Andrey P. Babenko

The C Terminus of SUR1 Is Required for Trafficking of KATP Channels

In beta cells from the pancreas, ATP-sensitive potassium channels, or KATP channels, are composed of two subunits, SUR1 and KIR6.2, assembled in a (SUR1/KIR6.2)4 stoichiometry. The correct stoichiometry of channels at the cell surface is tightly regulated by the presence of novel endoplasmic reticulum (ER) retention signals in SUR1 and KIR6.2; incompletely assembled KATPchannels fail to exit the ER/cis-Golgi compartments. In addition to these retrograde signals, we show that the C terminus of SUR1 has an anterograde signal, composed in part of a dileucine motif and downstream phenylalanine, which is required for KATPchannels to exit the ER/cis-Golgi compartments and transit to the cell surface. Deletion of as few as seven amino acids, including the phenylalanine, from SUR1 markedly reduces surface expression of KATP channels. Mutations leading to truncation of the C terminus of SUR1 are one cause of a severe, recessive form of persistent hyperinsulinemic hypoglycemia of infancy. We propose that the complete loss of beta cell KATP channel activity seen in this form of hyperinsulinism is a failure of KATPchannels to traffic to the plasma membrane.

Pharmaco-topology of Sulfonylurea Receptors SEPARATE DOMAINS OF THE REGULATORY SUBUNITS OFK ATP CHANNEL ISOFORMS ARE REQUIRED FOR SELECTIVE INTERACTION WITH K+ CHANNEL OPENERS

The differential responsiveness of (SUR1/KIR6.2)4 pancreatic β-cellversus (SUR2A/KIR6.2)4 sarcolemmal or (SUR2B/KIR6.0)4 smooth muscle cellK ATP channels to K+ channel openers (KCOs) is the basis for the selective prevention of hyperinsulinemia, myocardial infarction, and acute hypertension. KCO-stimulation ofK ATP channels is a unique example of functional coupling between a transport ATPase and a K+ inward rectifier. KCO binding to SUR is Mg-ATP-dependent and antagonizes the inhibition of (KIR6.0)4 pore opening by nucleotides. Patch-clamping of matched chimeric human SUR1-SUR2A/KIR6.2 channels was used to identify the SUR regions that specify the selective response of sarcolemmalversus β-cell channels to cromakalim or pinacidilversus diazoxide. The SUR2 segment containing the 12th through 17th predicted transmembrane domains, TMD12–17, confers sensitivity to the benzopyran, cromakalim, and the pyridine, pinacidil, whereas an SUR1 segment which includes TMD6–11 and the first nucleotide-binding fold, NBF1, controls responsiveness to the benzothiadiazine, diazoxide. These data are incorporated into a functional topology model for the regulatory SUR subunits ofK ATP channels.

Insulin Secretagogues, Sulfonylurea Receptors and KATP Channels

ATP-sensitive K+ channels, termed K(ATP) channels, provide a link between cellular metabolism and membrane electrical activity in a variety of tissues. Channel isoforms have been identified and are targets for compounds that both stimulate and inhibit their activity resulting in membrane hyperpolarization and depolarization, respectively. Examples include relaxation of vascular smooth muscle and stimulation of insulin secretion. This article reviews the cloning, molecular biology, and structure of K(ATP) channels, with particular focus on the SUR1/K(IR)6.2 neuroendocrine channels that are important for the regulation of insulin secretion. We integrate the extensive pharmacologic structure-activity-relationship data on these channels, which defines a bipartite drug binding pocket in the SUR (sulfonylurea receptor), with recent structure-function studies that identify domains of SUR and K(IR)6.2, the channel pore, which are critical for channel assembly, for gating, and for the ligand-receptor interactions that modulate channel activity. The atomic structure of a sulfonylurea in a protein pocket is used to develop insight into the recognition of these compounds. A homology model of K(ATP) channels, based on VC-MsbA, another member of the ABC protein family, is described and used to position amino acids important for the action of channel openers and blockers within the core of SUR. The model has a central chamber which could serve as a multifaceted binding pocket.

Two Regions of Sulfonylurea Receptor Specify the Spontaneous Bursting and ATP Inhibition of KATP Channel Isoforms

KATP channels are heteromultimers of KIR6.2 and a sulfonylurea receptor, SUR, an ATP binding cassette (ABC) protein with several isoforms. KIR6.2 forms a channel pore whose spontaneous activity and ATP sensitivity are modulated by the receptor via an unknown interaction(s). Side by side comparison of single-channel kinetics and steady-state ATP inhibition of human β-cell, SUR1/KIR6.2,versus cardiac, SUR2A/KIR6.2 channels demonstrate that the latter have a greater mean burst duration and open probability in the absence of nucleotides and ∼4-fold higher IC50(ATP). We have used matched chimeras of SUR1 and SUR2A to show that the kinetics, which determine the maximal open probability (Pomax), and the ATP sensitivity are functionally separable and to identify the two segments of SUR responsible for these isoform differences. A region within the first five transmembrane domains specifies the interburst kinetics, whereas a C-terminal segment determines the sensitivity to inhibitory ATP. The separable effects of SUR on ATP inhibition and channel kinetics implies that the cytoplasmic C terminus of SUR either directly modulates the affinity of a weak ATP binding site on the inward rectifier or affects linkage between the binding site and the gate. This is the first identification of parts of an ABC protein that interact with an ion channel subunit to modulate the spontaneous activity and ATP sensitivity of the heteromeric channel.

The tolbutamide site of SUR1 and a mechanism for its functional coupling to KATP channel closure

Micromolar concentrations of tolbutamide will inhibit (SUR1/KIR6.2)4 channels in pancreatic β‐cells, but not (SUR2A/KIR6.2)4 channels in cardiomyocytes. Inhibition does not require Mg2+ or nucleotides and is enhanced by intracellular nucleotides. Using chimeras between SUR1 and SUR2A, we show that transmembrane domains 12–17 (TMD12‐17) are required for high‐affinity tolbutamide inhibition of KATP channels. Deletions demonstrate involvement of the cytoplasmic N‐terminus of KIR6.2 in coupling sulfonylurea‐binding with SUR1 to the stabilization of an interburst closed configuration of the channel. The increased efficacy of tolbutamide by nucleotides results from an impairment of their stimulatory action on SUR1 which unmasks their inhibitory effects. The mechanism of inhibition of β‐cell KATP channels by sulfonylureas during treatment of non‐insulin‐dependent diabetes mellitus thus involves two components, drug‐binding and conformational changes within SUR1 which are coupled to the pore subunit through its N‐terminus and the disruption of nucleotide‐dependent stimulatory effects of the regulatory subunit on the pore. These findings uncover a molecular basis for an inhibitory influence of SUR1, an ATP‐binding cassette (ABC) protein, on KIR6.2, a ion channel subunit.

Sulfonylurea receptors set the maximal open probability, ATP sensitivity and plasma membrane density of KATP channels

KATP channels are heteromultimers of SUR and KIR6.2. C‐terminal truncation of KIR6.2 allows surface expression of the pore. KIR6.2ΔC35 channels display ∼7‐fold lower maximal open probability, ∼35‐fold reduced ATP sensitivity, reduced mean open time, a markedly increased transition rate from a burst into a long‐lived closed state, and have no counterpart in vivo. SUR1 and SUR2A restore wild‐type bursting, ATP sensitivity and increase channel density in the plasma membrane. The high IC50(ATP) of ∼4 mM for KIR6.2ΔCK185Q channels results from the additive effects of SUR removal and KIR6.2 modification. The results demonstrate allosteric interaction(s) are essential for normal intrinsic activity, ATP inhibition, and trafficking of KATP channels.

Hetero-concatemeric KIR6.X4/SUR14 channels display distinct conductivities but uniform ATP inhibition.

KIR6.1 and KIR6.2 are the pore-forming subunits ofK NDP , the nucleotide-diphosphate-activated K ATP channels, and classical K ATP channels, respectively. “Hybrid” channels, in which the structure is predetermined by concatemerizing KIR6.1 and KIR6.2, exhibit distinct conductivities specified by subunit number and position. Inclusion of one KIR6.2 is sufficient to open KIR6.X-X-X-X/SUR14 in the absence of nucleotide stimulation through sulfonylurea receptor-1 (SUR1). ATP inhibited the spontaneous bursting of hybrid channels with an IC50(ATP) ∼10− 5 m, similar to that of KIR6.24-containing channels. These findings and a transient increase in K NDP channel activity following rapid wash-out of MgATP suggested that KIR6.1 is not ATP-insensitive as previously believed. We propose that SUR-dependent, inhibitory ATP-enhanced interactions of the cytoplasmic domains of both KIR6.1 and KIR6.2 stabilize a closed form of the M2 bundle in the gating apparatus.

A Novel ABCC8 (SUR1)-dependent Mechanism of Metabolism-Excitation Uncoupling

ATP/ADP-sensing (sulfonylurea receptor (SUR)/KIR6)4 KATP channels regulate the excitability of our insulin secreting and other vital cells via the differential MgATP/ADP-dependent stimulatory actions of their tissue-specific ATP-binding cassette regulatory subunits (sulfonylurea receptors), which counterbalance the nearly constant inhibitory action of ATP on the K+ inwardly rectifying pore. Mutations in SUR1 that abolish its stimulation have been found in infants persistently releasing insulin. Activating mutations in SUR1 have been shown to cause neonatal diabetes. Here, analyses of KIR6.2-based channels with diabetogenic receptors reveal that MgATP-dependent hyper-stimulation of mutant SUR can compromise the ability of KATP channels to function as metabolic sensors. I demonstrate that the channel hyperactivity rises exponentially with the number of hyperstimulating subunits, so small subpopulations of channels with more than two mutant SUR can dominate hyperpolarizing currents in heterozygous patients. I uncovered an attenuated tolbutamide inhibition of the hyperstimulated mutant, which is normally sensitive to the drug under non-stimulatory conditions. These findings show the key role of SUR in sensing the metabolic index in humans and urge others to (re)test mutant SUR/KIR6 channels from probands in physiologic MgATP.

The Predominant Mechanisms of Pathogenic Hyperactivity of Mutant ABCC8/KCNJ11-Coded KATP Channels

We discovered numerous congenital diabetes (CD), insulin release-inhibiting mutations in ABCC8/KCNJ11 genes coding for the pancreatic beta-cell (SUR1/Kir6.2)4 KATP channel (Diabetes 53:2719; N Engl J Med 355:456; Diabetes 56:1737; J Biol Chem 283:8778; reviewed in Endocr Rev 29:265). Based on FEBS Lett 445:131; 459:367 and subsequent structure-activity analyses and mechanistic models of KATP channels (reviewed in J Mol Cell Cardiol 39:79), Babenko predicted three possible mechanisms of KATP hyperactivation by mutations in ABCC8 and started testing the hypothesis (DK077827 R01 project ABCC8/KCNJ11 Mechanisms and Diabetes). Following the analysis described in N Engl J Med 355:456 and J Biol Chem 283:8778, we established that the majority of CD mutations which map to the canonical ABC exporter core increase the open channel probability (Po) in intact cells by enhancing the MgATP/ADP-dependent stimulatory action of SUR1 on the channel, without affecting its maximal Po in the absence of nucleotides (Pomax) or its Mg-independent sensitivity to inhibitory ATP, IC50(ATP). This is called the A-type mechanism of KATP hyperactivation. The majority of CD mutations which map to the non-canonical, TMD0-L0 “gatekeeper” (J Biol Chem 278:41577) domain of SUR1 increase Pomax and apparent IC50(ATP). Single-channel kinetics analysis reveals that this effect is due to reciprocal changes in the rates of channel transitions to and from its long-lived closed state with the lowest apparent Kd(ATP) for the inhibitory nucleotide. This establishes the second, or B-type, mechanism of KATP hyperactivity. Although our search for the third, Kd(ATP)-increasing type mutations in ABCC8 continues, we are in the position to conclude that A/B-mechanisms cause the majority of CD-KATP cases and our refined Sav1866/MsbA / KcsA/Kir3.1/BacKir/Chimera-based models of KATP channels help correctly predict the principal effect of the majority of diabetogenic mutations in ABCC8/KCNJ11 on KATP gating.