A fluorescent probe for the enzymatic activity of K_ATP

The neuroendocrine ATP-sensitive K⁺(K_ATP) channel comprises four pore-forming subunits (Kir6.2), and four modulatory sulfonylurea receptor subunits (SUR1). ATP/ADP binding to Kir6.2 inhibits K_ATP, whereas MgATP/MgADP binding to two sites on SUR1 promotes activation. As SUR1 is part of the ABC transporter family, it can hydrolyze MgATP to MgADP. Whether or not enzymatic activity is required for K_ATPactivation remains controversial. 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, 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,¹H NMR, and mass spectrometry. Purity was assessed using TLC and reversed-phase HPLC. We can measure real-time binding of fluorescent nucleotide derivatives to intact K_ATPchannels 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 measured TNP-ATP binding to nucleotide binding site 1 on SUR1 in fluorescently labeled Kir6.2/SUR1 channels in unroofed membranes of HEK293T cells. 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_ATPdoes 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.