Andras Szollosi

Shortcomings of current models of glucose-induced insulin secretion.

Glucose‐induced insulin secretion by pancreatic β‐cells is generally schematized by a ‘consensus model’ that involves the following sequence of events: acceleration of glucose metabolism, closure of ATP‐sensitive potassium channels (KATP channels) in the plasma membrane, depolarization, influx of Ca2+ through voltage‐dependent calcium channels and a rise in cytosolic‐free Ca2+ concentration that induces exocytosis of insulin‐containing granules. This model adequately depicts the essential triggering pathway but is incomplete. In this article, we first make a case for a model of dual regulation in which a metabolic amplifying pathway is also activated by glucose and augments the secretory response to the triggering Ca2+ signal under physiological conditions. We next discuss experimental evidence, largely but not exclusively obtained from β‐cells lacking KATP channels, which indicates that these channels are not the only possible transducers of glucose effects on the triggering Ca2+signal. We finally address the identity of the widely neglected background inward current (Cl− efflux vs. Na+ or Ca2+ influx through voltage‐independent channels) that is necessary to cause β‐cell depolarization when glucose closes KATP channels. More attention should be paid to the possibility that some components of this background current are influenced by glucose metabolism and have their place in a model of glucose‐induced insulin secretion.

The structure of the KtrAB potassium transporter.

In bacteria, archaea, fungi and plants the Trk, Ktr and HKT ion transporters are key components of osmotic regulation, pH homeostasis and resistance to drought and high salinity. These ion transporters are functionally diverse: they can function as Na+ or K+ channels and possibly as cation/K+ symporters. They are closely related to potassium channels both at the level of the membrane protein and at the level of the cytosolic regulatory domains. Here we describe the crystal structure of a Ktr K+ transporter, the KtrAB complex from Bacillus subtilis. The structure shows the dimeric membrane protein KtrB assembled with a cytosolic octameric KtrA ring bound to ATP, an activating ligand. A comparison between the structure of KtrAB–ATP and the structures of the isolated full-length KtrA protein with ATP or ADP reveals a ligand-dependent conformational change in the octameric ring, raising new ideas about the mechanism of activation in these transporters.

Overnight culture unmasks glucose-induced insulin secretion in mouse islets lacking ATP-sensitive K+ channels by improving the triggering Ca2+ signal.

A current model ascribes glucose-induced insulin secretion to the interaction of a triggering pathway (KATP channel-dependent Ca2+ influx and rise in cytosolic [Ca2+]c) and an amplifying pathway (KATP channel-independent augmentation of secretion without further increase of [Ca2+]c). However, several studies of sulfonylurea receptor 1 null mice (Sur1KO) failed to measure significant effects of glucose in their islets lacking KATP channels. We addressed this issue that challenges the model. Compared with controls, fresh Sur1KO islets showed slightly elevated basal [Ca2+]c and insulin secretion. In 15 mm glucose, the absolute rate of secretion was ∼3-fold lower in Sur1KO than control islets, with only poor increase above base line. Overnight culture of Sur1KO islets in 10 mm glucose (not in 5 mm) augmented basal insulin secretion and considerably improved the response to 15 mm glucose, which reached higher values than in control islets, in which culture had little impact. Glucose stimulation during KCl depolarization showed that the amplifying pathway is functional in fresh and cultured Sur1KO islets. The differences in insulin secretion between fresh and cultured Sur1KO islets and between Sur1KO and control islets were not attributable to differences in insulin content, glucose oxidation rate, or synchronization of [Ca2+]c oscillations. The unmasking of glucose-induced insulin secretion in β-cells lacking KATP channels is paradoxically due to improvement in the production of a triggering signal (elevated [Ca2+]c). The results show that KATP channels are not the only transducer of glucose effects on [Ca2+]c in β-cells. They explain controversies in the literature and refute arguments raised against the model implicating an amplifying pathway in glucose-induced insulin secretion.

Mutant cycles at CFTR's non-canonical ATP-binding site support little interface separation during gating

Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel belonging to the adenosine triphosphate (ATP)-binding cassette (ABC) superfamily. ABC proteins share a common molecular mechanism that couples ATP binding and hydrolysis at two nucleotide-binding domains (NBDs) to diverse functions. This involves formation of NBD dimers, with ATP bound at two composite interfacial sites. In CFTR, intramolecular NBD dimerization is coupled to channel opening. Channel closing is triggered by hydrolysis of the ATP molecule bound at composite site 2. Site 1, which is non-canonical, binds nucleotide tightly but is not hydrolytic. Recently, based on kinetic arguments, it was suggested that this site remains closed for several gating cycles. To investigate movements at site 1 by an independent technique, we studied changes in thermodynamic coupling between pairs of residues on opposite sides of this site. The chosen targets are likely to interact based on both phylogenetic analysis and closeness on structural models. First, we mutated T460 in NBD1 and L1353 in NBD2 (the corresponding site-2 residues become energetically coupled as channels open). Mutation T460S accelerated closure in hydrolytic conditions and in the nonhydrolytic K1250R background; mutation L1353M did not affect these rates. Analysis of the double mutant showed additive effects of mutations, suggesting that energetic coupling between the two residues remains unchanged during the gating cycle. We next investigated pairs 460–1348 and 460–1375. Although both mutations H1348A and H1375A produced dramatic changes in hydrolytic and nonhydrolytic channel closing rates, in the corresponding double mutants these changes proved mostly additive with those caused by mutation T460S, suggesting little change in energetic coupling between either positions 460–1348 or positions 460–1375 during gating. These results provide independent support for a gating model in which ATP-bound composite site 1 remains closed throughout the gating cycle.

Insulin secretion in islets from mice with a double knockout for the dense core vesicle proteins islet antigen-2 (IA-2) and IA-2beta.

Islet antigen-2 (IA-2 or ICA 512) and IA-2beta (or phogrin) are major autoantigens in type 1 diabetes. They are located in dense core secretory vesicles including insulin granules, but their role in beta-cell function is unclear. Targeted disruption of either IA-2 or IA-2beta, or both, impaired glucose tolerance, an effect attributed to diminution of insulin secretion. In this study, we therefore characterized the dynamic changes in cytosolic Ca2+([Ca2+](c)) and insulin secretion in islets from IA-2/IA-2beta double knockout (KO) mice. High glucose (15 mM) induced biphasic insulin secretion in IA-2/IA-2beta KO islets, with a similar first phase and smaller second phase compared with controls. Since the insulin content of IA-2/IA-2beta KO islets was approximately 45% less than that of controls, fractional insulin secretion (relative to content) was thus increased during first phase and unaffected during second phase. This peculiar response occurred in spite of a slightly smaller rise in [Ca2+](c), could not be attributed to an alteration of glucose metabolism (NADPH fluorescence) and also was observed with tolbutamide. The dual control of insulin secretion via the K(ATP) channel-dependent triggering pathway and K(ATP) channel-independent amplifying pathway was unaltered in IA-2/IA-2beta KO islets, and so were the potentiations by acetylcholine or cAMP (forskolin). Intriguingly, amino acids, in particular the cationic arginine and lysine, induced larger fractional insulin secretion in IA-2/IA-2beta KO than control islets. In conclusion, IA-2 and IA-2beta are dispensable for exocytosis of insulin granules, but are probably more important for cargo loading and/or stability of dense core vesicles.

Pharmacological stimulation and inhibition of insulin secretion in mouse islets lacking ATP-sensitive K+ channels.

Background and purpose:  ATP‐sensitive potassium channels (KATP channels) in beta cells are a major target for insulinotropic drugs. Here, we studied the effects of selected stimulatory and inhibitory pharmacological agents in islets lacking KATP channels.

Involvement of F1296 and N1303 of CFTR in induced-fit conformational change in response to ATP binding at NBD2

The chloride ion channel cystic fibrosis transmembrane conductance regulator (CFTR) displays a typical adenosine trisphosphate (ATP)-binding cassette (ABC) protein architecture comprising two transmembrane domains, two intracellular nucleotide-binding domains (NBDs), and a unique intracellular regulatory domain. Once phosphorylated in the regulatory domain, CFTR channels can open and close when supplied with cytosolic ATP. Despite the general agreement that formation of a head-to-tail NBD dimer drives the opening of the chloride ion pore, little is known about how ATP binding to individual NBDs promotes subsequent formation of this stable dimer. Structural studies on isolated NBDs suggest that ATP binding induces an intra-domain conformational change termed “induced fit,” which is required for subsequent dimerization. We investigated the allosteric interaction between three residues within NBD2 of CFTR, F1296, N1303, and R1358, because statistical coupling analysis suggests coevolution of these positions, and because in crystal structures of ABC domains, interactions between these positions appear to be modulated by ATP binding. We expressed wild-type as well as F1296S, N1303Q, and R1358A mutant CFTR in Xenopus oocytes and studied these channels using macroscopic inside-out patch recordings. Thermodynamic mutant cycles were built on several kinetic parameters that characterize individual steps in the gating cycle, such as apparent affinities for ATP, open probabilities in the absence of ATP, open probabilities in saturating ATP in a mutant background (K1250R), which precludes ATP hydrolysis, as well as the rates of nonhydrolytic closure. Our results suggest state-dependent changes in coupling between two of the three positions (1296 and 1303) and are consistent with a model that assumes a toggle switch–like interaction pattern during the intra-NBD2 induced fit in response to ATP binding. Stabilizing interactions of F1296 and N1303 present before ATP binding are replaced by a single F1296-N1303 contact in ATP-bound states, with similar interaction partner toggling occurring during the much rarer ATP-independent spontaneous openings.

Conformational changes in the catalytically inactive nucleotide-binding site of CFTR

A central step in the gating of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is the association of its two cytosolic nucleotide-binding domains (NBDs) into a head-to-tail dimer, with two nucleotides bound at the interface. Channel opening and closing, respectively, are coupled to formation and disruption of this tight NBD dimer. CFTR is an asymmetric adenosine triphosphate (ATP)-binding cassette protein in which the two interfacial-binding sites (composite sites 1 and 2) are functionally different. During gating, the canonical, catalytically active nucleotide-binding site (site 2) cycles between dimerized prehydrolytic (state O1), dimerized post-hydrolytic (state O2), and dissociated (state C) forms in a preferential C→O1→O2→C sequence. In contrast, the catalytically inactive nucleotide-binding site (site 1) is believed to remain associated, ATP-bound, for several gating cycles. Here, we have examined the possibility of conformational changes in site 1 during gating, by studying gating effects of perturbations in site 1. Previous work showed that channel closure is slowed, both under hydrolytic and nonhydrolytic conditions, by occupancy of site 1 by N6-(2-phenylethyl)-ATP (P-ATP) as well as by the site-1 mutation H1348A (NBD2 signature sequence). Here, we found that P-ATP prolongs wild-type (WT) CFTR burst durations by selectively slowing (>2×) transition O1→O2 and decreases the nonhydrolytic closing rate (transition O1→C) of CFTR mutants K1250A (∼4×) and E1371S (∼3×). Mutation H1348A also slowed (∼3×) the O1→O2 transition in the WT background and decreased the nonhydrolytic closing rate of both K1250A (∼3×) and E1371S (∼3×) background mutants. Neither P-ATP nor the H1348A mutation affected the 1:1 stoichiometry between ATP occlusion and channel burst events characteristic to WT CFTR gating in ATP. The marked effect that different structural perturbations at site 1 have on both steps O1→C and O1→O2 suggests that the overall conformational changes that CFTR undergoes upon opening and coincident with hydrolysis at the active site 2 include significant structural rearrangement at site 1.

Dissecting the Molecular Mechanism of Nucleotide-Dependent Activation of the KtrAB K+ Transporter

KtrAB belongs to the Trk/Ktr/HKT superfamily of monovalent cation (K+ and Na+) transport proteins that closely resemble K+ channels. These proteins underlie a plethora of cellular functions that are crucial for environmental adaptation in plants, fungi, archaea, and bacteria. The activation mechanism of the Trk/Ktr/HKT proteins remains unknown. It has been shown that ATP stimulates the activity of KtrAB while ADP does not. Here, we present X-ray structural information on the KtrAB complex with bound ADP. A comparison with the KtrAB-ATP structure reveals conformational changes in the ring and in the membrane protein. In combination with a biochemical and functional analysis, we uncover how ligand-dependent changes in the KtrA ring are propagated to the KtrB membrane protein and conclude that, despite their structural similarity, the activation mechanism of KtrAB is markedly different from the activation mechanism of K+ channels.

Functional Characterization of the KtrAB Potassium Transport System

The Ktr transport system belongs to the large superfamily of Trk/Ktr/HKT ion transporters that share structural similarities with potassium and sodium channels. The KtrAB complex is composed by a dimer of the membrane-integrant KtrB and an octameric ring of the cytosolic KtrA protein, which is an RCK-domain. Each KtrB subunit has a K+ selective pore. We tested several nucleotides for eluting KtrA from a high-affinity resin and found that ADP and ATP are the most potent suggesting higher affinities towards the protein. The crystal structure of the complex, recently solved by our group, revealed that the ring conformation of KtrA changes upon nucleotide binding. To determine the functional effects of these nucleotides we reconstituted the KtrAB complex from B. subtilis into liposomes and measured the transport activity reflected by uptake of 86Rb+. Our results show that ATP activates the transporter, while the flux mediated by KtrAB-ADP is similar to the flux of KtrB reconstituted without KtrA. Increasing amounts of extraliposomal K+ or Na+ showed that Na+ permeates through the transporter, yet KtrAB favors K+ over Na+. In addition, the cytosolic ring regulates the activity of the transmembrane pore, however, it does not render selectivity towards ions as there are no selectivity differences between the KtrAB complex, either with ADP or ATP, and KtrB alone. In conclusion, our data shows that KtrA regulates the activity of KtrB and raises new ideas about the mechanism of activation by RCK domains in the superfamily. Current investigation attempts to characterize the molecular basis of activation.