Biochemical characterization of $P2X_2$, an ATP-gated ion channel

Abstract

$P2X_2$ receptor functions as cation-selective ATP-gated ion channel, which is thought to be activated by ATP binding to the extracellular domain of $P2X_2$. We have produced the putative extracellular domain (ECD) of $P2X_2$, in a bacterial expression system. High amount of $P2X_2$-ECD was induced as a form of inclusion bodies by 200μM IPTG at 30℃ for 3 hours at a level of ~100 mg liter^{-1}$ of bacterial culture. The purified $P2X_2$-ECD by immobilized metal affinity chromatography was greater than 95% pure based on Coomassie staining and migrated with the expected mobility of 31 kDa protein. For protein refolding, cysteine sulfhydryl groups were blocked by sulfitolysis reaction and the protein was refolded by the gradual removal of urea. To remove hexahistidine tag, thrombin cleavage of $P2X_2$-ECD was achieved and finally, a single band of the predicted molecular weight of 29 kDa shifted from 31 kDa on Coomassie stained SDS-PAGE. The functionality of refolded $P2X_2$-ECD was assayed using rapid filtration ATP filter binding. In saturation experiment, the Scatchard plot derived from the data gave a $K_d$ value of 11.3±0.30nM, $B_max$ was $221±0.34pmolmg^{-1}$ protein, and Hill slope was 1.05±0.16. In competition binding studies using antagonists, suramin and cibacron blue inhibited the specific binding of [$3^H$]ATP to $P2X_2$-ECD. Several divalent cations affected the binding of ATP to $P2X_2$-ECD directly. $Ca^{2+}, Mg^2$ and $Zn^{2+}$ at concentrations between 0.1 μM and 10 mM produce a concentration-dependent increase in the binding of [$3^H$]ATP. The magnitude of the increase in binding was the greatest with $ZnCl_2$. But $MnCl_2$ did not affect the binding of [$3^H$]ATP at any concentration we have tested. In order to demonstrate that ATP directly binds to the refolded $P2X_2$-ECD, we carried out photoaffinity labelling by [$\alpha ^{32}P$]ATP to $P2X_2$-ECD. $P2X_2$-ECD of 29 kDa was specifically labeled by 2 nM of [$α^32P$]ATP. Denatured $P2X_2$-EC...