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-Methylene ATP-Sensitive Phenotype
Glaxo Institute of Applied Pharmacology, Cambridge, United Kingdom (C.A.J., L.A.S., P.P.A.H., I.P.C.); and Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, United Kingdom (C.V., R.J.E.)
Investigation of rat recombinant P2X6 receptors has been limited because of the difficulty in obtaining functional expression in heterologous systems. In this study, we demonstrate glycosylation-dependent regulation of recombinant P2X6 receptor function and associated conferral of a novel phenotype that is sensitive to the P2X1 and P2X3 receptor agonist, 
-methylene ATP. In cells functionally expressing P2X6 receptors, ATP and -methylene ATP evoked slowly desensitizing inward currents (EC50 values, 0.5 and 0.6 µM, respectively) with slow kinetics of current decay on agonist washout. 2',3'-O-(2,4,6-trinitrophenyl ATP) and iso-pyridoxalphosphate-6-azophenyl-2'-5'-disulfonate were effective antagonists (IC50 values, 0.8 and 22 µM, respectively); however, suramin was relatively ineffective. Reverse transcription-polymerase chain reaction analysis confirmed the absence of other P2X receptor subunits. Western analysis of membrane fractions from functional and nonfunctional clones confirmed the presence of P2X6 at the cell membrane but revealed a difference in apparent molecular mass of immunoreactive products (
70 and 60 kDa, respectively). N-glycosidase F treatment of both functional and nonfunctional receptor cell membranes increased the electrophoretic mobilities of immunoreactive products, with both proteins migrating at 55 kDa, demonstrating an increased level of glycosylation of the P2X6 receptor in functional compared with nonfunctional cells. This study demonstrates that nonfunctional rat recombinant P2X6 receptors 1) are expressed on the membrane surface of human embryonic kidney cells and 2) are glycosylated. Expression of the novel functional receptor phenotype is associated with further glycosylation, resulting in an apparently larger molecular mass. These results suggest that P2X6 receptor subunits contribute to -methylene ATP sensitivity.
Address correspondence to: Iain Chessell, Neurology CEDD, GlaxoSmithKline, New Frontiers Science Park-North, Coldharbour Road, The Pinnacles, Harlow, Essex CM19 5AW, UK. E-mail: iain.p.chessell{at}gsk.com
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