ATP binding without hydrolysis switches sulfonylurea receptor 1 (SUR1) to outward-facing conformations that activate KATP channels

Abstract

Edited by Norma M. AllewellNeuroendocrine-type ATP-sensitive K ؉ (K ATP ) channels are metabolite sensors coupling membrane potential with metabolism, thereby linking insulin secretion to plasma glucose levels. They are octameric complexes, (SUR1/Kir6.2) 4 , comprising sulfonylurea receptor 1 (SUR1 or ABCC8) and a K ؉ -selective inward rectifier (Kir6.2 or KCNJ11). Interactions between nucleotide-, agonist-, and antagonist-binding sites affect channel activity allosterically. Although it is hypothesized that opening these channels requires SUR1-mediated MgATP hydrolysis, we show here that ATP binding to SUR1, without hydrolysis, opens channels when nucleotide antagonism on Kir6.2 is minimized and SUR1 mutants with increased ATP affinities are used. We found that ATP binding is sufficient to switch SUR1 alone between inward-or outward-facing conformations with low or high dissociation constant, K D , values for the conformation-sensitive channel antagonist [ 3 H]glibenclamide ([ 3 H]GBM), indicating that ATP can act as a pure agonist. Assembly with Kir6.2 reduced SUR1's K D for [ 3 H]GBM.ThisreductionrequiredtheKirNterminus(KNtp), consistent with KNtp occupying a "transport cavity," thus positioning it to link ATP-induced SUR1 conformational changes to channel gating. Moreover, ATP/GBM site coupling was constrained in WT SUR1/WT Kir6.2 channels; ATP-bound channels had a lower K D for [ 3 H]GBM than ATP-bound SUR1. This constraint was largely eliminated by the Q1179R neonatal diabetes-associated mutation in helix 15, suggesting that a "swapped" helix pair, 15 and 16, is part of a structural pathway connecting the ATP/GBM sites. Our results suggest that ATP binding to SUR1 biases K ATP channels toward open states, consistent with SUR1 variants with lower K D values causing neonatal diabetes, whereas increased K D values cause congenital hyperinsulinism.Neuroendocrine-type ATP-sensitive K ϩ (K ATP ) 2 channels comprise an ATP-binding cassette (ABC) protein (1), ABCC8/ SUR1, and a K ϩ -selective inward rectifier (2), KCNJ11/Kir6.2, assembled as heterooctamers (3-5), (SUR1/Kir6.2) 4 . In pancreatic ␤-cells, these channels are metabolite sensors that couple cellular metabolism with membrane electrical activity to link insulin secretion with blood glucose levels. This coupling is critical for normal physiology; loss of channel function is a cause of congenital hyperinsulinism (Ref. 6; for reviews, see Refs. 7 and 8), whereas gain-of-function mutations in Kir6.2 (9) and SUR1 (10) cause neonatal diabetes (ND) (for reviews, see Refs. 11 and 12). Gain of function is one cause of mature-onset diabetes of the young (13), whereas a ABCC8/SUR1 polymorphism, the Ala amino acid allele at p.A1369S, is associated with an increased risk for type 2 diabetes (14).Channel activity is regulated positively by ATP and ADP binding to SUR1 and negatively by nucleotide binding to . Additionally, multiple metabolites, including phosphoinositides (21-23) and long-chain acyl-CoA esters (24 -26), and phosphorylation (27-29) positively modulate ch...

Activation mechanism of ATP-sensitive K+ channels explored with real-time nucleotide binding

“…As lack of Mg 2+ or mutation of the catalytic Walker B motif do not support MgATP hydrolysis, this suggests that ATP can activate K ATP directly. No Mg 2+ -free ATP activation was demonstrated when wild-type SUR1 was expressed with Kir6.2-G334D and we see no indication in our data that nucleotides caused SUR1 to change conformation in the absence of Mg 2+ (Figure 2) (Sikimic et al, 2018).…”

“…Furthermore, high concentrations of ATP have been shown, in radioligand binding assays, to cause SUR1 to change conformation in the absence of Mg 2+ , under which conditions ATP hydrolysis is not expected (Ortiz et al, 2013). Mg 2+ -free ATP, at high concentrations, was also recently demonstrated to activate K ATP formed by SUR1-Q1179R, a gain-of-function SUR1 mutation, paired with Kir6.2-G334D, as well as SUR constructs in which the Walker B glutamate was mutated to glutamine (Sikimic et al, 2018). As lack of Mg 2+ or mutation of the catalytic Walker B motif do not support MgATP hydrolysis, this suggests that ATP can activate K ATP directly.…”

Mechanism of pharmacochaperoning in a mammalian KATP channel revealed by cryo-EM

“…The location of the KNt density observed in our structures, including the ATP-only structure, also illuminates how Kir6.2 and SUR1 interact to modulate channel activity. First, by occupying the central cavity of the SUR1 ABC core, KNt prevents NBD dimerization and stabilizes SUR1 in an inward-facing conformation, thereby abolishing the ability of Mg-nucleotides to stimulate channel activity (Sikimic et al, 2019). Second, by trapping KNt in the central cavity, SUR1 prevents the Kir6.2 tetramer from undergoing conformational changes needed for channel opening.…”

Mechanism of pharmacochaperoning in KATP channels revealed by cryo-EM

“…A critical missing piece of the puzzle was the KNt, which we have shown to be involved in pharmacochaperone rescue of TMD0 mutations (Devaraneni et al, 2015). KNt has not been resolved in previously determined structures (Li et al, 2017;Martin et al, 2017a;Martin et al, 2017b), although weak and disconnected cryoEM densities in the central cavity of the SUR1 ABC core have fueled speculation (Sikimic et al, 2018;Wu et al, 2018). Here, through improved focused refinement algorithms, cross-correlation of multiple structures, and biochemical as well as functional crosslinking experiments, we present evidence necessary to properly assign the KNt density adjacent to the pharmacochaperone binding site within the central cavity of the SUR1 ABC core.…”