Effects of extracellular pH on agonism and antagonism at a recombinant P2X(2) receptor

1 Under voltage-clamp conditions, the activity of agonists and antagonists at a recombinant P2X(2) receptor expressed in Xenopus oocytes was examined at different levels of extracellular pH (pH(e)). 2 In normal Ringer (Mg2(+) ions absent), the amplitude of submaximal inward currents to ATP was increased by progressively lowering pH(e) (8.0-5.5). ATP-responses reached a maximum at pH 6.5 with a 5 fold increase in ATP-affinity; the apparent pK(a) was 7.05+/-0.05. 3 Receptor affinity for ATP was lowered when extracellular Ca2+ ions were replaced with equimolar Mg2+ ions. However, the amplitude of the ATP-responses was still enhanced under acidic conditions, reaching maximal activity at pH 6.5 with a 5 fold increase in ATP-affinity; the apparent pK(a) was 7.35+/-0.05. 4 ATP species present in the superfusate (for the above ionic conditions and pH levels) were calculated to determine the forms of ATP which activate P2X(2) receptors: possible candidates include HATP, CaHATP and MgHATP. However, levels of these protonated species increase below pH 6.5, suggesting that receptor protonation rather than agonist protonation is more important. 5 The potency order for agonists of P2X(2) receptors was: ATP>2-MeS-ATP greater than or equal to ATP gamma S> ATP alpha S>>CTP greater than or equal to BzATP, while other nucleotides were inactive. EC50 and n(H) values for full agonists were determined at pH 7.4 and re-examined at pH 6.5. Extracellular acidification increased the affinity by approximately 5 fold for full agonists (ATP, 2-MeSATP, ATP gamma S and ATP alpha S), without altering the potency order. 6 The potency order for antagonists at P2X(2) receptors was: Reactive blue-2> trinitrophenol ATP greater than or equal to Palatine fast black greater than or equal to Coomassie brilliant blue greater than or equal to PPADS>suramin (at pH 7.4). IC50 values and slopes of the inhibition curves were re-examined at different pH levels. Only blockade by suramin was affected significantly by extracellular acidification (ICS, values: 10.4+/-2 mu M, at pH 7.4; 78+/-5 nM, at pH 6.5; 30+/-6 nM, at pH 5.5). 7 In summary, a lowered pH, enhanced the activity of all agonists at P2X(2) receptors but, with the exception of suramin, not antagonists. Since a lowered pH(e) is also known to enhance agonist activity at P-2X receptors on sensory neurones containing P2X(2) transcripts, the sensitization by metabolic acidosis of native P-2X receptors containing P2X(2) subunits may have a significant effect on purinergic cell-to-cell signalling.