Conserved negatively charged residues are not required for ATP action at P2X(1) receptors.
The role of conserved negatively charged aspartic (D) and glutamic (E) acid residues within the ectodomain of the human P2X(1) receptor were examined by alanine substitution mutagenesis. Effects on ATP potency and cell surface localisation were assessed in Xenopus oocytes using the two electrode voltage clamp technique and cell surface biotinylation. Of the eleven residues tested no major shifts in ATP potency were observed with EC(50) values for ATP ranging from 0.8 to 4.3 microM (compared to 1 microM ATP for wild-type P2X(1) receptors). Peak current amplitudes for mutants D86A and D264A where reduced by approximately 90% due to a corresponding reduction in both total protein and cell surface expression. These results demonstrate that individual conserved negatively charged amino acids are not essential for ATP recognition by the human P2X(1) receptor and coordinated binding of the positive charge on magnesium complexed ATP by negatively charged amino acids is not required.
Ennion
S J
SJ
Department of Cell Physiology and Pharmacology, University of Leicester, Leicester LE1 9HN, United Kingdom.
Ritson
J
J
Evans
R J
RJ
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics / Q000502:physiology*; D019921:Biotinylation; D002462:Cell Membrane / Q000378:metabolism; D017124:Conserved Sequence; D004305:Dose-Response Relationship, Drug; D004553:Electric Conductivity; D018698:Glutamic Acid / Q000235:genetics / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D018048:Receptors, Purinergic P2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D058469:Receptors, Purinergic P2X; D017386:Sequence Homology, Amino Acid; D014981:Xenopus
D018048:Receptors, Purinergic P2; D058469:Receptors, Purinergic P2X; D001224:Aspartic Acid; D018698:Glutamic Acid; D000255:Adenosine Triphosphate