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Data from: A fluorescent non-hydrolyzable probe for the nucleotide binding sites of KATP

Rubio, Prisicilla1; Whitaker, Shayla Q.1; Ashby, Jonathan1; Puljung, Michael1

Published Sep 10, 2026 on Dryad. https://doi.org/10.5061/dryad.rv15dv4mw

Data files

Sep 10, 2026 version files 312.70 MB

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Abstract

The neuroendocrine ATP-sensitive K+ channel (KATP) comprises four pore-forming subunits (Kir6.2), each associated with a modulatory sulfonylurea receptor subunit (SUR1). ATP/ADP binding to Kir6.2 inhibits KATP, whereas 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 or not enzymatic activity is required for KATP activation remains controversial. Previous studies have 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 β,γ-methyleneadenosine 5’-triphosphate (AMP-PCP). Correct synthesis was verified by UV-visible absorbance, fluorescence spectroscopy, 1H nuclear magnetic resonance, and mass spectrometry. Purity was assessed using thin-layer chromatography and reversed-phase high-performance liquid chromatography. We can measure real-time nucleotide binding to intact KATP channels in cell membranes using FRET between channels labeled with a fluorescent, non-canonical amino acid and TNP-nucleotide derivatives. Importantly, this technique provides us with 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, leaving open the possibilities that they are unable to induce an activating conformational change in SUR1 or that nucleotide hydrolysis by SUR1 is a prerequisite for channel activation.