Wilhelm G Lachnit , Ian B Oglesby , Joel R Gever , Marina Gever , Chiao-Chain Huang , Xing Cheng Li , Hong Jin , Joseph G McGivern , Anthony P.D.W Ford
{"title":"大鼠重组P2X3受体在稳定转染CHO-K1 tTA细胞中的表达调控","authors":"Wilhelm G Lachnit , Ian B Oglesby , Joel R Gever , Marina Gever , Chiao-Chain Huang , Xing Cheng Li , Hong Jin , Joseph G McGivern , Anthony P.D.W Ford","doi":"10.1016/S0165-1838(00)00120-X","DOIUrl":null,"url":null,"abstract":"<div><p>In this report, the regulatible expression by tetracycline of the rat recombinant P2X<sub>3</sub> receptor in stably transfected Chinese hamster ovary (CHO-K1) expressing the tetracycline-controlled transactivator (tTA) is described. cDNA encoding the rat P2X<sub>3</sub>-receptor was subcloned into pTRE (a tetracycline-repressible expression vector) which was used to transfect stably CHO-K1 tTA cells. Using whole cell patch clamp techniques, 100 μM ATP evoked inward currents of 2.9±1.6 nA in transfected cells grown in the absence of tetracycline (<em>tet−</em>). The P2X<sub>3</sub> receptor protein was detectable by immunoblot as early as 24 h and protein expression levels continued to increase as much as 192 h following activation of tTA by the removal of the antibiotic. Saturation binding isotherms using [<sup>35</sup>S]ATPγS yielded a p<em>K</em><sub>d</sub> of 8.2±0.1 and a <em>B</em><sub>max</sub> of 31.9±3.5 pmol/mg protein in <em>tet−</em> cell membranes and a p<em>K</em><sub>d</sub> of 8.1±0.1 and a <em>B</em><sub>max</sub> of 5.8±0.8 pmol/mg protein in <em>tet+</em> cell membranes. The agonist ligands 2MeSATP and αβMeATP displaced the binding of [<sup>35</sup>S]ATPγS in <em>tet−</em> cell membranes with very high affinity, yielding pIC<sub>50</sub> values of 9.4±0.2 and 7.5±0.2, respectively. In <em>tet+</em> cell membrane, displacement of [<sup>35</sup>S]ATPγS by 2MeSATP and αβMeATP was of much lower affinity (pIC<sub>50</sub> values of 7.8 and 6.2, respectively). ATP, ADP and UTP showed similar displacement of [<sup>35</sup>S]ATPγS binding in <em>tet−</em> and <em>tet+</em> cell membranes. In other experiments, cytosolic Ca<sup>2+</sup> was monitored using the fluorescent indicator, fluo-3. Increases in cytosolic Ca<sup>2+</sup> were elicited by 100 nM αβMeATP in <em>tet−</em> cells while no increases in cytosolic Ca<sup>2+</sup> were detected below 100 μM αβMeATP in either <em>tet+</em> cells or untransfected cells. These calcium responses to αβMeATP had a pEC<sub>50</sub> of 6.7 and were transient, returning to baseline within 120 s. Suramin produced concentration-dependent, parallel, dextral shifts of <em>E</em>/[<em>A</em>] curves to αβMeATP yielding a p<em>K</em><sub>B</sub> of 5.6. PPADS produced non-parallel, dextral shifts of <em>E</em>/[<em>A</em>] curves to αβMeATP which were insurmountable. These results show for the first time, expression of a functional, homomeric recombinant rat P2X<sub>3</sub> receptor which is under regulated expression in a stably transfected mammalian cell line.</p></div>","PeriodicalId":17228,"journal":{"name":"Journal of the autonomic nervous system","volume":"81 1","pages":"Pages 75-81"},"PeriodicalIF":0.0000,"publicationDate":"2000-07-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/S0165-1838(00)00120-X","citationCount":"8","resultStr":"{\"title\":\"Regulated expression of the rat recombinant P2X3 receptor in stably transfected CHO-K1 tTA cells\",\"authors\":\"Wilhelm G Lachnit , Ian B Oglesby , Joel R Gever , Marina Gever , Chiao-Chain Huang , Xing Cheng Li , Hong Jin , Joseph G McGivern , Anthony P.D.W Ford\",\"doi\":\"10.1016/S0165-1838(00)00120-X\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>In this report, the regulatible expression by tetracycline of the rat recombinant P2X<sub>3</sub> receptor in stably transfected Chinese hamster ovary (CHO-K1) expressing the tetracycline-controlled transactivator (tTA) is described. cDNA encoding the rat P2X<sub>3</sub>-receptor was subcloned into pTRE (a tetracycline-repressible expression vector) which was used to transfect stably CHO-K1 tTA cells. Using whole cell patch clamp techniques, 100 μM ATP evoked inward currents of 2.9±1.6 nA in transfected cells grown in the absence of tetracycline (<em>tet−</em>). The P2X<sub>3</sub> receptor protein was detectable by immunoblot as early as 24 h and protein expression levels continued to increase as much as 192 h following activation of tTA by the removal of the antibiotic. Saturation binding isotherms using [<sup>35</sup>S]ATPγS yielded a p<em>K</em><sub>d</sub> of 8.2±0.1 and a <em>B</em><sub>max</sub> of 31.9±3.5 pmol/mg protein in <em>tet−</em> cell membranes and a p<em>K</em><sub>d</sub> of 8.1±0.1 and a <em>B</em><sub>max</sub> of 5.8±0.8 pmol/mg protein in <em>tet+</em> cell membranes. The agonist ligands 2MeSATP and αβMeATP displaced the binding of [<sup>35</sup>S]ATPγS in <em>tet−</em> cell membranes with very high affinity, yielding pIC<sub>50</sub> values of 9.4±0.2 and 7.5±0.2, respectively. In <em>tet+</em> cell membrane, displacement of [<sup>35</sup>S]ATPγS by 2MeSATP and αβMeATP was of much lower affinity (pIC<sub>50</sub> values of 7.8 and 6.2, respectively). ATP, ADP and UTP showed similar displacement of [<sup>35</sup>S]ATPγS binding in <em>tet−</em> and <em>tet+</em> cell membranes. In other experiments, cytosolic Ca<sup>2+</sup> was monitored using the fluorescent indicator, fluo-3. Increases in cytosolic Ca<sup>2+</sup> were elicited by 100 nM αβMeATP in <em>tet−</em> cells while no increases in cytosolic Ca<sup>2+</sup> were detected below 100 μM αβMeATP in either <em>tet+</em> cells or untransfected cells. These calcium responses to αβMeATP had a pEC<sub>50</sub> of 6.7 and were transient, returning to baseline within 120 s. Suramin produced concentration-dependent, parallel, dextral shifts of <em>E</em>/[<em>A</em>] curves to αβMeATP yielding a p<em>K</em><sub>B</sub> of 5.6. PPADS produced non-parallel, dextral shifts of <em>E</em>/[<em>A</em>] curves to αβMeATP which were insurmountable. These results show for the first time, expression of a functional, homomeric recombinant rat P2X<sub>3</sub> receptor which is under regulated expression in a stably transfected mammalian cell line.</p></div>\",\"PeriodicalId\":17228,\"journal\":{\"name\":\"Journal of the autonomic nervous system\",\"volume\":\"81 1\",\"pages\":\"Pages 75-81\"},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2000-07-03\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://sci-hub-pdf.com/10.1016/S0165-1838(00)00120-X\",\"citationCount\":\"8\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Journal of the autonomic nervous system\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S016518380000120X\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"\",\"JCRName\":\"\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of the autonomic nervous system","FirstCategoryId":"1085","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S016518380000120X","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
Regulated expression of the rat recombinant P2X3 receptor in stably transfected CHO-K1 tTA cells
In this report, the regulatible expression by tetracycline of the rat recombinant P2X3 receptor in stably transfected Chinese hamster ovary (CHO-K1) expressing the tetracycline-controlled transactivator (tTA) is described. cDNA encoding the rat P2X3-receptor was subcloned into pTRE (a tetracycline-repressible expression vector) which was used to transfect stably CHO-K1 tTA cells. Using whole cell patch clamp techniques, 100 μM ATP evoked inward currents of 2.9±1.6 nA in transfected cells grown in the absence of tetracycline (tet−). The P2X3 receptor protein was detectable by immunoblot as early as 24 h and protein expression levels continued to increase as much as 192 h following activation of tTA by the removal of the antibiotic. Saturation binding isotherms using [35S]ATPγS yielded a pKd of 8.2±0.1 and a Bmax of 31.9±3.5 pmol/mg protein in tet− cell membranes and a pKd of 8.1±0.1 and a Bmax of 5.8±0.8 pmol/mg protein in tet+ cell membranes. The agonist ligands 2MeSATP and αβMeATP displaced the binding of [35S]ATPγS in tet− cell membranes with very high affinity, yielding pIC50 values of 9.4±0.2 and 7.5±0.2, respectively. In tet+ cell membrane, displacement of [35S]ATPγS by 2MeSATP and αβMeATP was of much lower affinity (pIC50 values of 7.8 and 6.2, respectively). ATP, ADP and UTP showed similar displacement of [35S]ATPγS binding in tet− and tet+ cell membranes. In other experiments, cytosolic Ca2+ was monitored using the fluorescent indicator, fluo-3. Increases in cytosolic Ca2+ were elicited by 100 nM αβMeATP in tet− cells while no increases in cytosolic Ca2+ were detected below 100 μM αβMeATP in either tet+ cells or untransfected cells. These calcium responses to αβMeATP had a pEC50 of 6.7 and were transient, returning to baseline within 120 s. Suramin produced concentration-dependent, parallel, dextral shifts of E/[A] curves to αβMeATP yielding a pKB of 5.6. PPADS produced non-parallel, dextral shifts of E/[A] curves to αβMeATP which were insurmountable. These results show for the first time, expression of a functional, homomeric recombinant rat P2X3 receptor which is under regulated expression in a stably transfected mammalian cell line.