Purification and recognition of recombinant mouse P2X1 receptors expressed in a baculovirus system

The hexahistidine-tagged mouse P2X(1) receptor (H-mP2X(1)R), an ATP-gated ion channel receptor, was expressed in a baculovirus system using the pAcHLT-B transfer vector containing a hexahistidine tag. Both widely used denaturing (8M urea) acid nondenaturing (such as 1% Triton X-100) solubilization conditions were compared, resulting in about 30% of the P2X(1) receptors being solubilized (S1). However, at pH 13 most of the n-mP2X(1)R from the initially insoluble pellet fraction was solubilized (S2) and remained in the soluble fraction (S3) after dialyzing against a nondenaturing buffer. H-mP2X(1)Rs were purified sequentially through cobalt and ATP affinity columns. Receptors purified from S3 had higher purity than those from S1 (i.e., similar to 90% vs. similar to 75%). Circular dichroism spectra indicated identical protein secondary structures of the receptors from both sources. Autoradiographic data showed that the purified receptors from S3 had higher affinity for 8-azido-ATP-gamma-P-32 than the receptors from S1. The binding of 8-azido-ATP-gamma-P-32 to H-mP2X(1)R was inhibited by ATP-gamma -S, alpha,beta -me-ATP, and PPADS, but not by a nucleoside analog (N-6-methyl-2'-deoxy-adenosine). In the presence of 2 mM Ca2+ or Mg2+ the binding was increased, but not when using a partially purified receptor fraction, in which unidentified proteins bound 8-azido ATP-gamma-P-32 or were phosphorylated at 4 degreesC in the presence of 2 mM Mg2+. These data suggest that the decrease in potency of ATP in the presence of Ca2+ and Mg2+, as observed in functional studies, is not due to a direct effect of the cations on the binding of ATP to the receptor. Both cyanogen bromide and hydroxylamine cleavage further confirmed the peptide structure of the purified H-mP2X1R. Autoradiographic analysis of the cleavage products showed that 8-azido-ATP-gamma-P-32 was crosslinked to the carboxyl side of the extracellular domain of the receptor. Drug Dev. Res. 51:7-19, 2000. Published 2000 Wiley-Liss, Inc.dagger