A fluorescent non-hydrolyzable probe for the nucleotide binding sites of K _ATP

Neuroendocrine ATP-sensitive K+ channels (K _ATP ) comprise four pore-forming subunits (Kir6.2), each associated with a modulatory sulfonylurea receptor subunit (SUR1). ATP/ADP binding to Kir6.2 inhibits K _ATP ; MgATP/MgADP binding to two different sites on SUR1 promotes activation. As SUR1 is part of the ABC transporter family of proteins, it can potentially hydrolyze MgATP to MgADP. Whether this activity is required for K _ATP activation remains controversial. Previous studies demonstrated that non-hydrolyzable ATP analogs do not activate K _ATP , which may reflect an inability of these compounds to bind to SUR1, their inability to promote a conformational change in SUR1 that leads to channel activation, or a requirement for ATP hydrolysis during channel gating. To explore this further, we synthesized a fluorescent trinitrophenyl (TNP) derivative of the non-hydrolyzable ATP analog β,γ-methyleneadenosine 5′-triphosphate (AMP-PCP). Synthesis was verified by UV-visible absorbance, fluorescence spectroscopy, 1H nuclear magnetic resonance, and mass spectrometry. Purity was assessed by reversed-phase high-performance liquid chromatography. We can measure real-time nucleotide binding to intact K _ATP channels in cell membranes using FRET between channels labeled with a fluorescent, non-canonical amino acid and TNP-nucleotide derivatives. This technique provides us with sufficient spatial resolution to discriminate between binding to each site on K _ATP . Using this approach we first established that TNP-ATP can bind to nucleotide binding site 1 on SUR1 in fluorescently labeled Kir6.2/SUR1 channels in unroofed membranes of HEK293T cells. We subsequently demonstrated that TNP-AMP-PCP binds to both nucleotide binding sites on SUR1 in the absence of Mg2+. AMP-PCP was able to compete with TNP-ATP for binding to NBS2, suggesting that it, too, binds NBS2. We conclude that the failure of non-hydrolyzable ATP analogs to activate K _ATP does not stem from an inability of these nucleotides to bind to the channel.