大鼠重组P2X3受体在稳定转染CHO-K1 tTA细胞中的表达调控

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
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引用次数: 8

摘要

本报告描述了四环素在稳定转染的表达四环素控制的转激活物(tTA)的中国仓鼠卵巢(CHO-K1)中调节大鼠重组P2X3受体的表达。编码大鼠p2x3受体的cDNA被亚克隆到pTRE(四环素抑制表达载体)中,该载体用于稳定转染CHO-K1 tTA细胞。使用全细胞膜片钳技术,100 μM ATP在没有四环素(tet−)的转染细胞中产生2.9±1.6 nA的内向电流。P2X3受体蛋白早在24小时就可以通过免疫印迹检测到,在tTA被去除抗生素激活后的192小时内,蛋白表达水平继续增加。用[35S]ATPγS进行饱和结合等温线测定,tet -细胞膜的pKd为8.2±0.1,Bmax为31.9±3.5 pmol/mg, tet+细胞膜的pKd为8.1±0.1,Bmax为5.8±0.8 pmol/mg。激动剂配体2MeSATP和αβMeATP以非常高的亲和力取代了[35S] atp - γ - s在tet -细胞膜上的结合,pIC50值分别为9.4±0.2和7.5±0.2。在tet+细胞膜中,2MeSATP和αβMeATP对[35S] atp - γ - s的置换亲和力较低(pIC50值分别为7.8和6.2)。ATP、ADP和UTP在tet -和tet+细胞膜上对[35S]ATP - γ s结合表现出类似的位移。在其他实验中,使用荧光指示剂fluo-3监测胞质Ca2+。100 μM αβMeATP诱导tet+细胞胞质Ca2+升高,而100 μM αβMeATP诱导tet+细胞和未转染tet+细胞胞质Ca2+均未升高。这些钙对αβMeATP的反应pEC50为6.7,并且是短暂的,在120秒内恢复到基线。苏拉明产生浓度依赖性的E/[A]曲线向αβMeATP的平行右移,产生5.6的pKB。PPADS产生了E/[A]曲线向αβMeATP的非平行、右移,这是不可克服的。这些结果首次表明,在稳定转染的哺乳动物细胞系中,具有功能的、同源的重组大鼠P2X3受体得到了调控表达。
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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.

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