Regulated expression of the rat recombinant P2X(3) receptor in stably transfected CHO-K1 tTA cells

In this report, the regulatible expression by tetracycline of the rat recom binant 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 tetracycli ne-repressible expression vector) which was used to transfect stably CHO-KI tTA cells. Using whole cell patch clamp techniques, 100 mu M ATP evoked in ward currents of 2.9 +/- 1.6 nA in transfected cells grown in the absence o f tetracycline (tet(-)). The P2X(3) receptor protein was detectable by immu noblot 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 antibio tic. Saturation binding isotherms using [S-35]ATP gamma S yielded a pK(d) o f 8.2 +/- 0.1 and a B-max of 31.9 +/- 3.5 pmol/mg protein in tet- cell memb ranes 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 d isplaced the binding of [S-35]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, resp ectively. In tet+ cell membrane, displacement of [S-35]ATP gamma S by 2MeSA TP and alpha beta MeATP was of much lower affinity (pIC(50) values of 7.8 a nd 6.2, respectively). ATP, ADP and UTP showed similar displacement of [S-3 5]ATP gamma S binding in tet- and tet+ cell membranes. In other experiments , cytosolic Ca2+ was monitored using the fluorescent indicator, fluo-3. Inc reases in cytosolic Ca2+ were elicited by 100 nM alpha beta MeATP in tet- c ells while no increases in cytosolic Ca2+ were detected below 100 mu M alph a beta MeATP in either tet+ cells or untransfected cells. These calcium res ponses to alpha beta MeATP had a pEC(50) of 6.7 and were transient, returni ng to baseline within 120 s. Suramin produced concentration-dependent, para llel, dextral shifts of EI[A] curves to alpha beta MeATP yielding a pK(B) o f 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 tim e, expression of a functional, homomeric recombinant rat P2X(3) receptor wh ich is under regulated expression in a stably transfected mammalian cell li ne. (C) 2000 Elsevier Science B.V. All rights reserved.