Vol. 62, Issue 4, 957-966, October 2002

Sensitization by Extracellular Ca²⁺ of Rat P2X₅ Receptor and Its Pharmacological Properties Compared with Rat P2X₁.

Scott S. Wildman, Sean G. Brown, Mary Rahman, Carole A. Noel, Linda Churchill, Geoffrey Burnstock, Robert J. Unwin, and Brian F. King

Department of Physiology (Centre for Nephrology) (S.S.W., C.A.N., L.C., R.J.U., B.F.K), and Autonomic Neuroscience Institute (G.B., R.J.U., B.F.K.), Royal Free and University College Medical School, London, United Kingdom; and Department of Clinical Pharmacology, The Rayne Institute (S.G.B.) and Department of Anatomy and Developmental Biology (M.R., G.B.), University College London, London, United Kingdom

The recombinant rat P2X₅ (rP2X₅) receptor, a poorly understood ATP-gated ion channel, was studied under voltage-clamp conditions and compared with the better understood homomeric rP2X₁ receptor with which it may coexist in vivo. Expressed in defolliculated Xenopus laevis oocytes, rP2X₅ responded to ATP with slowly desensitizing inward currents that, for successive responses, ran down in the presence of extracellular Ca²⁺ (1.8 mM). Replacement of Ca²⁺ with either Ba²⁺ or Mg²⁺ prevented rundown, although agonist responses were very small, whereas reintroduction of Ca²⁺ for short periods of time (<300 s) before and during agonist application yielded consistently larger responses. Using this Ca²⁺-pulse conditioning, rP2X₅ responded to ATP and other nucleotides (ATP, 2-methylthio-ATP, adenosine-5'-O-(thiotriphosphate), 2'-&-3'-O-(4-benzoylbenzoyl)-ATP, ,-methylene-ATP, P¹-P⁽⁴⁾-diadenosine-5'-phosphate, and more) with pEC₅₀ values within 1 log unit of respective determinations for rP2X₁. Only GTP was selective for rP2X₅, although 60-fold less potent than ATP. At rP2X₅, lowering extracellular pH reduced the potency and efficacy of ATP, whereas extracellular Zn²⁺ ions (0.1-1000 µM) potentiated then inhibited ATP responses in a concentration-dependent manner. However, these modulators affected rP2X₁ receptors in subtly different ways-with increasing H⁺ and Zn²⁺ ion concentrations reducing agonist potency. For P2 receptor antagonists, the potency order at rP2X₅ was pyridoxal-5-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) > 2',3'-O-(2,4,6-trinitrophenyl)ATP (TNP-ATP) > suramin > reactive blue 2 (RB-2) > diinosine pentaphosphate (Ip₅I). In contrast, the potency order at rP2X₁ was TNP-ATP = Ip₅I > PPADS > suramin = RB-2. Thus, the Ca²⁺-sensitized homomeric rP2X₅ receptor is similar in agonist profile to homomeric rP2X₁although it can be distinguished from the latter by GTP agonism, antagonist profile, and the modulatory effects of H⁺ and Zn²⁺ ions.