Inactivation of P2X2 purinoceptors by divalent cations

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Abstract
1 P2X2 channels are activated by extracellular ATP. Despite being commonly described as non-desensitizing, P2X2 receptors do desensitize or inactivate. In the unspliced, 472 amino acid isoform of the P2X2 receptor, inactivation required membrane disruption and the presence of extracellular Ca2+. 2 The ability to inactivate whole-cell currents developed slowly after breaking in. In contrast, currents from excised patches exhibited rapid (∼100 ms) inactivation with a dependence on extracellular Ca2+, ATP and voltage. 3 The inactivation rate increased with the fourth power of [Ca2+] suggesting that the functional channel may be a tetramer. Ca2+ had both a higher affinity and a larger Hill coefficient for inactivation than Mg2+, Ba2+ or Mn2+. Trivalent cations at concentrations up to the solubility product of ATP had no effect. The change in apparent co-operativity with ionic species suggests the presence of experimentally unresolved ligand-insensitive kinetic steps. 4 Based on the weak voltage dependence of inactivation and the lack of effect of intracellular Ca2+ buffers, the Ca2+-binding sites are probably located near the extracellular surface of the membrane. 5 The recovery from inactivation was slow, with a time constant of ∼7 min. 6 Ca2+-sensitive inactivation only appeared when the membrane was disrupted in some manner. Treatment with actin and microtubule reagents did not induce inactivation, suggesting that an intact cytoskeleton is not necessary. 7 Inactivation rates observed in different patch configurations suggest that the induction of Ca2+-dependent inactivation was due to the loss of a diffusible cofactor located in the membrane or the cytoplasm.