Drosophila taste neurons as an agonist-screening platform for P2X receptors.

The P2X receptor family of ATP-gated cation channels are attractive drug targets for pain and inflammatory disease, but no subtype-selective agonists, and few partially selective agonists have been described to date. As proof-of-concept for the discovery of novel P2X receptor agonists, here we demonstrate the use of Drosophila taste neurons heterologously expressing rat P2X2 receptors as a screening platform. We demonstrate that wild-type rat P2X2 expressed in Drosophila is fully functional (ATP EC50 8.7 µM), and that screening of small (2 µl) volumes of a library of 80 adenosine nucleotide analogues is rapid and straightforward. We have determined agonist potency and specificity profiles for rat P2X2 receptors; triphosphate-bearing analogues display broad activity, tolerating a number of substitutions, and diphosphate and monophosphate analogues display very little activity. While several ATP analogues gave responses of similar magnitude to ATP, including the previously identified agonists ATPγS and ATPαS, we were also able to identify a novel agonist, the synthetic analogue 2-fluoro-ATP, and to confirm its agonist activity on rat P2X2 receptors expressed in human cells. These data validate our Drosophila platform as a useful tool for the analysis of agonist structure-activity relationships, and for the screening and discovery of novel P2X receptor agonists.

Purinergic P2X receptors: structural and functional features depicted by X-ray and molecular modelling studies.

The publication of the first crystal structures of the zebrafish P2X4 receptor in 2009 was a pivotal moment; for the first time, researchers were able to interpret their experimental data in a structural context. Several research groups immediately set about using the data to make molecular models of the better-understood mammalian P2X receptors, in order to design and interpret the results of new, more focused structure-function experiments. In 2012, the publication of the crystal structure of zebrafish P2X4 in the ATP-bound state gave further insights into the mechanism of ligand binding and its coupling to ion channel activation, initiating a new cycle of modelling, experimentation and interpretation. The purpose of this review is to describe our current understanding of the 3D-structure of P2X receptors, by highlighting the strengths and limitations of the zebrafish P2X4 crystal structures, discussing how the molecular models derived from them have been made, and what they have been used for, and explaining why crystal structures of mammalian P2X receptors are still needed to uncover the molecular mechanisms of differential agonist/antagonist potency, allosteric modulation, pore dilatation and desensitisation.