Abstract
P2X2 receptors (P2X2R) exhibit a slow desensitization during the initial ATP application and a progressive, calcium-dependent increase in rates of desensitization during repetitive stimulation. This pattern is observed in whole-cell recordings from cells expressing recombinant and native P2X2R. However, desensitization is not observed in perforated-patched cells and in two-electrode voltage clamped oocytes. Addition of ATP, but not ATPγS or GTP, in the pipette solution also abolishes progressive desensitization, whereas intracellular injection of apyrase facilitates receptor desensitization. Experiments with injection of alkaline phosphatase or addition of staurosporine and ATP in the intracellular solution suggest a role for a phosphorylation-dephosphorylation in receptor desensitization. Mutation of residues that are potential phosphorylation sites identified a critical role of the S363 residue in the intracellular ATP action. These findings indicate that intracellular calcium and ATP have opposing effects on P2X2R gating: calcium allosterically facilitates receptor desensitization and ATP covalently prevents the action of calcium. Single cell measurements further revealed that intracellular calcium stays elevated after washout in P2X2R-expressing cells and the blockade of mitochondrial sodium/calcium exchanger lowers calcium concentrations during washout periods to basal levels, suggesting a role of mitochondria in this process. Therefore, the metabolic state of the cell can influence P2X2R gating.
Highlights
Extracellular ATP-gated P2X receptor channels (P2XRs) are composed of three homologous or heterologous subunits; in mammals seven subunits, designated P2X1-7, have been identified
The cysteine-rich head domain located above the ATP-binding cleft of P2X1R [3] and the negatively charged aspartate residues located below the ATP-binding site of P2X3R [4] play important roles in the transition from the open to the desensitized state
We recently reported that P2X2 receptors (P2X2R) undergoes a progressive calcium-dependent desensitization during repetitive ATP application, a phenomenon termed use-dependent desensitization (UDD)
Summary
Extracellular ATP-gated P2X receptor channels (P2XRs) are composed of three homologous or heterologous subunits; in mammals seven subunits, designated P2X1-7, have been identified. It is well established that the N- and C-terminal domains provide the structural elements involved in controlling the kinetics of receptor desensitization These studies included work with splice forms of P2X2Rs [7,8,9], and mutagenesis studies focused on identification of the residues that account for those effects [10,11]. P2XRs are highly regulated channels and can respond to changes in their extracellular and intracellular environment, which further contributes to the complexity in their gating properties [2] This includes the roles of bath and intracellular calcium in receptor gating; its main effect is observed in a millimolar concentration range causing inhibition of agonist-induced currents [19], but it has been suggested that Ca2+ facilitates recovery from desensitization of P2X3Rs [20]. These data suggest that P2X2Rs represent an important functional crosslink between extracellular and intracellular ATP and calcium signaling and provide integration of cellular responses to these ligands
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