Specific inhibition of the rat ligand-gated ion channel P2X3 function via methoxyethoxy-modified phosphorothioated antisense oligonucleotides

P2X3 is one receptor of a family of seven ligand-gated ion channels respond ing to purines, Increasing evidence indicates its involvement in neuronal s ignaling and in pain. However, there is currently no selective inhibitor kn own for this subtype, In order to obtain such a specific inhibitor, a varie ty of antisense oligonucleotides (ASO) against rat P2X3 was tested, and dos e-dependent, sequence-specific downregulation of the rat P2X3 receptor (exp ressed in a Chinese hamster ovary cell line [CHO-K1]) on the mRNA, protein, and functional levels was observed. Using real-time quantitative PCR, a do se-dependent downregulation of P2X3 mRNA by ASO, as compared with untreated and mismatch controls, was demonstrated. Subsequently, downregulation by t he two most potent ASO was confirmed at the protein level by Western blot. Sequence specificity was shown by titration of mismatches to the original s elected oligonueleotide, 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 muM of a nonspecific a gonist, alpha,beta -methylene-ATP (alpha beta meATP), pretreatment with inc reasing amounts of the most active ASO 5037 correlated with a decrease in d epolarization. The ability to specifically downregulate the P2X3 receptor b y ASO treatment will allow investigation of the biologic role of this recep tor in neuronal tissues and eventually in in vivo models of chronic pain.