Regulated expression of the rat recombinant P2X(3) receptor in stably transfected CHO-K1 tTA cells.
In this report, the regulatable expression by tetracycline of the rat recombinant P2X(3) receptor in stably transfected Chinese hamster ovary (CHO-K1) expressing the tetracycline-controlled transactivator (tTA) is described. cDNA encoding the rat P2X(3)-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 microM ATP evoked inward currents of 2.9+/-1.6 nA in transfected cells grown in the absence of tetracycline (tet-). The P2X(3) 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]ATP gamma S yielded a pK(d) of 8.2+/-0.1 and a B(max) of 31.9+/-3.5 pmol/mg protein in tet- cell membranes and a pK(d) of 8.1+/-0.1 and a B(max) of 5.8+/-0.8 pmol/mg protein in tet+ cell membranes. The agonist ligands 2MeSATP and alpha beta MeATP displaced the binding of [35S]ATP gamma S in tet- cell membranes with very high affinity, yielding pIC(50) values of 9.4+/-0.2 and 7.5+/-0. 2, respectively. In tet+ cell membrane, displacement of [35S]ATP gamma S by 2MeSATP and alpha beta MeATP was of much lower affinity (pIC(50) values of 7.8 and 6.2, respectively). ATP, ADP and UTP showed similar displacement of [35S]ATP gamma S binding in tet- and tet+ cell membranes. In other experiments, cytosolic Ca(2+) was monitored using the fluorescent indicator, fluo-3. Increases in cytosolic Ca(2+) were elicited by 100 nM alpha beta MeATP in tet- cells while no increases in cytosolic Ca(2+) were detected below 100 microM alpha beta MeATP in either tet+ cells or untransfected cells. These calcium responses to alpha beta MeATP had a pEC(50) of 6.7 and were transient, returning to baseline within 120 s. Suramin produced concentration-dependent, parallel, dextral shifts of E/[A] curves to alpha beta MeATP yielding a pK(B) of 5.6. PPADS produced non-parallel, dextral shifts of E/[A] curves to alpha beta MeATP which were insurmountable. These results show for the first time, expression of a functional, homomeric recombinant rat P2X(3) receptor which is under regulated expression in a stably transfected mammalian cell line.
Lachnit
W G
WG
Neurobiology Unit, Center for Biological Research, Roche Bioscience, 3401 Hillview Avenue, Palo Alto, CA 94304, USA. wilhelm.lachnit@roche.com
Oglesby
I B
IB
Gever
J R
JR
Gever
M
M
Huang
C
C
Li
X C
XC
Jin
H
H
McGivern
J G
JG
Ford
A P
AP
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D006224:Cricetinae; D004594:Electrophysiology; D005727:Ganglia, Spinal / Q000187:drug effects / Q000502:physiology; D005786:Gene Expression Regulation / Q000502:physiology*; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D018408:Patch-Clamp Techniques; D011869:Radioligand Assay; D051381:Rats; D017207:Rats, Sprague-Dawley; D018048:Receptors, Purinergic P2 / Q000096:biosynthesis* / Q000235:genetics; D058477:Receptors, Purinergic P2X3; D013498:Suramin / Q000494:pharmacology; D013752:Tetracycline / Q000494:pharmacology; D014162:Transfection / Q000235:genetics; D014981:Xenopus
D000900:Anti-Bacterial Agents; C546410:P2rx3 protein, rat; D018048:Receptors, Purinergic P2; D058477:Receptors, Purinergic P2X3; D013498:Suramin; D013752:Tetracycline; D002118:Calcium