Structural identification of vasodilator binding sites on the SUR2 subunit

Abstract

ATP-sensitive potassium channels (KATP), composed of Kir6 and SUR subunits, convert the metabolic status of the cell into electrical signals. Pharmacological activation of SUR2- containing KATP channels by class of small molecule drugs known as KATP openers leads to hyperpolarization of excitable cells and to vasodilation. Thus, KATP openers could be used to treat cardiovascular diseases. However, where these vasodilators bind to KATP and how they activate the channel remains elusive. Here, we present cryo-EM structures of SUR2A and SUR2B subunits in complex with Mg-nucleotides and P1075 or levcromakalim, two chemically distinct KATP openers that are specific to SUR2. Both P1075 and levcromakalim bind to a common site in the transmembrane domain (TMD) of the SUR2 subunit, which is between TMD1 and TMD2 and is embraced by TM10, TM11, TM12, TM14, and TM17. These KATP openers synergize with Mg-nucleotides to stabilize SUR2 in the NBD-dimerized occluded state to activate the channel.

The Emerging Structural Pharmacology of ATP-Sensitive Potassium Channels

“…Despite the distinct chemical structures and selectivities of these three KCO, they all bind at a common KCOS in the middle of the transmembrane domain of SUR (Figure 2A), in agreement with the competitive binding behavior of KCO (Bray and Quast, 1992). KCOS is embraced by TM10, TM11, TM12, TM13, TM14, and TM17 helices (Figure 2C,2E,and 2G), and is about 20 Å away from the aforementioned ISBS (Ding et al, 2022). Based on the structures, we define the KCOS as three connecting regions on SUR: the A site, the Polar site (P site), and the B site (Figure 2B).…”

“…Their exact binding sites on the SUR subunit were elusive despite the enlightening work on chimeric SUR and mutagenesis (Ashcroft and Gribble, 2000;Babenko et al, 2000;D'Hahan et al, 1999a;D'Hahan et al, 1999b;Moreau et al, 2000;Reimann et al, 2001a;Uhde et al, 1999). To experimentally determine the KCO binding site, our lab has solved the structure of SUR2 in complex with SUR2-selective P1075 and Lev (Ding et al, 2022), and SUR1/Kir6.2 K ATP with SUR1-selective NN414 (Wang et al, 2022b), providing comprehensive views of KCO binding sites (KCOS) at near-atomic resolutions (Figure 2). The binding of KCO synergizes with Mg-nucleotides to stabilize the SUR in the NBD-dimerized occluded conformation (Figure 2A), in agreement with the fact that KCO bind to SUR with slower off-rates in the presence of Mgnucleotides (Gribble et al, 2000).…”

“…Structural information is consistent with previous studies showing that NH groups of pyridylamino group of P1075 can act as a hydrogen-bond donor and make a crucial contribution toward K ATP activation (Manley and Quast, 1992); the substitution of pyrrolidinone of Lev changed drug potency significantly (Mannhold, 2006); the substitution of the methyl group on the thiadiazine rings by alkylamino group enhanced the potency of dioxide derivatives (Hansen, 2006). Mutation of these two residues greatly decreased the activation effect of both SUR1-selective opener NN414 (Wang et al, 2022b) and SUR2-selective KCO, including P1075 and Lev (Ding et al, 2022), emphasizing that the polar interactions between the P site of SUR and KCO are essential for channel activation.…”

“…The A site is formed by residues in TMD2 and these residues are not identical between SUR1 and SUR2 (Ding et al, 2022). I1004 and T1253 in SUR2 are L1027 and M1290 in SUR1, respectively.…”

“…I1004 of TM12 and T1253 of TM17 of SUR2 are close to the cyano group and pyridine ring of P1075. Mutations of I1004L and T1253M introduce steric hindrance to the binding of P1075 (Ding et al, 2022). Rb + efflux assay showed that I1004L in SUR2B reduced the activating effect of P1075 significantly and T1253M in SUR2B had a more profound effect (Ding et al, 2022).…”