Functional Clustering of Mutations in the Dimer Interface of the Nucleotide Binding Folds of the Sulfonylurea Receptor

Abstract

ATP-sensitive potassium (K ATP )2 channels link cellular metabolism to membrane excitability by varying their activity in accordance with changes in cellular [ATP]/[ADP], thus controlling Ca 2ϩ entry via voltage-dependent Ca 2ϩ channels. In the pancreatic ␤-cell, K ATP channels regulate insulin secretion, and mutations that increase or decrease channel activity result in insulin hyper-or hyposecretion, respectively (1, 2).K ATP channels are hetero-octamers of two distinct subunits (3): four pore-forming Kir6 subunits combine with four regulatory SUR subunits, members of the ATP-binding cassette (ABC) transporter family of proteins. SUR, like all other ABC transporters, consists of two transmembrane domains (TMD1 and TMD2) and two cytoplasmic domains (nucleotide binding folds 1 and 2; NBF1 and NBF2). In addition, SUR also contains an N-terminal transmembrane domain (TMD0), the putative site of SUR-Kir6 interaction (4, 5). While ATP inhibits the K ATP channel by direct binding to the cytoplasmic domains of Kir6 (6, 7), SUR mediates channel regulation by other ligands, including high affinity inhibition by sulfonylureas (8), stimulation by potassium channel openers (KCOs) (9), and stimulation by Mg-nucleotides (10).Channel stimulation by MgADP is hypothesized to reflect ATP hydrolysis at the NBFs: MgADP binds to, and stabilizes, a post-hydrolytic, activated configuration of the NBFs, which is then transduced to the TMDs and ultimately to the Kir6 pore (10 -13). Importantly, abnormalities in MgADP stimulation cause disease in humans: in SUR1, NBF mutations that decrease MgADP stimulation underlie Persistent Hyperinsulinemic Hypoglycemia of Infancy (PHHI) (10, 14), while NBF mutations that result in hyperstimulation by MgADP have recently been shown to underlie both transient and persistent forms of neonatal diabetes (15-17).Based on crystallographic and biochemical studies of prokaryotic NBFs, a common mechanism of ABC transporter function has been proposed: ATP binding to the NBF domains induces formation of a NBF dimer, such that the two ATP binding sites are sandwiched between the two NBFs. ATP hydrolysis by the NBF dimer induces a conformational change that is transduced to the TMDs, fueling the function of the ABC transporter (18,19); in the case of SUR1, stabilization of the open state of the Kir6.2 pore. Each ATPbinding site (ABS) in the NBF dimer is composed of sequence elements that are highly conserved among ABC transporters: Walker A and Walker B sequences from one NBF form most of the contact points, while the Signature sequence of the other NBF provides additional interactions with the nucleotide (20 -23). In SUR, mutation of catalytic Walker A and Walker B residues (which are required for in vitro ATPase activity of prokaryotic NBFs) leads to decreased MgADP stimulation of K ATP channels (10, 12, 13). Azido-ATP labeling studies of SUR1 indicate that the binding site formed by NBF1 Walker A and Walker B does not significantly hydrolyze ATP, while the binding site formed by . Consistent with this, isolat...

Domain Organization of the ATP-sensitive Potassium Channel Complex Examined by Fluorescence Resonance Energy Transfer

“…Co-expression of nSUR1/cSUR1 FRET constructs together with untagged Kir6.2 exhibited ϳ15% apparent FRET (Fig. 6), consistent with NBD1 and NBD2 forming heterodimers in vivo (15,33).…”

Structural basis for allosteric cross-talk between the asymmetric nucleotide binding sites of a heterodimeric ABC exporter

“…However, there are only a few reports that describe its functional role. A systematic cysteine-scanning study of residues lining the NBD interface of the sulfonylurea receptor SUR1 highlighted D-loop residues to play a key role in MgADP stimulation of its associated potassium channel subunit (25). Of special note, substituting the degenerate site D-loop aspartate by a cysteine (D1513C) completely abolished MgADP stimulated potassium gating, whereas the functional impact of the corresponding consensus site mutant (D861C) was less severe.…”

Inwardly Rectifying Potassium Channels: Their Structure, Function, and Physiological Roles

“…When mutations equivalent to those that permit MJ0796 to be dimerized by Na ϩ -bound ATP were introduced into NBDs of SUR2, Na i ϩ increased the activity of K ATP channels in the presence of ATP i (852). Homology models of the dimerized structure of the NBDs in SUR1 were generated and mutations introduced into the STRUCTURE AND PHYSIOLOGICAL FUNCTION OF Kir CHANNELS putative interface of the dimerized NBDs altered their affinity for Mg-NDP (75,503). These results indicate that the two NBDs form a dimer in SUR proteins to contribute to gating of the ion channels.…”