was proposed to represent a postfusion conformation based on the similarity with the postfusion form of G.Fusion peptides of class I and II fusion proteins are rich in hydrophobic and aromatic residues and directly insert into the membrane after the conformational change is triggered. The residues critical for the ability of VSV G protein to cause fusion fall within two internal regions and give rise to a bipartite fusion peptide made of WY [72][73] and YA 116-117 (7, 35, 37). The conformation of the two fusion loops resembles the typical hairpin fold adopted by fusion peptides of class II fusion proteins (16). Regions structurally homologous to the fusion peptide of G were proposed to form putative fusion loops in HSV-1 gB (11). Most of the corresponding residues in HSV-1 gB, however, are not hydrophobic an...

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[21] Selective labeling of neurotransmitter transporters at the cell surface

Cited by 54 publications

References 7 publications

Adaptation of Connexin 43-Hemichannel Prostaglandin Release to Mechanical Loading

Adaptation of Connexin 43-Hemichannel Prostaglandin Release to Mechanical Loading

Disease-Causing Dysfunctions of Barttin in Bartter Syndrome Type IV

Hydrophobic Residues That Form Putative Fusion Loops of Epstein-Barr Virus Glycoprotein B Are Critical for Fusion Activity

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