Analysis of novel P2X subunit-specific antibodies in rat cardiac and smooth muscle

Abstract

P2X receptors are cation-selective channels gated by extracellular adenosine triphosphate (ATP). There are relatively few known types of ligand-gated receptors. In vertebrates they include acetylcholine (Ach), 5-hydroxytryptamine (5-HT), γ-aminobutyric acid (GABA), glycine, and glutamate as well as ATP. Ach, 5-HT, GABA and glycine ligand-gated receptors are related in evolutionary terms, while glutamate and ATP receptors form separate groups. There have been seven cloned proteins identified to date as members of the P2X receptor family in a wide range of cells and species. We have carried out hydropathy investigations and sequence comparisons of each of the seven subunits in order to examine the putative transmembrane and cysteine-rich extracellular domains. Probable locations of disulphide bridges are consistent with there being two separate extracellular folding domains. Assessment of the putative surface-accessible regions was used to select small localised amino acid segments in nonglycosylated regions for raising antibodies against each of the P2X receptor subunits. To test the specificity of these novel P2X receptor antibodies and their presence in cardiac and smooth muscle, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)/Western blotting was undertaken in homogenised rat heart, bladder, kidney, and vas deferens.