Specific Inhibition of the Rat Ligand-Gated Ion Channel P 2 X 3 Function Via Methoxyethoxy-Modified Phosphorothioated Antisense Oligonucleotides

Abstract

P₂X₃ is one receptor of a family of seven ligand-gated ion channels responding to purines. Increasing evidence indicates its involvement in neuronal signaling and in pain. However, there is currently no selective inhibitor known for this subtype. In order to obtain such a specific inhibitor, a variety of antisense oligonucleotides (ASO) against rat P₂X₃ was tested, and dose-dependent, sequence-specific downregulation of the rat P₂X₃ receptor (expressed in a Chinese hamster ovary cell line [CHO-K1]) on the mRNA, protein, and functional levels was observed. Using real-time quantitative PCR, a dose-dependent downregulation of P₂X₃ mRNA by ASO, as compared with untreated and mismatch controls, was demonstrated. Subsequently, downregulation by the two most potent ASO was confirmed at the protein level by Western blot. Sequence specificity was shown by titration of mismatches to the original selected oligonucleotide, and this correlated with progressive loss of P₂X₃ inhibition. The functional response of the P₂X₃ receptor was examined using whole-cell voltage clamping. Upon application of 10 µM of a nonspecific agonist, α,β-methylene-ATP (αβ meATP), pretreatment with increasing amounts of the most active ASO 5037 correlated with a decrease in depolarization. The ability to specifically downregulate the P₂X₃ receptor by ASO treatment will allow investigation of the biologic role of this receptor in neuronal tissues and eventually in in vivo models of chronic pain.