Desensitization masks nanomolar potency of ATP for the P2X1 receptor.
ATP-gated P2X1 receptors feature fast activation and fast desensitization combined with slow recovery from desensitized states. Here, we exploited a non-desensitizing P2X2/P2X1 chimera that includes the entire P2X1 ectodomain (Werner, P., Seward, E. P., Buell, G. N., and North, R. A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 15485-15490) to obtain a macroscopic representation of intrinsic receptor kinetics without bias arising from the overlap of channel activation and desensitization. From the stationary currents made amenable to analysis by this chimera, an EC50 for ATP of 3.3 nM was derived, representing a >200- and >7000-fold higher ATP potency than observed for the parental P2X1 and P2X2A receptors, respectively. Also, other agonists activated the P2X2/P2X1 chimera with nanomolar EC50 values ranging from 3.5 to 73 nM in the following rank order: 2-methylthio-ATP, 2',3'-O-(4-benzoylbenzoyl)-ATP, alpha,beta-methylene-ATP, adenosine 5'-O-(3-thiotriphosphate). Upon washout, the P2X2/P2X1 chimera deactivated slowly with a time constant (ranging from 63 to 2.5 s) that is inversely related to the EC50 value for the corresponding agonist. This suggests that deactivation time courses reflect unbinding rates, which by themselves define agonist potencies. The P2X2/P2X1 chimera and the P2X1 receptor possess virtually identical sensitivity to inhibition by the P2X1 receptor-selective antagonist NF279, a suramin analog. These results suggest that the P2X1 ectodomain confers nanomolar ATP sensitivity, which, within the wild-type P2X1 receptor, is obscured by desensitization such that only a micromolar ATP potency can be deduced from peak current measurements, representing an amalgam of activation and desensitization.
Rettinger
Jürgen
J
Max Planck Institute of Biophysics, Marie-Curie-Strasse 13-15, D-60439 Frankfurt am Main, Germany.
Schmalzing
Günther
G
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000494:pharmacology; D000345:Affinity Labels / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D007700:Kinetics; D008954:Models, Biological; D008956:Models, Chemical; D009865:Oocytes / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D018048:Receptors, Purinergic P2 / Q000737:chemistry / Q000378:metabolism*; D058469:Receptors, Purinergic P2X; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013498:Suramin / Q000031:analogs & derivatives* / Q000494:pharmacology; D013873:Thionucleotides / Q000494:pharmacology; D013997:Time Factors; D014981:Xenopus
D000345:Affinity Labels; C114657:NF 279; D018048:Receptors, Purinergic P2; D058469:Receptors, Purinergic P2X; D011993:Recombinant Fusion Proteins; D013873:Thionucleotides; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; D013498:Suramin; D000255:Adenosine Triphosphate; C002630:alpha,beta-methyleneadenosine 5'-triphosphate; C008501:2-methylthio-ATP