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Pharmacological and Biophysical Properties of the Human P2X₅ Receptor

Institute of Molecular Physiology, University of Sheffield, Sheffield, United Kingdom (X.B., L.-H.J., H.L.W., M.K., A.S., R.A.N.); and Autonomic Neuroscience Institute, Royal Free and University College Medical School, London, United Kingdom (G.B.)

We constructed a full-length human P2X₅ purinoceptor cDNA by incorporating a sequence corresponding to exon 10, which is missing in cDNAs cloned previously from human tissues. We studied the functional properties by patch-clamp recording and fluorescence imaging after expression in human embryonic kidney 293 cells. ATP (1–100 µM; half-maximal current at 4 µM) elicited inward currents at –60 mV; these persisted during brief (2 s) applications but declined during longer applications. The peak current was dependent on the holding potential and showed little rectification; however, both the desensitization during the application and the decline in the current when ATP was washed out were slower at +30 mV than at –60 mV. 2',3'-O-(4-Benzoyl)-benzoyl-ATP and -methylene-ATP mimicked the action of ATP (half-maximal concentrations 6 and 161 µM, respectively). The currents were inhibited by suramin, pyridoxal-5-phosphate-6-azo-2',4'-disulfonic acid and Brilliant Blue G, with half-maximal inhibition at 3, 0.2, and 0.5 µM, respectively; 2',3'-O-(2',4',6'-trinitrophenol)-ATP (1 µM) was ineffective. Removing divalent cations did not significantly alter ATP concentration-response curves. Reversal potential measurements showed that the human P2X₅ receptor was permeable to calcium (P_Ca/P_Na = 1.5) and N-methyl-D-glucamine (NMDG) (P_NMDG/P_Na = 0.4); it was also permeable to chloride (P_Cl/P_Na = 0.5) but not gluconate (P_gluc/P_Na = 0.01) ions. The permeability to NMDG developed as quickly as the channel opened, in contrast to the P2X₇ receptor where the NMDG permeability develops over several seconds. Cells expressing human P2X₅ receptors also rapidly accumulated the propidium dye YO-PRO-1 in response to ATP.

Address correspondence to: R. Alan North, Institute of Molecular Physiology, University of Sheffield, Alfred Denny Building, Western Bank, Sheffield S10 2TN, UK. E-mail: r.a.north{at}sheffield.ac.uk

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