Effects of extracellular pH on agonism and antagonism at a recombinant P2X₂ receptor

Abstract

1 Under voltage-clamp conditions, the activity of agonists and antagonists at a recombinant P2X 2 receptor expressed in Xenopus oocytes was examined at di erent levels of extracellular pH (pH e ). 2 In normal Ringer (Mg 2+ 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-a nity; the apparent pK a was 7.05+0.05. 3 Receptor a nity for ATP was lowered when extracellular Ca 2+ ions were replaced with equimolar Mg 2+ 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-a nity; 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: ATP42-MeS-ATP5ATPgS4 ATPaS44CTP5BzATP, while other nucleotides were inactive. EC 50 and n H values for full agonists were determined at pH 7.4 and re-examined at pH 6.5. Extracellular acidi®cation increased the a nity by approximately 5 fold for full agonists (ATP, 2-MeSATP, ATPgS and ATPaS), without altering the potency order. 6 The potency order for antagonists at P2X 2 receptors was: Reactive blue-24trinitrophenol-ATP5Palatine fast black5Coomassie brilliant blue5PPADS4suramin (at pH 7.4). IC 50 values and slopes of the inhibition curves were re-examined at di erent pH levels. Only blockade by suramin was a ected signi®cantly by extracellular acidi®cation (IC 50 values: 10.4+2 mM, at pH 7.4; 78+5 nM, at pH 6.5; 30+6 nM, at pH 5.5). 7 In summary, a lowered pH e 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 signi®cant e ect on purinergic cell-to-cell signalling.

Receptor-Like Activity Evoked by Extracellular ADP in Arabidopsis Root Epidermal Plasma Membrane

“…The pH dependence of animal ionotropic purinoreceptors varies with isoform (King et al, 1997;Wildman et al, 1999;Wirkner et al, 2008). Here, the extracellular ADP-induced [Ca 2+ ] cyt response was operative over a wide pH range but activity at more neutral to alkaline pH suggests that extracellular ADP (and ATP) signaling could operate during stress responses in which the cytosol acidifies and apoplastic pH elevates above its normal acid value, such as hypoosmotic stress or elicitor challenge (Mathieu et al, 1996;Pugin et al, 1997;Beffagna et al, 2005).…”

Acid potentiation of the capsaicin receptor determined by a key extracellular site

“…An important component of this proalgesic response is local acidosis, namely, a reduction in extracellular pH to levels below the physiological norm of Ϸ7.6 (2). Protons are capable of modulating the activity of a number of receptors and ion channels expressed by primary afferent nociceptors, including acid-sensitive channels of the degenerin family (3), ATPgated channels (4,5), and vanilloid receptors (6,7). Analyses of native and cloned vanilloid receptors suggest that they play a role in mediating sustained proton responses in vivo (8)(9)(10)(11)(12).…”

In search of selective P2 receptor ligands: interaction of dihydropyridine derivatives at recombinant rat P2X2 receptors

“…Antagonist activity at recombinant P2X receptors-Xenopus oocytes were harvested and prepared as previously described (King et al, 1997). Defolliculated oocytes were injected cytosolically with 40 nl of a solution of cRNA of rat P2X 4 receptors (1 μg/ml) or rat P2X 2 receptors (0.002 μg/ml) incubated for 24 h at 18°C in Barth's solution and kept for up to 12 days at 4°C until used in electrophysiological experiments.…”