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• W2012165680 abstract "Sequential stages in the life cycle of the ionotropic 5-HT3 receptor (5-HT3R) were resolved temporally and spatially in live cells by multicolor fluorescence confocal microscopy. The insertion of the enhanced cyan fluorescent protein into the large intracellular loop delivered a fluorescent 5-HT3R fully functional in terms of ligand binding specificity and channel activity, which allowed for the first time a complete real-time visualization and documentation of intracellular biogenesis, membrane targeting, and ligand-mediated internalization of a receptor belonging to the ligand-gated ion channel superfamily. Fluorescence signals of newly expressed receptors were detectable in the endoplasmic reticulum about 3 h after transfection onset. At this stage receptor subunits assembled to form active ligand binding sites as demonstrated in situ by binding of a fluorescent 5-HT3R-specific antagonist. After novel protein synthesis was chemically blocked, the 5-HT3 R populations in the endoplasmic reticulum and Golgi cisternae moved virtually quantitatively to the cell surface, indicating efficient receptor folding and assembly. Intracellular 5-HT3 receptors were trafficking in vesicle-like structures along microtubules to the cell surface at a velocity generally below 1 μm/s and were inserted into the plasma membrane in a characteristic cluster distribution overlapping with actin-rich domains. Internalization of cell surface 5-HT3 receptors was observed within minutes after exposure to an extracellular agonist. Our orchestrated use of spectrally distinguishable fluorescent labels for the receptor, its cognate ligand, and specific organelle markers can be regarded as a general approach allowing subcellular insights into dynamic processes of membrane receptor trafficking. Sequential stages in the life cycle of the ionotropic 5-HT3 receptor (5-HT3R) were resolved temporally and spatially in live cells by multicolor fluorescence confocal microscopy. The insertion of the enhanced cyan fluorescent protein into the large intracellular loop delivered a fluorescent 5-HT3R fully functional in terms of ligand binding specificity and channel activity, which allowed for the first time a complete real-time visualization and documentation of intracellular biogenesis, membrane targeting, and ligand-mediated internalization of a receptor belonging to the ligand-gated ion channel superfamily. Fluorescence signals of newly expressed receptors were detectable in the endoplasmic reticulum about 3 h after transfection onset. At this stage receptor subunits assembled to form active ligand binding sites as demonstrated in situ by binding of a fluorescent 5-HT3R-specific antagonist. After novel protein synthesis was chemically blocked, the 5-HT3 R populations in the endoplasmic reticulum and Golgi cisternae moved virtually quantitatively to the cell surface, indicating efficient receptor folding and assembly. Intracellular 5-HT3 receptors were trafficking in vesicle-like structures along microtubules to the cell surface at a velocity generally below 1 μm/s and were inserted into the plasma membrane in a characteristic cluster distribution overlapping with actin-rich domains. Internalization of cell surface 5-HT3 receptors was observed within minutes after exposure to an extracellular agonist. Our orchestrated use of spectrally distinguishable fluorescent labels for the receptor, its cognate ligand, and specific organelle markers can be regarded as a general approach allowing subcellular insights into dynamic processes of membrane receptor trafficking. A fundamental concern in neurobiology is the study of processes involved in expression, assembly and subcellular trafficking of neuroreceptors. Of particular interest are members of the large family of ligand-gated ion channels (LGIC) 1The abbreviations used are: LGIC, ligand-gated ion channel; 5-HT, serotonin; 5-HT3R, serotonin 5-HT3A receptor; nACh, nicotinic acetylcholine; nAChR, nicotinic acetylcholine receptor; GABAA, γ-aminobutyric acid receptor; GR65630, 3-(5-methyl-1H-imidazol-4-yl)-1-(1-methyl-1H-indol-3-yl)propanone; GR-Cy5, 1,2,3,9-tetrahydro-3-[(5-methyl-1H-imidazol-4-yl)methyl]-9-[3-amino-[N-(Cy5)amido]propyl]-4H-carbazol-4-one; GR-Flu, 1,2,3,9-tetrahydro-3-[(5-methyl-1H-imidazol-4-yl)methyl]-9-(3-amino-(N-fluoresceinthiocarbamoyl)propyl)-4H-carbazol-4-one; GR-Rho, 1,2,3,9-tetrahydro-3-[(5-methyl-1H-imidazol-4-yl)methyl]-9-[3-amino-[N-(rhodamine B)thiocarbamoyl]propyl]-4H-carbazol-4-one; mCPBG, 1-(m-chlorophenyl)biguanide; GFP, green fluorescent protein; ECFP, enhanced cyan fluorescent protein; EYFP, enhanced yellow fluorescent protein; ER, endoplasmic reticulum; PBS, phosphate-buffered saline. that include nicotinic acetylcholine (nAChR), serotonin (5-HT3R), γ-aminobutyric acid (GABAA), glycine, and ionotropic glutamate receptors (1Betz H. Neuron. 1990; 5: 383-392Abstract Full Text PDF PubMed Scopus (351) Google Scholar, 2Barnard E.A. Trends Biochem. Sci. 1992; 17: 368-374Abstract Full Text PDF PubMed Scopus (145) Google Scholar). All of them are oligomeric membrane proteins composed of subunits, which surround an ion channel that opens upon neurotransmitter binding to the receptor. The structural relationship between the different LGICs suggests that their assembly and trafficking involves similar molecular events (3Green W.N. Millar N.S. Trends Neurosci. 1995; 18: 280-287Abstract Full Text PDF PubMed Scopus (175) Google Scholar). In general, it is thought that subunit assembly and ligand binding site formation occurs shortly after biosynthesis in the endoplasmic reticulum (ER) followed by trafficking to the plasma membrane (4Blount P. Merlie J.P. J. Cell Biol. 1990; 111: 2613-2622Crossref PubMed Scopus (93) Google Scholar, 5Gu Y. Forsayeth J.R. Verrall S. Yu X.M. Hall Z.W. J. Cell Biol. 1991; 114: 799-807Crossref PubMed Scopus (91) Google Scholar, 6Chavez R.A. Maloof J. Beeson D. Newsom-Davis J. Hall Z.W. J. Biol. Chem. 1992; 267: 23028-23034Abstract Full Text PDF PubMed Google Scholar, 7Hall Z.W. Trends Cell Biol. 1992; 2: 66-68Abstract Full Text PDF PubMed Scopus (18) Google Scholar, 8Boyd G.W. Low P. Dunlop J.I. Robertson L.A. Vardy A. Lambert J.J. Peters J.A. Connolly C.N. Mol. Cell. Neurosci. 2002; 21: 38-50Crossref PubMed Scopus (67) Google Scholar). Methodologies allowing the dynamic observation of these processes as well as a direct probing of receptor functions in intracellular compartments have still to be elaborated. In the present study, we focus on the ionotropic 5-HT3R as a representative member of the LGICs and explore new strategies to monitor receptor biogenesis in real time starting with the delivery of their coding DNA into living cells. We will resolve events, occurring after the receptors “birth” from those leading to cell membrane insertion of mature receptors and finally to ligand-induced re-absorption of receptors into the cell reflecting the end point of the receptors lifespan. Improved understanding of these processes can provide valuable information for the therapeutic targeting of LGICs at specific stages in their life cycle. The ionotropic 5-HT3R is known to mediate fast signal transduction across synapses in the nervous system. Homopentameric complexes of recombinant 5-HT3 receptors exhibit high cell surface expression levels in heterologous systems (9Pick H. Preuss A.K. Mayer M. Wohland T. Hovius R. Vogel H. Biochemistry. 2003; 42: 877-884Crossref PubMed Scopus (44) Google Scholar) and share substantial pharmacological and functional properties with native neuronal 5-HT3R (10Maricq A.V. Peterson A.S. Brake A.J. Myers R.M. Julius D. Science. 1991; 254: 432-437Crossref PubMed Scopus (884) Google Scholar, 11Boess F.G. Beroukhim R. Martin I.L. J. Neurochem. 1995; 64: 1401-1405Crossref PubMed Scopus (123) Google Scholar, 12Green T. Stauffer K.A. Lummis S.C. J. Biol. Chem. 1995; 270: 6056-6061Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). To date the direct intracellular analysis of 5-HT3 receptors has been limited to antibody labeling methods (8Boyd G.W. Low P. Dunlop J.I. Robertson L.A. Vardy A. Lambert J.J. Peters J.A. Connolly C.N. Mol. Cell. Neurosci. 2002; 21: 38-50Crossref PubMed Scopus (67) Google Scholar). However, these technologies are not adequate for visualizing receptors in live cells and cannot be used to label intracellular receptors in non-permeabilized cells. Here we fuse the enhanced cyan fluorescent protein (ECFP) to the 5-HT3Rto visualize the entire sequence of stages in the life of the receptor. The green fluorescent protein and its spectral variants have been widely used as molecular reporters to monitor gene expression, localization, and trafficking of proteins in living cells (13Chalfie M. Tu Y. Euskirchen G. Ward W.W. Prasher D.C. Science. 1994; 263: 802-805Crossref PubMed Scopus (5504) Google Scholar, 14Cubitt A.B. Heim R. Adams S.R. Boyd A.E. Gross L.A. Tsien R.Y. Trends Biochem. Sci. 1995; 20: 448-455Abstract Full Text PDF PubMed Scopus (1204) Google Scholar, 15Kain S.R. Adams M. Kondepudi A. Yang T.T. Ward W.W. Kitts P. BioTechniques. 1995; 19: 650-655PubMed Google Scholar). GFP-tagged proteins often retain their biological activity and have the same trafficking pattern as native proteins (16Ogawa H. Inouye S. Tsuji F.I. Yasuda K. Umesono K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11899-11903Crossref PubMed Scopus (235) Google Scholar, 17Stauber R. Gaitanaris G.A. Pavlakis G.N. Virology. 1995; 213: 439-449Crossref PubMed Scopus (104) Google Scholar, 18Naray-Fejes-Toth A. Fejes-Toth G. J. Biol. Chem. 1996; 271: 15436-15442Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 19Sengupta P. Chou J.H. Bargmann C.I. Cell. 1996; 84: 899-909Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar, 20Htun H. Barsony J. Renyi I. Gould D.L. Hager G.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4845-4850Crossref PubMed Scopus (327) Google Scholar). The combination of tagging 5-HT3 receptors with autofluorescent proteins and the parallel use of fluorescent 5-HT3R specific ligands (21Tairi A.P. Hovius R. Pick H. Blasey H. Bernard A. Surprenant A. Lundstrom K. Vogel H. Biochemistry. 1998; 37: 15850-15864Crossref PubMed Scopus (52) Google Scholar) will be demonstrated as a powerful tool allowing the multicolor analysis of receptor biosynthesis, trafficking, and ligand-dependent receptor internalization. Materials—Synthetic oligonucleotides were purchased from MWG-Biotech AG (Ebersberg, Germany), kits for plasmid and DNA fragment purification were from Qiagen GmbH (Hilden, Germany), restriction endonucleases (ClaI, HindIII and NotI) were from New England Biolabs. The radioligand 3-(5-methyl-1H-imidazol-4-yl)-1-(1-[3H]methyl-1H-indol-3-yl)propanone ([3H]GR65630; 85.5 Ci/mmol) was from PerkinElmer Life Sciences; the agonist serotonin (5-HT) was obtained from Sigma (Buchs, Switzerland), and quipazine and 1-(m-chlorophenyl)biguanide (mCPBG) were from Tocris-Cookson (Langford, UK). The fluorescent ligands GR-Cy5 (1,2,3,9-tetrahydro-3-[(5-methyl-1H-imidazol-4-yl)methyl]-9-[3-amino-[N-(Cy5)amido]propyl]-4H-carbazol-4-one), GR-Flu (1,2,3,9-tetrahydro-3-[(5-methyl-1H-imidazol-4-yl)methyl]-9-(3-amino-(N-fluorescein-thiocarbamoyl)-propyl)-4H-carbazol-4-one) and GR-Rho (1,2,3,9-tetrahydro-3-[(5-methyl-1H-imidazol-4-yl)methyl]-9-[3-amino-[N-(rhodamine B)thiocarbamoyl]-propyl]-4H-carbazol-4-one) were prepared as described elsewhere (21Tairi A.P. Hovius R. Pick H. Blasey H. Bernard A. Surprenant A. Lundstrom K. Vogel H. Biochemistry. 1998; 37: 15850-15864Crossref PubMed Scopus (52) Google Scholar, 22Wohland T. Friedrich K. Hovius R. Vogel H. Biochemistry. 1999; 38: 8671-8681Crossref PubMed Scopus (117) Google Scholar). Triton-X100 was from Fluka (Buchs, Switzerland). The plasmids pEYFP-ER, pEYFP-Golgi, pEYFP-Tub, and pEYFP-Actin were purchased from Clontech. Other chemicals were from Sigma. DNA Constructs—All constructs are based on a vector containing the short splicing variant of the murine 5-hydroxytryptamine type 3A subunit cDNA (23Werner P. Kawashima E. Reid J. Hussy N. Lundstrom K. Buell G. Humbert Y. Jones K.A. Brain Res. Mol. Brain Res. 1994; 26: 233-241Crossref PubMed Scopus (77) Google Scholar) preceded by the human cytomegalovirus gene promoter, as described before (9Pick H. Preuss A.K. Mayer M. Wohland T. Hovius R. Vogel H. Biochemistry. 2003; 42: 877-884Crossref PubMed Scopus (44) Google Scholar). The vector p5HT3R-ECFP, containing the ECFP-labeled receptor, was obtained as follows: the original vector was first mutated using the oligonucleotides 5′-CTG ATG ACT GCT CAA TCG ATG CCA TGG GAA ACC-3′ and 5′-GGT TTC CCA TGG CAT CGA TTG AGC AGT CAT CAG-3′, adding a ClaI restriction site in the large cytoplasmic loop sequence between the third and fourth predicted membrane-spanning domains. ECFP was introduced between Ser359 and Ala360 of the receptor (corresponding to Swiss-Prot entry p23979, mature sequence numbering) to give 5-HT3R-ECFP. The ECFP insert was obtained by PCR amplification on the template pECFP-N1 (Clontech) using the synthetic oligonucleotides 5′-CCA TCG ATA TGG TGA GCA AGG GCG AGG-3′ and 5′-CCA TCG ATC TTG TAC AGC TCG TCC ATG CCG-3′ and ligated into the receptor ClaI restriction site. The restriction sites HindIII and NotI were added to the 5′ and 3′ ends of the 5-HT3R-EGFP sequence by PCR amplification using the oligonucleotides 5′-CGA TAA GCT TCA CCA TGC GGC TCT GCA TCC CGC AGG TG-3′ and 5′-GCT GTG CCC ACG CGG CCG CTC AAG AAT AAT GCC AAA TGG ACC AGA G-3′, and the purified 2197-bp fragment was subcloned into the HindIII/NotI cut vector pEAK8 (Edge BioSystems, Gaithersburg, MD), yielding the vector p5HT3R-ECFP. The vector p5HT3R was obtained by subcloning the non-mutated receptor gene into the plasmid pEAK8 between HindIII and NotI, using the same oligonucleotides as for constructing p5HT3R-ECFP. The plasmid constructs were confirmed by restriction mapping and DNA sequencing. Cell Culture, Transfections, and Permeabilization—Adherent human embryonic kidney (HEK293) and N1E-115 cells were grown in Dulbecco's modified Eagle medium/F-12 (Invitrogen) supplemented with 2.2 and 10% fetal calf serum (Invitrogen), respectively, using plastic flasks from TPP AG (Trasadingen, Switzerland). The cultures were split regularly and kept at 37 °C in a humidified atmosphere with 5% CO2. For electrophysiology and confocal microscopy measurements, respectively, cells were seeded in 35-mm cell culture dishes and 6-well plates containing 22-mm diameter glass coverslips at a density of 150,000 cells/ml and transfected using LipofectAMINE 2000 reagent (Invitrogen). Transfection efficiencies were determined by confocal fluorescence microscopy on cell samples which were cotransfected with an enhanced GFP (pEGFP-N1; Clontech) reporter DNA. Fixation of cells was achieved by 10-min incubation at room temperature in a solution containing 3.7% formaldehyde in PBS. Subsequent permeabilization was performed by 5-min incubation in the presence of 0.1% Triton X-100 in PBS. Triton X-100 was used for cell permeabilization because this detergent was shown not to interfere with ligand binding affinity (12Green T. Stauffer K.A. Lummis S.C. J. Biol. Chem. 1995; 270: 6056-6061Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 24Hovius R. Tairi A.P. Blasey H. Bernard A. Lundstrom K. Vogel H. J. Neurochem. 1998; 70: 824-834Crossref PubMed Scopus (44) Google Scholar). Cells were washed three times with PBS between incubations. Radioactive Binding Assays—Receptor concentrations as well as ligand affinities were measured by radioligand binding assay. 100 μl containing ∼1 × 106 cells resuspended in 10 mm Hepes, pH 7.4, were incubated for 30 min at room temperature in solutions of 10 mm Hepes, pH 7.4, with varying concentrations of the specific antagonist [3H]GR65630 in a final volume of 1 ml. A rapid filtration through Whatman GF/B filters (presoaked for 15 min in 0.5% (w/v) polyethylenimine) followed by two washes with 3 ml of ice-cold 10 mm Hepes at pH 7.4 terminated the incubation. Filters were then transferred into scintillation vials and 4 ml of Ultima Gold (Packard, Meridan, CT) was added. The radioactivity was measured in a Tri-Carb 2200CA liquid scintillation counter (Packard). Nonspecific binding was determined in the presence of 1 μm quipazine. Binding assay on permeabilized cells were processed as above, except that the cells were pre-treated with 0.1% saponin in 10 mm Hepes pH 7.4 for 5 min at room temperature before radioligand binding. All experiments were done in triplicate. The dissociation constant Kd of [3H]GR65630 and the Hill coefficient n were calculated by fitting the experimental data to the following binding equation.[GR65630]bound=[5-HT3R]1+(Kd/[GR65630]free)n(Eq. 1) Electrophysiology—We used the standard patch-clamp technique in whole-cell voltage clamp to –60 mV employing an EPC-9 patch clamp amplifier (HEKA Elektronik GmbH, Lambrecht, Germany). For data acquisition and storage the software PULSE 8.3 (HEKA Elektronik GmbH) was used. Borosilicate glass pipettes were heat polished and had resistances of 2–5 MΩ. Pipettes were filled with 140 mm KCl, 5 mm MgCl2, 10 mm EGTA, 10 mm Hepes-KOH, pH 7.3. The external solution was 147 mm NaCl, 12 mm glucose, 2 mm KCl, 2 mm CaCl2, 1 mm MgCl2, 10 mm Hepes-NaOH, pH 7.4. Ligands dissolved in the external solution were applied with a RSC-200 or the MSC-200 perfusion system (BioLogic, Claix, France). During experiments the cells were continuously perfused. All experiments were performed at room temperature. Data were evaluated by fitting to the Hill following equations,I=Imax/{1+(EC50/[agonist])n}(Eq. 2) I=I0/{1+(IC50/[antagonist])−n}(Eq. 3) where I is the peak current at a certain ligand concentration, Imax is the maximal peak current achievable, I0 is the peak current in absence of any antagonist, EC50 and IC50 are the half-maximal effective and inhibitory concentrations, respectively, and n is the Hill coefficient. For antagonist-agonist competition experiments, cells were perfused with granisetron-containing solutions for 3 min before the addition of serotonin (5-HT) at a concentration of 30 μm. Laser Scanning Confocal Microscopy—Laser scanning confocal micrographs were recorded using 458/488-, 543-, and 633-nm laser lines on a Zeiss LSM 510 microscope (Carl Zeiss AG) equipped with a 63× water objective (1.2 numerical aperture). Detection and distinction of fluorescence signals was achieved by appropriate filter sets using a multitracking mode. Scanning speed and laser intensity were adjusted to avoid photobleaching of the fluorescent probes and damage or morphological changes of the cells. The microscope was equipped with a microcultivation system (Incubator S, CTI controller 3700 digital, Zeiss) to control temperature, humidity, and CO2 for maintaining physiological conditions during long-term experiments. Image analysis and fluorescence signal quantification was performed using Zeiss LSM software. We investigated the time course of 5-HT3R plasma membrane expression by analyzing non-permeabilized HEK293 cell samples at different times after transfection via radioligand binding assay. 5-HT3R cell surface expression increased over time and reached a maximum number of 2.6 × 106 receptors per cell 34 h after transfection (Fig. 1). When these cells were permeabilized with saponin it was possible to detect in addition to cell surface receptors also 5-HT3R-specific ligand binding activity in the cytoplasm, which accounted for about 40% of the total ligand binding sites. In N1E-115 cells, which endogenously express 5-HT3 receptors, about 60% of the ligand binding activity was localized inside the cell (Table I). These results indicate that a substantial fraction of intracellular receptors is capable of binding 5-HT3R-specific ligands before plasma membrane integration. We then analyzed intracellular 5-HT3R formation and trafficking in more detail. To observe in real-time 5-HT3R biosynthesis after cell delivery of its coding DNA, we inserted the coding sequence of the ECFP into the large cytoplasmic loop between the third and fourth predicted membrane-spanning domains of the receptor (Fig. 2A). The preserved functionality of the fusion construct was confirmed by radioligand binding assay and whole cell patch clamp measurements (Table II).Table IRadioactive binding of [3H]GR65630 to 5-HT3R on whole cells (1Betz H. Neuron. 1990; 5: 383-392Abstract Full Text PDF PubMed Scopus (351) Google Scholar) and on saponin-permeabilized cells (2Barnard E.A. Trends Biochem. Sci. 1992; 17: 368-374Abstract Full Text PDF PubMed Scopus (145) Google Scholar)N1E-115 cellsHEK293 cells1) Receptor binding sites per cell on membrane5.0 ± 0.6 × 1042.6 ± 0.2 × 1062) Total number of receptor binding sites per cell1.2 ± 0.2 × 1054.4 ± 0.2 × 106Percentage of receptor binding sites localized on membrane42 ± 12%59 ± 7% Open table in a new tab Fig. 2Fusion of ECFP to the 5-HT3R and cellular trafficking visualized by confocal fluorescence microscopy. A, scheme of the folding of the membrane integrated 5-HT3R-ECFP. Extramembraneous regions are shown as thin lines; parts spanning the membrane (gray) are depicted as black bars. The position of the ECFP in the 5-HT3R-ECFP is shown correspondingly. The ECFP coding sequence is genetically fused into the large cytoplasmic loop between the third and fourth transmembrane domains, replacing the amino acids present in the long splicing variant of the receptor. B–D, trafficking of 5-HT3 R-ECFP in HEK293 cells. Images were acquired 3 h (B), 4 h (C), and 5 h (D) after transfection. The endoplasmic reticulum and Golgi apparatus were labeled by cotransfection with pEYFP-ER (B) and pEYFP-Golgi (C), respectively. During image acquisition the confocal plane was adjusted to achieve best optical resolution for the organelle under observation. The inset in D shows the membrane-integrated receptors by labeling the non-permeabilized cell with the fluorescent ligand GR-Cy5 (red). Regions where ECFP (blue) and EYFP (yellow) colocalized are indicated in white. Size bars represent 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IIRadioligand binding and electrophysiology on wild-type and ECFP-labeled 5-HT3R5-HT3R5-HT3R-ECFPBinding assay[3H]GR65630Kd (nm)0.3 ± 0.10.6 ± 0.1Hill0.8 ± 0.21.1 ± 0.2ElectrophysiologySerotoninEC50 (μm)0.97 ± 0.020.62 ± 0.06Hill3.1 ± 0.21.8 ± 0.3mCPBGEC50 (μm)0.4 ± 0.10.20 ± 0.03Hill1.0 ± 0.21.5 ± 0.3Granisetron inhibitionIC50 (nm)0.17 ± 0.010.26 ± 0.02Hill0.92 ± 0.031.04 ± 0.05 Open table in a new tab Using laser scanning confocal microscopy we found that the ECFP-labeled receptors start to form in the endoplasmic reticulum typically 3 h after transfection onset. The receptor-derived fluorescence signal could be colocalized with a spectrally distinguishable enhanced yellow fluorescent ER marker (Fig. 2B), which was transfected together with the ECFP-tagged receptor DNA. In parallel experiments, we were able to overlap fluorescence images arising from the receptor with EYFP targeted to the Golgi apparatus at ∼4 h after transfection (Fig. 2C). Receptor trafficking from the appearance in the Golgi to plasma membrane insertion occurred in about 30 min. The integration of mature receptors into the plasma membrane could be confirmed by binding of a fluorescence-labeled 5-HT3R-specific antagonist (Fig. 2D). Although the fluorescence emitted from the 5-HT3R-ECFP is primarily an indication of a folded ECFP marker protein, it allows also monitoring of 5-HT3 receptor biogenesis, since the ECFP label is directly integrated into the receptor sequence. Information about the assembly of 5-HT3R subunits can be assessed via detection of the formation of the ligand binding sites. It has been shown for the structurally related acetylcholine receptor (25Brejc K. van Dijk W.J. Klaassen R.V. Schuurmans M. van Der Oost J. Smit A.B. Sixma T.K. Nature. 2001; 411: 269-276Crossref PubMed Scopus (1579) Google Scholar) as well as by amino acid sequence homology studies on the 5-HT3R (26Maksay G. Bikadi Z. Simonyi M. J. Recept. Signal Transduct. Res. 2003; 23: 255-270Crossref PubMed Scopus (44) Google Scholar, 27Reeves D.C. Sayed M.F. Chau P.L. Price K.L. Lummis S.C. Biophys. J. 2003; 84: 2338-2344Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar) that the ligand binding sites are located between neighboring subunits; in consequence, ligand binding can only be observed once the subunits are fully assembled to a functional pentameric receptor. After monitoring 5-HT3R biosynthesis in cytoplasmic compartments we investigated the presence of intracellular 5-HT3-specific ligand binding sites, which indicate the functional assembly of receptor subunits. Ligand binding to intracellular 5-HT3 receptors was first investigated by permeabilizing fixed HEK293 cells at different times after transfection and subsequent labeling with fluorescent ligands. The binding specificity of those ligands to 5-HT3 receptors in intracellular compartments was confirmed by ligand displacement experiments (see supplemental data). ECFP-labeled 5-HT3Rs were localized in the endoplasmic reticulum or Golgi apparatus by comparing fluorescence images of the receptor with those of organelles labeled with EYFP as described above. The diffusion of a Cy5-labeled 5-HT3R-specific antagonist (GR-Cy5) into permeabilized cells allowed us to detect ligand binding activity in ER and Golgi apparatus (Fig. 3). This reports on receptor assembly at an early stage of expression in the ER. The GR-Cy5 fluorescence signal located in the Golgi was stronger than in the ER indicating substantially higher amounts of assembled 5-HT3 receptors in the Golgi apparatus. The amount of 5-HT3R ligand binding sites on the cell surface receptors as compared to those formed inside the cell was then evaluated by double ligand binding experiments (Fig. 3, G–I). Fluorescent rhodamine-labeled antagonist GR-Rho was applied to non-permeabilized cells expressing 5-HT3R-ECFP to label the receptors located on the plasma membrane. After fixation and permeabilization of the cells, the antagonist GR-Cy5 was applied to label the receptors inside the cell. Due to the low off-rate of GR-Rho from 5-HT3R, it was not replaced by GR-Cy5, as described before (9Pick H. Preuss A.K. Mayer M. Wohland T. Hovius R. Vogel H. Biochemistry. 2003; 42: 877-884Crossref PubMed Scopus (44) Google Scholar), and thus allowed us to visualize and to distinguish between extra- and intracellular ligand binding sites. Analyzing images arising from the fluorescence of Cy5- and rhodamine-labeled ligands in consecutive confocal cross-sections covering the whole cell revealed that 43 ± 14% of the receptor binding sites were located on the plasma membrane and the rest inside the cell (value averaged on 20 cells). This value is in the same range as obtained by radioligand binding assays on cell populations (average on 1 × 106 cells). Receptor trafficking from the Golgi apparatus to the plasma membrane was then analyzed in detail by imaging 5-HT3R-ECFP-expressing HEK293 cells (frame rate of 2 images/s; Fig. 4, A and B). The ECFP label allowed us to detect vesicle-like structures carrying 5-HT3 receptors in their membrane, which moved inside the cytoplasm. The velocity of these carrier vesicles was generally below 1 μm/s, measured as the distance between the pixel coordinates in consecutive images. The receptor-carrying vesicles seemed to be located in tubulin-rich regions visualized by cotransfecting EYFP-labeled tubulin (pEYFP-Tub). To verify whether tubulin filaments are required for 5-HT3 receptor transport to the plasma membrane, we studied the influence of colchicine on receptor trafficking (Fig. 4, C and D); colchicine is known to inhibit microtubule formation (28Weisenberg R.C. Borisy G.G. Taylor E.W. Biochemistry. 1968; 7: 4466-4479Crossref PubMed Scopus (917) Google Scholar, 29Sherline P. Leung J.T. Kipnis D.M. J. Biol. Chem. 1975; 250: 5481-5486Abstract Full Text PDF PubMed Google Scholar, 30Sackett D.L. Varma J.K. Biochemistry. 1993; 32: 13560-13565Crossref PubMed Scopus (97) Google Scholar). 5 h after adding 100 μg/ml cycloheximide to cells, which interferes with the translation machinery (31Wettstein F.O. Noll H. Penman S. Biochim. Biophys. Acta. 1964; 87: 525-528PubMed Google Scholar), the receptors were almost completely located on the cell membrane. However, when the cells were treated 2 h before with 50 μg/ml colchicine, the receptors did not completely localize at the membrane, demonstrating that the tubulin filaments are required for proper receptor trafficking and final membrane insertion. We then investigated the effects of agonist application on the subcellular distribution of 5-HT3 receptors. 34 h after transient expression of 5-HT3R-ECFP, HEK293 cells were treated with 100 μg/ml cycloheximide for 5 h to stop new protein synthesis and in turn allowing delivery of intracellular receptors to the cell membrane and thus depleting intracellular receptor fluorescence. Subsequent incubation of non-permeabilized cells with 12 nm GR-Cy5 yielded a complete spatial overlap of the ECFP receptor and the GR-Cy5 ligand fluorescence signals on the cell surface (Fig. 5, A–E). The fluorescent antagonist could be replaced by adding an excess (500 nm) of the non-fluorescent 5-HT3R-specific agonist mCPBG. After incubation at room temperature for 30 min, the cells were intensively washed with PBS buffer. We then applied the fluorescent antagonist GR-Cy5 for a second time using the same conditions as before (Fig. 5, F–J). The absence of fluorescence labeling on the cell surface wa" @default.
• W2012165680 created "2016-06-24" @default.
• W2012165680 creator A5010408256 @default.
• W2012165680 creator A5040027813 @default.
• W2012165680 creator A5048516955 @default.
• W2012165680 creator A5052124820 @default.
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• W2012165680 date "2004-12-01" @default.
• W2012165680 modified "2023-09-30" @default.
• W2012165680 title "Noninvasive Imaging of 5-HT3 Receptor Trafficking in Live Cells" @default.
• W2012165680 cites W1487561672 @default.
• W2012165680 cites W1530704472 @default.
• W2012165680 cites W1575079901 @default.
• W2012165680 cites W1776988577 @default.
• W2012165680 cites W1788430178 @default.
• W2012165680 cites W1967667749 @default.
• W2012165680 cites W1979790654 @default.
• W2012165680 cites W1980093334 @default.
• W2012165680 cites W1980490360 @default.
• W2012165680 cites W1981415432 @default.
• W2012165680 cites W1984302804 @default.
• W2012165680 cites W1987466586 @default.
• W2012165680 cites W1999832893 @default.
• W2012165680 cites W2000129974 @default.
• W2012165680 cites W2011403954 @default.
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• W2012165680 cites W2032579724 @default.
• W2012165680 cites W2032675721 @default.
• W2012165680 cites W2041206504 @default.
• W2012165680 cites W2044768708 @default.
• W2012165680 cites W2049793619 @default.
• W2012165680 cites W2053338555 @default.
• W2012165680 cites W2053944362 @default.
• W2012165680 cites W2058416613 @default.
• W2012165680 cites W2061104342 @default.
• W2012165680 cites W2066027396 @default.
• W2012165680 cites W2068009732 @default.
• W2012165680 cites W2077776088 @default.
• W2012165680 cites W2078971679 @default.
• W2012165680 cites W2081488550 @default.
• W2012165680 cites W2082942756 @default.
• W2012165680 cites W2102409079 @default.
• W2012165680 cites W2105560565 @default.
• W2012165680 cites W2112890098 @default.
• W2012165680 cites W2128261107 @default.
• W2012165680 cites W2129488948 @default.
• W2012165680 cites W2143992953 @default.
• W2012165680 cites W2146000414 @default.
• W2012165680 cites W2148211715 @default.
• W2012165680 cites W2165992768 @default.
• W2012165680 cites W2397317987 @default.
• W2012165680 cites W4321317620 @default.
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