FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2002609528> ?p ?o ?g. }

• W2002609528 endingPage "15182" @default.
• W2002609528 startingPage "15177" @default.
• W2002609528 abstract "Western blots of Xenopus oocyte membrane preparations showed that the apparent molecular mass of the wild type P2X2 receptor (about 65 kDa) was reduced by pretreatment with endoglycosidase H. Mutagenesis of one or more of three potential asparagines (N182S, N239S, and N298S) followed by Western blots showed that each of the sites was glycosylated in the wild type receptor. Functional channels were formed by receptors lacking any single asparagine, but not by channels mutated in two or three positions. Artificial consensus sequences (N-X -S/T) introduced into the N-terminal region (asparagine at position 9, 16, or 26) were not glycosylated. Asparagines were glycosylated when introduced at the C-terminal end of the first hydrophobic domain (positions 62 and 66) and at the N-terminal end of the second hydrophobic domain (position 324). A protein in which the C terminus of one P2X2 subunit was joined to the N terminus of a second P2X2 subunit (from a concatenated cDNA) had twice the molecular mass of the P2X2 receptor subunit, and formed fully functional channels. The experiments provide direct evidence for the topology originally proposed for the P2X receptor, with intracellular N and C termini, two membrane-spanning domains, and a large extracellular loop. Western blots of Xenopus oocyte membrane preparations showed that the apparent molecular mass of the wild type P2X2 receptor (about 65 kDa) was reduced by pretreatment with endoglycosidase H. Mutagenesis of one or more of three potential asparagines (N182S, N239S, and N298S) followed by Western blots showed that each of the sites was glycosylated in the wild type receptor. Functional channels were formed by receptors lacking any single asparagine, but not by channels mutated in two or three positions. Artificial consensus sequences (N-X -S/T) introduced into the N-terminal region (asparagine at position 9, 16, or 26) were not glycosylated. Asparagines were glycosylated when introduced at the C-terminal end of the first hydrophobic domain (positions 62 and 66) and at the N-terminal end of the second hydrophobic domain (position 324). A protein in which the C terminus of one P2X2 subunit was joined to the N terminus of a second P2X2 subunit (from a concatenated cDNA) had twice the molecular mass of the P2X2 receptor subunit, and formed fully functional channels. The experiments provide direct evidence for the topology originally proposed for the P2X receptor, with intracellular N and C termini, two membrane-spanning domains, and a large extracellular loop. The extracellular signaling properties of nucleotides are mediated through two distinct families of membrane proteins. These are the P2Y receptors, coupled to G proteins and second messenger pathways, and the P2X receptors, which are ligand-gated ion channels (1North R.A. Barnard E.A. Curr. Opin. Neurobiol. 1997; 7: 346-357Crossref PubMed Scopus (426) Google Scholar). Seven subunits of the P2X receptor family (P2X1–7) have been characterized at the molecular level (reviewed in Refs. 2Buell G. Collo G. Rassendren F. Eur. J. Neurosci. 1996; 8: 2221-2228Crossref PubMed Scopus (240) Google Scholar and 3North R.A. Curr. Opin. Cell Biol. 1996; 8: 474-483Crossref PubMed Scopus (138) Google Scholar). These show a broad expression pattern compared with other ligand-gated channels, with the various forms being found in central and peripheral nervous system, different types of immune cells, glands, and smooth and skeletal muscles (4Collo G. North R.A. Kawashima E. Merlo-Pich E. Neidhart S. Surprenant A. Buell G. J. Neurosci. 1996; 16: 2495-2507Crossref PubMed Google Scholar). The P2X receptor subunits can form channels as homomultimers or, in some cases, as heteromultimers (5Lewis C. Neidhart S. Holy C. North R.A. Buell G. Surprenant A. Nature. 1995; 377: 432-434Crossref PubMed Scopus (889) Google Scholar, 6Radford K.M. Virginio C. Surprenant A. North R.A. Kawashima E. J. Neurosci. 1997; 17: 6529-6533Crossref PubMed Google Scholar). The number of subunits in each channel molecule is not known. P2X receptor subunits are 36–48% identical to one another at the amino acid level. All seven proteins have similar hydrophobicity profiles, with only two hydrophobic regions sufficiently long to span the plasma membrane. These regions display the features often seen in transmembrane segments such as aromatic residues at interfacial regions, and they have an excess of positively charged amino acids at their presumed cytoplasmic ends (7Von Heijne G. Gavel Y. Eur. J. Biochem. 1988; 97: 175-181Crossref Scopus (261) Google Scholar). Together with the absence of signal peptide sequence after the initiating methionine, this suggests that P2X receptors may have intracellular N and C termini, and two transmembrane domains separated by a large extracellular loop (8Valera S. Hussy N. Evans R.J. Adami N. North R.A. Surprenant A. Buell G. Nature. 1994; 371: 516-519Crossref PubMed Scopus (894) Google Scholar, 9Brake A.J. Wagenbach M.J. Julius D. Nature. 1994; 371: 519-523Crossref PubMed Scopus (837) Google Scholar). This proposed topology differs from that of the nicotinic and glutamate superfamilies of ligand-gated ion channels (reviewed in Ref. 10Dani J.A. Mayer M.L. Curr. Opin. Neurobiol. 1995; 5: 310-317Crossref PubMed Scopus (58) Google Scholar), but resembles that shown for the pore-forming subunits of epithelial sodium channels (11Canessa C.M. Merillat A.M. Rossier B.C. Am. J. Physiol. 1994; 267: 1682-1690Crossref PubMed Google Scholar, 12Renard S. Lingueglia E. Voilley N. Lazdunski M. Barbry P. J. Biol. Chem. 1994; 269: 12981-12986Abstract Full Text PDF PubMed Google Scholar). There is, however, no detectable similarity of amino acid sequence between P2X receptors and epithelial sodium channels (3North R.A. Curr. Opin. Cell Biol. 1996; 8: 474-483Crossref PubMed Scopus (138) Google Scholar). There are other experimental results that are consistent with this topological model of P2X receptor subunit. First, P2X1receptors are activated by the ATP analog αβmeATP, 1The abbreviations used are: αβmeATP, adenosine 5′-(α,β-methylene)triphosphate; Endo H, endoglycosidase H; MTSET, methanethiosulfonate trimethylammonium; EGFP, enhanced green fluorescent protein. 1The abbreviations used are: αβmeATP, adenosine 5′-(α,β-methylene)triphosphate; Endo H, endoglycosidase H; MTSET, methanethiosulfonate trimethylammonium; EGFP, enhanced green fluorescent protein. whereas P2X2 receptors are not; transferring the putative extracellular loop (approximately residues 50–320) from the P2X1 receptor into the P2X2 receptor confers αβmeATP sensitivity (13Werner P. Seward E.P. Buell G.N. North R.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15485-15490Crossref PubMed Scopus (128) Google Scholar). Second, changing one amino acid within the loop of the P2X4 receptor (E249K) causes a large increase in the blocking action of the slowly reversible antagonist pyridoxal-5-phosphate-6-azophenyl-2′-4′-disulfonic acid (14Buell G. Lewis C. Collo G. North R.A. Surprenant A. EMBO J. 1996; 15: 26-55Crossref Scopus (375) Google Scholar). Third, the difference in sensitivity to pyridoxal-5-phosphate-6-azophenyl-2′-4′-disulfonic acid between human and rat P2X4 receptors can be transferred by exchange of a segment within the first half of this loop (15Garcia-Guzman M. Soto F. Gomez-Hernandez J.M. Lund P.-E. Stuhmer W. Mol. Pharmacol. 1997; 51: 109-118Crossref PubMed Scopus (190) Google Scholar). Fourth, evidence forN -glycosylation of Asn184 of the P2X1 receptor has been obtained, indicating that this must be located extracellularly (16Valera S. Talabot F. Evans R.J. Gos A. Antoarakis S.E. Morris M.A. Buell G. Receptors Channels. 1995; 3: 283-289PubMed Google Scholar). The recent identification of residues contributing to the pore of the P2X2 receptor has also provided evidence for an intracellular location for the C-terminal part of the protein (17Rassendren F. Buell G. Newbolt A. North R.A. Surprenant A. EMBO J. 1997; 16: 3446-3454Crossref PubMed Scopus (179) Google Scholar). Amino acids in and around the second hydrophobic domain of the P2X2 receptor (residues 316–354) were mutated individually to cysteine, the proteins were expressed, and ATP-activated currents were measured. Inhibition of the current by polar methanethiosulfonate derivatives was then used to determine whether the residue was likely exposed to the aqueous solution. One substitution was identified (D349C) at which extracellularly applied methanethiosulfonate ethylamine inhibited the ATP-evoked current. However, the inhibition required channel opening. Because methanethiosulfonate ethylamine can permeate the open channel, these results indicate that Asp349 normally lies internal to the channel “gate.” The residue is close to the C-terminal end of the second hydrophobic region; this therefore implies that the C terminus of the protein is within the cytoplasm, because there is no further hydrophobic domain long enough to span the plasma membrane. These results are consistent with the topology for P2X receptors initially proposed (8Valera S. Hussy N. Evans R.J. Adami N. North R.A. Surprenant A. Buell G. Nature. 1994; 371: 516-519Crossref PubMed Scopus (894) Google Scholar, 9Brake A.J. Wagenbach M.J. Julius D. Nature. 1994; 371: 519-523Crossref PubMed Scopus (837) Google Scholar), but alternative models are also possible. For example, the data generated so far on cloned P2X receptors might as well be explained with a model in which only the second hydrophobic domain spans the plasma membrane, placing the entire N-terminal part of the protein extracellularly. Therefore, we have used theN -glycosylation site tagging approach to determine the precise topology of P2X2 receptors. During protein biosynthesis, extracellular domains of the protein are facing the lumen of the endoplasmic reticulum where they are able to be glycosylated. Due to the strict compartmentalization of N -glycosylating enzymes at the luminal face of the endoplasmic reticulum, it is possible to assess the extracellular location of different region of a given protein by N -glycosylation site tagging. This approach has been used to determine the topology of other membrane proteins including channels and transporters (18Hollmann M. Maron C. Heinemann S.F. Neuron. 1994; 13: 1331-1343Abstract Full Text PDF PubMed Scopus (382) Google Scholar, 19Bennett E.R. Kanner B.I. J. Biol. Chem. 1997; 272: 1203-1210Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). We first identified the endogenous N -glycosylation of P2X2 receptor, and then by mutagenesis obtained a form in which no natural N -glycosylation of the protein remained. The cDNA for this modified form of the receptor was then used as a background plasmid to engineer artificial N -glycosylation sites at different locations in the protein, with respect to the two hydrophobic domains. The extent of N -glycosylation was assayed by gel shift assay after functional expression of epitope-tagged forms of the P2X2 receptor inXenopus oocytes. Finally, we concatenated cDNAs and expressed the tandem constructs to show that the N and C termini reside on the same side of the plasma membrane. In all experiments, a P2X2receptor cDNA was used that carried a C-terminal epitope (DPGLNEYMPME), cloned into the expression vector pcDNA3 (Invitrogen) (17Rassendren F. Buell G. Newbolt A. North R.A. Surprenant A. EMBO J. 1997; 16: 3446-3454Crossref PubMed Scopus (179) Google Scholar, 20Kawashima E. Estoppey D. Fahmi D. Virginio C. Rees S. Surprenant A. North R.A. Receptors Channels. 1998; (in press)PubMed Google Scholar). Point mutations in P2X2 receptor cDNA were introduced by full-length polymerase chain reaction amplification of plasmid DNA with sense and antisense mutated primers.Pfu polymerase (Stratagene) was used to reduce the rate of contaminating mutations. Parental (wild type) DNA was digested with 10 units of Dpn I (New England Biolabs) for 1 h at 37 °C; 2.5 μl of the reaction was then directly used for transformation of Escherichia coli DH5α strain. DNA fragments carrying the mutation were digested with appropriate restriction enzymes and subcloned in a background vector that had not been amplified. All mutants were sequenced on both strands. A concatenated tandem P2X2 receptor cDNA was constructed in pBluescript vector. Briefly, two P2X2 cDNAs were modified by in-frame addition of an Eco RI site either at the 5′ or 3′ end of the coding sequence, respectively. These two cDNAs were ligated through the Eco RI site and a unique site from the backbone of the vector. The resulting construct contains two P2X2 subunits linked from the C terminus of one (-KGLAQL) to the N terminus of another (MVRRLA-); the deduced amino acid sequence at the junction site is -KGLGIRLA-. The mutant subunit P2X2-T336C (17Rassendren F. Buell G. Newbolt A. North R.A. Surprenant A. EMBO J. 1997; 16: 3446-3454Crossref PubMed Scopus (179) Google Scholar) was also constructed in pcDNA3; it also had the C terminus tag. To create the wild type-mutant dimer, a fragment from the Bluescript dimer was obtained by Bst EII digestion and subcloned into the Bst EII site of P2X2-T336C. The resulting plasmid encodes a wild type subunit followed by a mutant subunit and has the C-terminal epitope tag. Stage V-VI oocytes were removed from ovaries of anesthetized Xenopus laevis as described (21Evans R.J. Lewis C. Buell G. North R.A. Surprenant A. Mol. Pharmacol. 1995; 48: 178-183PubMed Google Scholar). Ovarian lobules were treated with 1 mg/ml collagenase (Sigma) for 2 h at room temperature in calcium-free solution (96 mm NaCl, 2 mm KCl, 1 mmMgCl2, 5 mm sodium pyruvate, 5 mmHepes, 10 units/ml penicillin, and 10 units/ml streptomycin). Healthy oocytes were selected and allowed to recover overnight prior to injection. Plasmid DNA was directly injected into oocyte nuclei. All plasmids were co-injected with an enhanced green fluorescent protein (EGFP)-containing plasmid (pEGFP, Life Technologies, Inc.). From 24 to 48 h after injection, EGFP-positive oocytes were sorted using a fluorescent microscope. Crude membrane fractions were prepared from batches of 12 oocytes; oocytes were homogenized in 240 μl of buffer H (100 mm NaCl, 20 mm Tris-HCl (pH 7.4), 1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, 20 μg/ml benzamidine hydrochloride, 200 μg/ml pepstatin A, 250 μg/ml aprotinin, 260 μg/ml α1-antitrypsin, 100 ng/ml soybean trypsin inhibitor, 200 μg/ml bestatin). Homogenates were shaken for 15 min at 4 °C and centrifuged at 14,000 × g for 2 min. After denaturation, membrane-containing supernatants were separated by SDS-polyacrylamide gel electrophoresis on a 8% Tris glycine gel (Novex). Separated proteins were electro-transferred onto nitrocellulose membranes (2 h, 25 V, 4 °C). Membranes were blocked with 5% milk, 0.2% Tween 20 in phosphate-buffered saline for 1 h at 37 °C. Monoclonal antibody against EYMPME (22Grussenmeyer T. Scheidtmann K.H. Hutchinson M.A. Eckhart W. Walter G. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 7952-7954Crossref PubMed Scopus (188) Google Scholar) was added at 1:1000 dilution and incubated for 16 h at 4 °C, in 2.5% milk, phosphate-buffered saline solution. A secondary antibody (goat anti-mouse IgG) conjugated to horseradish peroxidase was added at a 1:1000 dilution in 2.5% milk and incubated for 1 h at 37 °C. Proteins were detected by a chemiluminescent assay (ECL detection kit, Amersham Pharmacia Biotech). Membrane extracts (15 μl) were incubated with 10 μl of endoglycosidase H for 1 h at 37 °C in the presence of 0.2% SDS and 0.4% 2-β-mercaptoethanol. Transfection of human embryonic kidney 293 (HEK) cells, and electrophysiological recording from oocytes and HEK cells has been described previously (17Rassendren F. Buell G. Newbolt A. North R.A. Surprenant A. EMBO J. 1997; 16: 3446-3454Crossref PubMed Scopus (179) Google Scholar, 21Evans R.J. Lewis C. Buell G. North R.A. Surprenant A. Mol. Pharmacol. 1995; 48: 178-183PubMed Google Scholar). HEK cells were co-transfected with EGFP cDNA (0.5 μg/ml;CLONTECH) and the relevant P2X2receptor cDNA (1 μg/ml), and recordings were made 18–48 h later. All EGFPpositive (i.e. fluorescent) cells exhibited ATP-evoked current. We determined if it was possible to detect tagged P2X2 protein by Western blot from a crude oocyte membrane preparation. As shown in Fig.1, when oocytes were injected with the wild type P2X2 cDNA, the anti-EE antibody recognized a protein of apparent molecular mass around 65 kDa. The calculated size of the P2X2 receptor protein is 52.6 kDa; the observed difference is due to N -glycosylation because a mutated subunit lacking all three asparagines found at consensus glycosylation sites (N-X -S/T) migrated at approximately 50 kDa. The broad smear of the wild type Western blot may result from the fact that crude membrane preparations were used; these would include not only plasma membrane but also the membrane of intracellular organelles (endoplasmic reticulum and Golgi) in which the identity and length of sugars added to proteins might be variable. Uninjected or EGFP-injected oocytes showed no staining or very weak bands (Fig.1 B ). The P2X2 receptor primary sequence contains three consensus sites for N -glycosylation (N-X -S/T); these are Asn182, Asn239, and Asn298. To determine if any or all of these were glycosylated, we constructed a series of mutants in which one, two, or three sites were removed. As shown in Fig. 1 (C and D ), all three sites were glycosylated in Xenopus oocytes. This was evidenced by the progressive reduction of the apparent molecular mass of P2X2 protein as the number of N -glycosylation sites decreases (Fig. 1 C ). No differences were obvious among the three single mutants (N182S, N239S, and N298S) or among the three double mutants (N182S/N239S, N182S/N298S, and N239S/N298S), suggesting that all three asparagines are modified (Fig. 1 C ). This was further demonstrated by the effect of endoglycosidase H (Endo H). Membrane preparations of oocytes expressing wild type P2X2receptors and the three double mutants were treated with Endo H; in each case, this led to the appearance of a band that co-migrated with the triply mutant receptor (Δ3N-P2X2) (Fig.1 D ). A fraction of the protein in each case was resistant to Endo H; this may be because Endo H only hydrolyzes high mannose oligosaccharides specific to the endoplasmic reticulum and does not affect sugars added in other compartments such as the Golgi. Higher concentrations of enzyme or longer incubation times did not change the pattern of action of Endo H. In the case of the Δ3N-P2X2receptor, Endo H had no effect on the apparent molecular mass of the protein (data not shown). Mutants lacking one, two, or three N -glycosylation sites were transfected in HEK293 cell and tested electrophysiologically for the presence of ATP-induced currents. In HEK293 cells expressing each of the single mutants, ATP (30 μm) evoked inward currents that were not obviously different from those observed in cells expressing wild type receptors (Fig. 2). These results indicate both that full glycosylation is not required for normal function, and that the point mutations introduced do not critically disrupt the overall structure of the protein. However, in cells expressing the double mutations ATP evoked only very small currents, and on the triple mutant receptor (Δ3N-P2X2) ATP had no detectable effect." @default.
• W2002609528 created "2016-06-24" @default.
• W2002609528 creator A5007992856 @default.
• W2002609528 creator A5018570925 @default.
• W2002609528 creator A5027552825 @default.
• W2002609528 creator A5037029834 @default.
• W2002609528 creator A5045949797 @default.
• W2002609528 creator A5058480540 @default.
• W2002609528 creator A5084712627 @default.
• W2002609528 date "1998-06-01" @default.
• W2002609528 modified "2023-10-14" @default.
• W2002609528 title "Membrane Topology of an ATP-gated Ion Channel (P2X Receptor)" @default.
• W2002609528 cites W1590547533 @default.
• W2002609528 cites W1709464822 @default.
• W2002609528 cites W171632912 @default.
• W2002609528 cites W1775244163 @default.
• W2002609528 cites W1871656259 @default.
• W2002609528 cites W1974642350 @default.
• W2002609528 cites W1992660876 @default.
• W2002609528 cites W2018934217 @default.
• W2002609528 cites W2021184142 @default.
• W2002609528 cites W2022310648 @default.
• W2002609528 cites W2035189018 @default.
• W2002609528 cites W2038602542 @default.
• W2002609528 cites W2044120209 @default.
• W2002609528 cites W2048082309 @default.
• W2002609528 cites W2048201868 @default.
• W2002609528 cites W2049122170 @default.
• W2002609528 cites W2050821290 @default.
• W2002609528 cites W2052517937 @default.
• W2002609528 cites W2081355823 @default.
• W2002609528 cites W2123835637 @default.
• W2002609528 cites W2128693861 @default.
• W2002609528 cites W2156955619 @default.
• W2002609528 cites W2301973105 @default.
• W2002609528 cites W4211044690 @default.
• W2002609528 doi "https://doi.org/10.1074/jbc.273.24.15177" @default.
• W2002609528 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9614131" @default.
• W2002609528 hasPublicationYear "1998" @default.
• W2002609528 type Work @default.
• W2002609528 sameAs 2002609528 @default.
• W2002609528 citedByCount "115" @default.
• W2002609528 countsByYear W20026095282012 @default.
• W2002609528 countsByYear W20026095282013 @default.
• W2002609528 countsByYear W20026095282014 @default.
• W2002609528 countsByYear W20026095282015 @default.
• W2002609528 countsByYear W20026095282016 @default.
• W2002609528 countsByYear W20026095282017 @default.
• W2002609528 countsByYear W20026095282018 @default.
• W2002609528 countsByYear W20026095282019 @default.
• W2002609528 countsByYear W20026095282020 @default.
• W2002609528 countsByYear W20026095282021 @default.
• W2002609528 countsByYear W20026095282022 @default.
• W2002609528 countsByYear W20026095282023 @default.
• W2002609528 crossrefType "journal-article" @default.
• W2002609528 hasAuthorship W2002609528A5007992856 @default.
• W2002609528 hasAuthorship W2002609528A5018570925 @default.
• W2002609528 hasAuthorship W2002609528A5027552825 @default.
• W2002609528 hasAuthorship W2002609528A5037029834 @default.
• W2002609528 hasAuthorship W2002609528A5045949797 @default.
• W2002609528 hasAuthorship W2002609528A5058480540 @default.
• W2002609528 hasAuthorship W2002609528A5084712627 @default.
• W2002609528 hasBestOaLocation W20026095281 @default.
• W2002609528 hasConcept C119599485 @default.
• W2002609528 hasConcept C12554922 @default.
• W2002609528 hasConcept C127162648 @default.
• W2002609528 hasConcept C127413603 @default.
• W2002609528 hasConcept C145148216 @default.
• W2002609528 hasConcept C170493617 @default.
• W2002609528 hasConcept C178790620 @default.
• W2002609528 hasConcept C181911157 @default.
• W2002609528 hasConcept C184720557 @default.
• W2002609528 hasConcept C185592680 @default.
• W2002609528 hasConcept C31258907 @default.
• W2002609528 hasConcept C41008148 @default.
• W2002609528 hasConcept C41625074 @default.
• W2002609528 hasConcept C50254741 @default.
• W2002609528 hasConcept C55493867 @default.
• W2002609528 hasConcept C82617044 @default.
• W2002609528 hasConcept C86803240 @default.
• W2002609528 hasConcept C95444343 @default.
• W2002609528 hasConceptScore W2002609528C119599485 @default.
• W2002609528 hasConceptScore W2002609528C12554922 @default.
• W2002609528 hasConceptScore W2002609528C127162648 @default.
• W2002609528 hasConceptScore W2002609528C127413603 @default.
• W2002609528 hasConceptScore W2002609528C145148216 @default.
• W2002609528 hasConceptScore W2002609528C170493617 @default.
• W2002609528 hasConceptScore W2002609528C178790620 @default.
• W2002609528 hasConceptScore W2002609528C181911157 @default.
• W2002609528 hasConceptScore W2002609528C184720557 @default.
• W2002609528 hasConceptScore W2002609528C185592680 @default.
• W2002609528 hasConceptScore W2002609528C31258907 @default.
• W2002609528 hasConceptScore W2002609528C41008148 @default.
• W2002609528 hasConceptScore W2002609528C41625074 @default.
• W2002609528 hasConceptScore W2002609528C50254741 @default.
• W2002609528 hasConceptScore W2002609528C55493867 @default.
• W2002609528 hasConceptScore W2002609528C82617044 @default.
• W2002609528 hasConceptScore W2002609528C86803240 @default.

• next