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

Abstract

ATP-sensitive potassium (KATP) channels composed of a pore-forming Kir6.2 potassium channel and a regulatory ABC transporter sulfonylurea receptor 1 (SUR1) regulate insulin secretion in pancreatic β-cells to maintain glucose homeostasis. Mutations that impair channel folding or assembly prevent cell surface expression and cause congenital hyperinsulinism. Structurally diverse KATP inhibitors are known to act as pharmacochaperones to correct mutant channel expression, but the mechanism is unknown. Here, we compare cryoEM structures of a mammalian KATP channel bound to pharmacochaperones glibenclamide, repaglinide, and carbamazepine. We found all three drugs bind within a common pocket in SUR1. Further, we found the N-terminus of Kir6.2 inserted within the central cavity of the SUR1 ABC core, adjacent the drug binding pocket. The findings reveal a common mechanism by which diverse compounds stabilize the Kir6.2 N-terminus within SUR1’s ABC core, allowing it to act as a firm ‘handle’ for the assembly of metastable mutant SUR1-Kir6.2 complexes.

Structural Determinants of Insulin Release: Disordered N-Terminal Tail of Kir6.2 Affects Potassium Channel Dynamics through Interactions with Sulfonylurea Binding Region in a SUR1 Partner

“…The SU region is important for binding antidiabetic drugs that keep the KATP channel closed. 6 , 18 , 63 …”

“…Only very recently has a putative position of the KN tail been proposed on the basis of cryo-EM data. 18 We asked a question regarding whether there is any “preferable” way for the binding of the KN tail into the SU binding region. We docked all tail-derived consecutive pentapeptides to our open form of human SUR1 (results are shown in SI Table 2).…”

“…In that way, three alternative variants of model KS , named model KNt26 , model KNt37 , and model KNt48 , were further investigated. Additionally, we performed a set of simulations in which the target position of the KN tail was based on the N-terminal fragment from the cryo-EM structure published by Martin et al (courtesy of Professor Shyng) 18 (model KNtM ). Details are described in SI .…”

Nucleotide inhibition of the pancreatic ATP-sensitive K+ channel explored with patch-clamp fluorometry

“…We designate this construct Kir6.2*. Based on the theoretical FRET efficiency calculated from the Förster equation and available cryo-EM structures (Martin et al, 2017;Martin et al, 2019), we expect 91% FRET efficiency between ANAP at position 311 and a TNP-ATP molecule bound to Kir6.2, and only 4% FRET efficiency to TNP-ATP bound to the closest nucleotide-binding site on SUR1 (nucleotide binding site 1, Figure 1D). We also expect very little FRET between ANAP at position 311 and TNP-ATP bound to neighbouring Kir6.2 subunits (15-25%).…”

“…The modal highest-scoring pose from the docking run was selected (PDB accession #5XW6, (Kasuya et al, 2017)) and distances were measured from a pseudo atom at the centre of the fluorescent moiety. TNP-ATP (PDB #3AR7, (Toyoshima et al, 2011)) was positioned into the first nucleotide binding domain of SUR1 (PDB #6PZI, (Martin et al, 2019)) using the alignment tool in Pymol (Schrödinger, LLC; New York, NY).…”