A fluorescent probe for the enzymatic activity of KATP

Abstract

Neuroendocrine ATP-sensitive K+ channels (KATP) comprise four pore-forming subunits (Kir6.2), each associated with a modulatory sulfonylurea receptor subunit (SUR1). ATP/ADP binding to Kir6.2 inhibits KATP; MgATP/MgADP binding to two different sites on SUR1 promotes activation. As SUR1 is a member of the ABC transporter family of proteins, it can potentially hydrolyze MgATP to MgADP. Whether this activity is required for KATP activation remains controversial. Previous studies demonstrated that non-hydrolyzable ATP analogs do not activate KATP, 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 {beta},{gamma}-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. Real-time nucleotide binding to intact KATP channels in cell membranes was measured using FRET between channels labeled with a fluorescent, non-canonical amino acid and TNP-nucleotide derivatives. This technique provides sufficient spatial resolution to discriminate between binding to each site on KATP. 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 KATP does not stem from an inability of these nucleotides to bind to the channel.