An aspartic acid residue near the second transmembrane segment of ATP receptor/channel regulates agonist sensitivity

Charged or polarized amino acid residues near or within the second transmembrane (M2) segment of neuronal ATP receptor/channels (P2X₂ receptors) were neutralized by site-directed mutagenesis, and the properties of the mutants were electrophysiologically characterized using Xenopus oocytes. When Asp³¹⁵ was substituted with Val (D315V), the sensitivity to ATP was reduced by about 60-fold. The sensitivity to ATP was not affected by the neutralization of Lys³²⁴, which is involved in a Walker type A ATP-binding sequence, Lys³⁶⁶, Tyr³³⁰, or Asn³³³. With D315V channels, the sensitivities to other agonists (ADP, ATPγS, and a-methylthio ATP) were also reduced. The sensitivities to antagonists (suramin and Cibacron Blue F3GA) were, however, not affected by this neutralization. The results suggest that Asp³¹⁵, which is assumed to be present in the extracellular region near the M2 segment of P2X₂ receptor/channels, serves to maintain agonist sensitivity.