Fluorescent, non-hydrolyzable nucleotide probes for ATP-activated ion channels.

Abstract

In addition to its role as a ubiquitous, intracellular energy source and signaling molecule, adenosine 5’-triphosphate (ATP) also impacts extracellular signaling as both a neurotransmitter and a product of tissue damage. We previously developed a real-time nucleotide binding assay to study the gating of ATP-sensitive K+ (KATP) channels. Nucleotide binding was measured as Förster resonance energy transfer (FRET) between fluorescent, trinitrophenyl nucleotide derivatives and KATP channels tagged with the fluorescent, non-canonical amino acid L-3-(6-acetylnaphthalen-2-ylamino)-2-aminopropionic acid (ANAP). In order to further probe the shape and chemistry of the nucleotide binding sites of ATP activated channels, we have synthesized and purified trinitrophenyl analogs of the weakly hydrolyzable adenosine-5'-(γ-thio)-triphosphate (ATP- γ-S) and the non-hydrolyzable nucleotides adenosine-5'-[(β,γ)-methyleno]triphosphate (AMP-PCP) and adenosine-5'-[(β,γ)-imido]triphosphate. The trinitrophenyl group was added to the ribose moiety of these nucleotides through an aqueous reaction with 2,4,6-trinitrobenzenesulfonic acid under basic conditions. The reaction products were purified by precipitation in ethanol followed by reversed-phase high-performance liquid chromatography. Products were verified using 1H and 31P nuclear magnetic resonance spectroscopy, fluorescence spectroscopy, and UV/visible absorbance. These compounds will be useful for exploring the requirement (if any) for ATP hydrolysis to activate KATP channels and the unique chemistry of the ATP binding sites of purinergic P2X-family receptors.