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 (P2X(2) receptors) were neutralized by site-directed mutagenesis, and the properties of the mutants were electrophysiologically characterized using Xenopus oocytes. When Asp(315) 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 Lys(324), which is involved in a Walker type A ATP-binding sequence, Lys(366), Tyr(330), or Asn(333). With D315V channels, the sensitivities to other agonists (ADP, ATP gamma S, and 2-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 Asp(315), which is assumed to be present in the extracellular region near the M2 segment of P2X(2) receptor/channels, serves to maintain agonist sensitivity. (C) 1998 Academic Press.