Nucleotide-binding Fold Research Articles

The Cystic Fibrosis Gene

It’s now well over a year since the cystic fibrosis gene was cloned and there is still much to be done before its localisation can be translated into an improvement in health care for affected people. I’m not going to go into any details on how the gene was located, for this information (which is rather technical) see ref. 1. However to put it rather bluntly, despite the fact that the gene has been localised and sequenced has been sequenced, no-one really knows what it does. The cystic fibrosis gene has been named the CFTR gene (cystic fibrosis transmembrane conductance regulator). It is located on the long arm of chromosome 7 and is composed of 27 extrons which code for 1,400 amino-acid residues. There seem to be several different final products of the CFTR gene which result from the removal of exons from the first nucleotide binding fold. The functional significance of these products is not known.

HCN C-Terminal Region Speeds Activation Independently of Autoinhibition

The C-terminal Cyclic Nucleotide Binding (CNB) fold of HCN channels preferentially stabilizes the closed state, and this is relieved by cAMP binding or by CNB fold truncation [Wainger et al. (2001) Nature 411: 805]. Whereas the autoinhibition model was discovered in excised-patch conditions, we investigated the ability of the C-terminal region to regulate gating thermodynamics and kinetics with two-electrode voltage clamp on Xenopus oocytes. We examined wildtype channels (HCN2) with endogenous cAMP bound, C-terminal-truncated channels (HCN2 ΔCNB), and channels with autoinhibition imposed due to a mutation preventing cAMP binding (HCN2 R591E). Hyperpolarized V1/2 and faster deactivation t1/2 values were observed for HCN2 R591E, verifying its autoinhibition in intact oocytes. Surprisingly, we found conditions where the autoinhibition model is insufficient to explain the hyperpolarization-dependent activation kinetics. First, channels with autoinhibition relieved by cAMP binding exhibited faster activation (HCN2, 81 ± 17 ms at −150 mV) than channels with autoinhibition wholly removed by CNB fold truncation (HCN2 ΔCNB, 215 ± 110 ms at same voltage). Second, autoinhibited channels opened at a relatively fast rate (HCN2 R591E, 77 ± 16 ms) similar to the autoinhibition-free HCN2. We therefore propose a new in the C-terminal region which works distinctly from and opposite to autoinhibition in determining activation kinetics. The quickening effect of this element in HCN2 and HCN2 R591E were observed for voltages from −110 mV through −160 mV when compared to HCN2 ΔCNB. At −100 mV, as HCN2 R591E opened slower (385 ± 46 ms) than HCN2 (238 ± 33 ms) as predicted by the autoinhibition model. Therefore in intact oocytes, the autoinhibition and the quickening element mechanisms exert two opposing influences on HCN activation speeds with a balance that changes with voltage.

A Single Point Mutation in the Distal C-Terminal of the Pore Forming Kir6.1 Subunit Modifies ATP-Sensitive Potassium (KATP) Channel Regulation

KATP channels are hetero-octamers composed of two subunits; a pore forming Kir6 and a sulphonylurea receptor (SUR). SUR subunits regulate KATP channel gating allosterically, in response to nucleotides and pharmacological agents.We have previously shown that residue D323 of Kir6.2 is central to interaction with the nucleotide-binding fold 2 of SUR2A (Rubaiy et al, Biophysical J. 2011: 100; P432a). The aim of this study was to assess whether E332, the corresponding residue in Kir6.1, plays an equally important role in Kir6.1/SUR2A complexes. Wild type Kir6.1/SUR2A channels expressed in HEK293 cells required activation by potassium channel opener pinacidil (EC50 = 43.90 ± 1.28 µM), in the presence of UDP (10 mM) before passing current. Introduction of the single point mutation E332K into full length Kir6.1 caused constitutive opening of Kir6.1/SUR2A channels in the absence of pinacidil. Reinstatement of putative inter-subunit salt bridges by expressing Kir6.1-E332K with charge reversal mutants SUR2A-Q1336E or SUR2A-K1322D failed to restore regulated opening. Channels containing the Kir6.1E332K mutant were also insensitive to block by high concentrations of glibenclamide (100 µM). However, co-expression of Kir6.1-E332K with SUR2A-K1322D restored glibenclamide sensitivity to wild type levels ((IC50 = 9.12±1.12 nM), p < 0.15 versus wild type Kir6.1/SUR2A channel (IC50 = 6.14±1.13 nM). Together, these data suggest a key functional role for inter-subunit salt bridges involving Kir6.1-E332K. Constitutive channel opening on mutation of this residue suggests that Kir6.1E332 and salt bridge(s) formed between it and the SUR subunit are crucial for stabilizing closed states of Kir6.1-containing KATP channels.

Regulation of Cardiac ATP-sensitive Potassium Channel Surface Expression by Calcium/Calmodulin-dependent Protein Kinase II

Cardiac ATP-sensitive potassium (K(ATP)) channels are key sensors and effectors of the metabolic status of cardiomyocytes. Alteration in their expression impacts their effectiveness in maintaining cellular energy homeostasis and resistance to injury. We sought to determine how activation of calcium/calmodulin-dependent protein kinase II (CaMKII), a central regulator of calcium signaling, translates into reduced membrane expression and current capacity of cardiac K(ATP) channels. We used real-time monitoring of K(ATP) channel current density, immunohistochemistry, and biotinylation studies in isolated hearts and cardiomyocytes from wild-type and transgenic mice as well as HEK cells expressing wild-type and mutant K(ATP) channel subunits to track the dynamics of K(ATP) channel surface expression. Results showed that activation of CaMKII triggered dynamin-dependent internalization of K(ATP) channels. This process required phosphorylation of threonine at 180 and 224 and an intact (330)YSKF(333) endocytosis motif of the K(ATP) channel Kir6.2 pore-forming subunit. A molecular model of the μ2 subunit of the endocytosis adaptor protein, AP2, complexed with Kir6.2 predicted that μ2 docks by interaction with (330)YSKF(333) and Thr-180 on one and Thr-224 on the adjacent Kir6.2 subunit. Phosphorylation of Thr-180 and Thr-224 would favor interactions with the corresponding arginine- and lysine-rich loops on μ2. We concluded that calcium-dependent activation of CaMKII results in phosphorylation of Kir6.2, which promotes endocytosis of cardiac K(ATP) channel subunits. This mechanism couples the surface expression of cardiac K(ATP) channels with calcium signaling and reveals new targets to improve cardiac energy efficiency and stress resistance.

Crystallography, Evolution, and the Structure of Viruses

My undergraduate education in mathematics and physics was a good grounding for graduate studies in crystallographic studies of small organic molecules. As a postdoctoral fellow in Minnesota, I learned how to program an early electronic computer for crystallographic calculations. I then joined Max Perutz, excited to use my skills in the determination of the first protein structures. The results were even more fascinating than the development of techniques and provided inspiration for starting my own laboratory at Purdue University. My first studies on dehydrogenases established the conservation of nucleotide-binding structures. Having thus established myself as an independent scientist, I could start on my most cherished ambition of studying the structure of viruses. About a decade later, my laboratory had produced the structure of a small RNA plant virus and then, in another six years, the first structure of a human common cold virus. Many more virus structures followed, but soon it became essential to supplement crystallography with electron microscopy to investigate viral assembly, viral infection of cells, and neutralization of viruses by antibodies. A major guide in all these studies was the discovery of evolution at the molecular level. The conservation of three-dimensional structure has been a recurring theme, from my experiences with Max Perutz in the study of hemoglobin to the recognition of the conserved nucleotide-binding fold and to the recognition of the jelly roll fold in the capsid protein of a large variety of viruses.

Using a Low Denaturant Model To Explore the Conformational Features of Translocation-Active SecA

The SecA molecular nanomachine in bacteria uses energy from ATP hydrolysis to drive post-translational secretion of preproteins through the SecYEG translocon. Cytosolic SecA exists in a dimeric, "closed" state with relatively low ATPase activity. After binding to the translocon, SecA undergoes major conformational rearrangement, leading to a state that is structurally more "open", has elevated ATPase activity, and is active in translocation. The structural details underlying this conformational change in SecA remain incompletely defined. Most SecA crystal structures report on the cytosolic form; only one structure sheds light on a form of SecA that has engaged the translocon. We have used mild destabilization of SecA to trigger conformational changes that mimic those in translocation-active SecA and thus study its structural changes in a simplified, soluble system. Results from circular dichroism, tryptophan fluorescence, and limited proteolysis demonstrate that the SecA conformational reorganization involves disruption of several domain-domain interfaces, partial unfolding of the second nucleotide binding fold (NBF) II, partial dissociation of the helical scaffold domain (HSD) from NBF I and II, and restructuring of the 30 kDa C-terminal region. These changes account for the observed high translocation SecA ATPase activity because they lead to the release of an inhibitory C-terminal segment (called intramolecular regulator of ATPase 1, or IRA1) and of constraints on NBF II (or IRA2) that allow it to stimulate ATPase activity. The observed conformational changes thus position SecA for productive interaction with the SecYEG translocon and for transfer of segments of its passenger protein across the translocon.

Role of the amino-terminal transmembrane domain of sulfonylurea receptor SUR2B for coupling to KIR6.2, ligand binding, and oligomerization

ATP-sensitive K(+) (K(ATP)) channels consist of two types of subunits, K(IR)6.x that form the pore, and sulfonylurea receptors (SURs) that serve as regulatory subunits. SURs are ATP-binding cassette (ABC) proteins and contain, in addition to two nucleotide binding folds, the binding sites for channel openers such as diazoxide and P1075 and channel inhibitors such as glibenclamide (GBC) and repaglinide. Structurally, SURs differ from most eukaryotic ABC proteins by an additional amino-terminal transmembrane domain (TMD0); in case of SUR1, the subunit of the pancreatic K(ATP) channel, TMD0 serves as a major domain for association with K(IR). In this study we sought to elucidate the roles of TMD0 in SUR2B, the smooth muscle gating subunit, in the coupling between SUR2B and K(IR)6.2, in the self-association of SUR2B and in channel modulator binding to SUR2B. SUR2B has a weaker affinity for sulfonylureas thus SUR2B(Y1206S), with a higher affinity for GBC, but an equivalent opener binding was used. Association of SUR2B(YS)Δ, lacking TMD0, with K(IR)6.2 was shown by immunoprecipitation; however, no evidence for formation of functional channels was obtained. SUR2B(YS)Δ self-associates like SUR2B(YS) and binds GBC, repaglinide, and P1075 with slightly reduced affinities. The binding profile of the SUR2B(YS)Δ/K(IR)6.2 complex differs slightly but significantly from that of SUR2B(YS)Δ alone showing impaired allosteric coupling of binding sites. We conclude that TMD0 is not required for oligomerization of SUR2B, is of only minor importance in ligand binding, but is essential for both functional and allosteric coupling of SUR2B to K(IR)6.2.

Syntaxin-1A inhibits K ATP channels by interacting with specific conserved motifs within sulfonylurea receptor 2A

We previously demonstrated that syntaxin (Syn)-1A is present in the sarcolemma of rat cardiomyocytes and binds sulfonylurea receptor (SUR) 2A nucleotide binding folds (NBFs) to inhibit ATP-sensitive potassium (K ATP) channel. Here, we examined for the precise domains within the NBFs of SUR2A that may interact with Syn-1A. Specifically, we tested truncated NBF protein segments encompassing the conserved motifs Walker A (W A), signature/Linker (L), and Walker B (W B). In vitro binding results indicate that the domains encompassing W A and L of NBF-1 and all three conserved motifs of NBF-2 bound Syn-1A. Electrophysiological studies, employing inside-out patch-clamp recordings from SUR2A/Kir6.2 expressing HEK cells and mouse cardiomyocytes, show that W B and L of NBF-1 and all three NBF-2 truncated protein segments reduced Syn-1A inhibition of SUR2A/K ATP channels. Remarkably, these same NBF-1 and -2 truncated proteins could independently disrupt the intimate FRET interactions of full length SUR2A (− mCherry) and Syn-1A (− EGFP). These results taken together indicate that Syn-1A possibly maintains inhibition of cardiac ventricular K ATP channels by binding to large regions of NBF-1 and NBF-2 to stabilize the NBF-1-NBF-2 heterodimer formation and prevent ATP-binding and ATP hydrolysis. Since K ATP channels are closely coupled to metabolic states, we postulate that these very intimate Syn-1A-SUR2A interactions are critically important for myocardial protection during stress, in which profound changes in metabolic factors (pH, ATP) could modulate these Syn-1A-SUR2A interactions.

Structural Analysis of the Essential Resuscitation Promoting Factor YeaZ Suggests a Mechanism of Nucleotide Regulation through Dimer Reorganization

BackgroundThe yeaZ gene product forms part of the conserved network YjeE/YeaZ/YgjD essential for the survival of many Gram-negative eubacteria. Among other as yet unidentified roles, YeaZ functions as a resuscitation promoting factor required for survival and resuscitation of cells in a viable but non-culturable (VBNC) state.Methodology/Principal FindingsIn order to investigate in detail the structure/function relationship of this family of proteins we have performed X-ray crystallographic studies of Vibrio parahaemolyticus YeaZ. The YeaZ structure showed that it has a classic actin-like nucleotide-binding fold. Comparisons of this crystal structure to that of available homologues from E. coli, T. maritima and S. typhimurium revealed two distinctly different modes of dimer formation. In one form, prevalent in the absence of nucleotide, the putative nucleotide-binding site is incomplete, lacking a binding pocket for a nucleotide base. In the second form, residues from the second subunit complete the nucleotide-binding site. This suggests that the two dimer architectures observed in the crystal structures correspond to a free and a nucleotide-bound form of YeaZ. A multiple sequence alignment of YeaZ proteins from different bacteria allowed us to identify a large conserved hydrophobic patch on the protein surface that becomes exposed upon nucleotide-driven dimer re-arrangement. We hypothesize that the transition between two dimer architectures represents the transition between the ‘on’ and ‘off’ states of YeaZ. The effect of this transition is to alternately expose and bury a docking site for the partner protein YgjD.Conclusions/SignificanceThis paper provides the first structural insight into the putative mechanism of nucleotide regulation of YeaZ through dimer reorganization. Our analysis suggests that nucleotide binding to YeaZ may act as a regulator or switch that changes YeaZ shape, allowing it to switch partners between YjeE and YgjD.

Syntaxin-1A Interacts with Distinct Domains within Nucleotide-binding Folds of Sulfonylurea Receptor 1 to Inhibit β-Cell ATP-sensitive Potassium Channels

The ATP-sensitive potassium (K(ATP)) channel regulates pancreatic β-cell function by linking metabolic status to electrical activity. Syntaxin-1A (Syn-1A), a SNARE protein mediating exocytotic fusion, binds and inhibits the K(ATP) channel via the nucleotide-binding folds (NBFs) of its sulfonylurea receptor-1 (SUR1) regulatory subunit. In this study, we elucidated the precise regions within the NBFs required for Syn-1A-mediated K(ATP) inhibition, using in vitro binding assays, whole cell patch clamp and FRET assay. Specifically, NBF1 and NBF2 were each divided into three subregions, Walker A (W(A)), signature sequence linker, and Walker B (W(B)), to make GST fusion proteins. In vitro binding assays revealed that Syn-1A associates with W(A) and W(B) regions of both NBFs. Patch clamp recordings on INS-1 and primary rat β-cells showed that Syn-1A-mediated channel inhibition was reversed by co-addition of NBF1-W(B) (not NBF1-W(A)), NBF2-W(A), and NBF2-W(B). The findings were corroborated by FRET studies showing that these truncates disrupted Syn-1A interactions with full-length SUR1. To further identify the binding sites, series single-site mutations were made in the Walker motifs of the NBFs. Only NBF1-W(A) (K719M) or NBF2-W(A) (K1385M) mutant no longer bound to Syn-1A; K1385M failed to disrupt Syn-1A-mediated inhibition of K(ATP) channels. These data suggest that NBF1-W(A) (Lys-719) and NBF2-W(A) (Lys-1385) are critical for Syn-1A-K(ATP) channel interaction. Taken together, Syn-1A intimately and functionally associates with the SUR1-NBF1/2 dimer via direct interactions with W(A) motifs and sites adjacent to W(B) motifs of NBF1 and NBF2 but transduces its inhibitory actions on K(ATP) channel activity via some but not all of these NBF domains.

Zebrafish: A Model System to Study Heritable Skin Diseases

Heritable skin diseases represent a broad spectrum of clinical manifestations due to mutations in ∼500 different genes. A number of model systems have been developed to advance our understanding of the pathomechanisms of genodermatoses. Zebrafish (Danio rerio), a freshwater vertebrate, has a well-characterized genome, the expression of which can be easily manipulated. The larvae develop rapidly, with all major organs having developed by 5-6 days post-fertilization, including the skin, consisting of the epidermis comprising two cell layers and separated from the dermal collagenous matrix by a basement membrane. This perspective highlights the morphological and ultrastructural features of zebrafish skin, in the context of cutaneous gene expression. These observations suggest that zebrafish provide a useful model system to study the molecular aspects of skin development, as well as the pathogenesis and treatment of select heritable skin diseases.

ATP Modulates Interaction of Syntaxin-1A with Sulfonylurea Receptor 1 to Regulate Pancreatic β-Cell KATP Channels

ATP-sensitive potassium (K(ATP)) channels are regulated by a variety of cytosolic factors (adenine nucleotides, Mg(2+), phospholipids, and pH). We previously reported that K(ATP) channels are also regulated by endogenous membrane-bound SNARE protein syntaxin-1A (Syn-1A), which binds both nucleotide-binding folds of sulfonylurea receptor (SUR)1 and 2A, causing inhibition of K(ATP) channel activity in pancreatic islet β-cells and cardiac myocytes, respectively. In this study, we show that ATP dose-dependently inhibits Syn-1A binding to SUR1 at physiological concentrations, with the addition of Mg(2+) causing a decrease in the ATP-induced inhibitory effect. This ATP disruption of Syn-1A binding to SUR1 was confirmed by FRET analysis in living HEK293 cells. Electrophysiological studies in pancreatic β-cells demonstrated that reduced ATP concentrations increased K(ATP) channel sensitivity to Syn-1A inhibition. Depletion of endogenous Syn-1A in insulinoma cells by botulinum neurotoxin C1 proteolysis followed by rescue with exogenous Syn-1A showed that Syn-1A modulates K(ATP) channel sensitivity to ATP. Thus, our data indicate that although both ATP and Syn-1A independently inhibit β-cell K(ATP) channel gating, they could also influence the sensitivity of K(ATP) channels to each other. These findings provide new insight into an alternate mechanism by which ATP regulates pancreatic β-cell K(ATP) channel activity, not only by its direct actions on Kir6.2 pore subunit, but also via ATP modulation of Syn-1A binding to SUR1.

Differential roles for SUR subunits in KATP channel membrane targeting and regulation

since their discovery over 25 years ago, ATP-sensitive K+ (KATP) channels have been studied extensively in many tissues, including heart, pancreas, brain, skeletal and smooth muscle, pituitary, and kidney ([6][1], [10][2], [18][3], [19][4]). KATP channels are critical sensors that couple cellular

Neonatal Diabetes Caused by Mutations in Sulfonylurea Receptor 1: Interplay between Expression and Mg-Nucleotide Gating Defects of ATP-Sensitive Potassium Channels

Abstract Context: ATP-sensitive potassium (KATP) channels regulate insulin secretion by coupling glucose metabolism to β-cell membrane potential. Gain-of-function mutations in the sulfonylurea receptor 1 (SUR1) or Kir6.2 channel subunit underlie neonatal diabetes. Objective: The objective of the study was to determine the mechanisms by which two SUR1 mutations, E208K and V324M, associated with transient neonatal diabetes affect KATP channel function. Design: E208K or V324M mutant SUR1 was coexpressed with Kir6.2 in COS cells, and expression and gating properties of the resulting channels were assessed biochemically and electrophysiologically. Results: Both E208K and V324M augment channel response to MgADP stimulation without altering sensitivity to ATP4− or sulfonylureas. Surprisingly, whereas E208K causes only a small increase in MgADP response consistent with the mild transient diabetes phenotype, V324M causes a severe activating gating defect. Unlike E208K, V324M also impairs channel expression at the cell surface, which is expected to dampen its functional impact on β-cells. When either mutation was combined with a mutation in the second nucleotide binding domain of SUR1 previously shown to abolish Mg-nucleotide response, the activating effect of E208K and V324M was also abolished. Moreover, combination of E208K and V324M results in channels with Mg-nucleotide sensitivity greater than that seen in individual mutations alone. Conclusion: The results demonstrate that E208K and V324M, located in distinct domains of SUR1, enhance transduction of Mg-nucleotide stimulation from the SUR1 nucleotide binding folds to Kir6.2. Furthermore, they suggest that diabetes severity is determined by interplay between effects of a mutation on channel expression and channel gating.

Crystal Structure of a Virus-Encoded Putative Glycosyltransferase

The chloroviruses (family Phycodnaviridae), unlike most viruses, encode some, if not most, of the enzymes involved in the glycosylation of their structural proteins. Annotation of the gene product B736L from chlorovirus NY-2A suggests that it is a glycosyltransferase. The structure of the recombinantly expressed B736L protein was determined by X-ray crystallography to 2.3-Å resolution, and the protein was shown to have two nucleotide-binding folds like other glycosyltransferase type B enzymes. This is the second structure of a chlorovirus-encoded glycosyltransferase and the first structure of a chlorovirus type B enzyme to be determined. B736L is a retaining enzyme and belongs to glycosyltransferase family 4. The donor substrate was identified as GDP-mannose by isothermal titration calorimetry and was shown to bind into the cleft between the two domains in the protein. The active form of the enzyme is probably a dimer in which the active centers are separated by about 40 Å.

Neonatal Diabetes Caused by Mutations in Sulfonylurea Receptor 1: Interplay between Expression and Mg-Nucleotide Gating Defects of ATP-Sensitive Potassium Channels

ATP-sensitive potassium (KATP) channels regulate insulin secretion by coupling glucose metabolism to β-cell membrane potential. Gain-of-function mutations in the sulfonylurea receptor 1 (SUR1) or Kir6.2 channel subunit underlie neonatal diabetes. The objective of the study was to determine the mechanisms by which two SUR1 mutations, E208K and V324M, associated with transient neonatal diabetes affect KATP channel function. E208K or V324M mutant SUR1 was coexpressed with Kir6.2 in COS cells, and expression and gating properties of the resulting channels were assessed biochemically and electrophysiologically. Both E208K and V324M augment channel response to MgADP stimulation without altering sensitivity to ATP4- or sulfonylureas. Surprisingly, whereas E208K causes only a small increase in MgADP response consistent with the mild transient diabetes phenotype, V324M causes a severe activating gating defect. Unlike E208K, V324M also impairs channel expression at the cell surface, which is expected to dampen its functional impact on β-cells. When either mutation was combined with a mutation in the second nucleotide binding domain of SUR1 previously shown to abolish Mg-nucleotide response, the activating effect of E208K and V324M was also abolished. Moreover, combination of E208K and V324M results in channels with Mg-nucleotide sensitivity greater than that seen in individual mutations alone. The results demonstrate that E208K and V324M, located in distinct domains of SUR1, enhance transduction of Mg-nucleotide stimulation from the SUR1 nucleotide binding folds to Kir6.2. Furthermore, they suggest that diabetes severity is determined by interplay between effects of a mutation on channel expression and channel gating.

PERSPECTIVES: Silencing vascular smooth muscle ATP-sensitive K+channels with caveolin-1

PERSPECTIVES: Silencing vascular smooth muscle ATP-sensitive K⁺channels with caveolin-1

Identification of an ABCB/P-glycoprotein-specific Inhibitor of Auxin Transport by Chemical Genomics

Plant development and physiology are widely determined by the polar transport of the signaling molecule auxin. This process is controlled on the cellular efflux level catalyzed by members of the PIN (pin-formed) and ABCB (ATP-binding cassette protein subfamily B)/P-glycoprotein family that can function independently and coordinately. In this study, we have identified by means of chemical genomics a novel auxin transport inhibitor (ATI), BUM (2-[4-(diethylamino)-2-hydroxybenzoyl]benzoic acid), that efficiently blocks auxin-regulated plant physiology and development. In many respects, BUM resembles the functionality of the diagnostic ATI, 1-N-naphtylphtalamic acid (NPA), but it has an IC(50) value that is roughly a factor 30 lower. Physiological analysis and binding assays identified ABCBs, primarily ABCB1, as key targets of BUM and NPA, whereas PIN proteins are apparently not directly affected. BUM is complementary to NPA by having distinct ABCB target spectra and impacts on basipetal polar auxin transport in the shoot and root. In comparison with the recently identified ATI, gravacin, it lacks interference with ABCB membrane trafficking. Individual modes or targets of action compared with NPA are reflected by apically shifted root influx maxima that might be the result of altered BUM binding preferences or affinities to the ABCB nucleotide binding folds. This qualifies BUM as a valuable tool for auxin research, allowing differentiation between ABCB- and PIN-mediated efflux systems. Besides its obvious application as a powerful weed herbicide, BUM is a bona fide human ABCB inhibitor with the potential to restrict multidrug resistance during chemotherapy.

Identification of a Heterozygous Sulfonylurea Receptor 1 Mutation that Exerts a Strong Dominant-Negative Effect on KATP Channel Response to MgADP

ATP-sensitive potassium (KATP) channels couple cell metabolism to cell excitability thus mediating a range of physiological responses to metabolic stress. In pancreatic β-cells, KATP channels regulate insulin secretion according to plasma glucose concentrations. Mutations in the channel genes ABCC8 encoding the regulatory sulfonylurea receptor 1 or KCNJ11 encoding the pore-forming inwardly rectifying potassium channel Kir6.2 that lead to loss of channel function are causes of congenital hyperinsulinism, characterized by inappropriate insulin secretion despite severe hypoglycemia. The disease-causing mutations can be recessively inherited, which are usually associated with severe disease phenotype, or dominantly inherited, which are commonly associated with less severe disease phenotype and are clinically responsive to the KATP channel opener diazoxide. The most prominent channel gating defects caused by mutations identified in congenital hyperinsulinism is loss of channel response to the stimulatory effect of MgADP and diazoxide. Here, we have identified a heterozygous in-frame insertion mutation in exon 37 of the ABCC8 gene that results in duplication of two amino acids ala-ser at position 1508 in the second nucleotide binding fold 2 (NBF2) from a patient with severe congenital hyperinsulinism unresponsive to diazoxide. Functional characterization of mutant channels reconstituted in COS cells show that the mutation does not disrupt surface expression of the channel but abolishes channel response to MgADP and diazoxide. Strikingly, in simulated heterozygous expression condition, the mutant SUR1 subunit exhibited a strong dominant negative effect on WT SUR1 subunit such that the MgADP and diazoxide response are nearly identical to homomeric mutant channels. This clinical and in-vitro strong dominant negative effect is distinct from other heterozygous mutations reported previously present an interesting case for understanding the structural mechanisms underlying channel response to MgADP and diazoxide.

Isolation and characterization of an ABC-transporter cDNA clone from wheat (Triticum aestivum L.)

The outmost layer of the surface of any land plant is covered with wax, which protects the inner tissues of plants. The Arabidopsis ATP binding cassette (ABC) transporter, Cer5, was found to be an exporter of wax molecules. Full-length wheat tacer5 cDNA, a sequence homologous to cer5, was isolated. Similar to other transporters of the ABCG subfamily, TaCer5 also has a typical basic structure of NBD-MSD (nucleotide-binding folds domain - membrane-spanning domain). Although the identity scores of TaCer5 and other ABCG transporters are not very high, amino acid residues of NBDs and MSDs are highly conserved among them. Tacer5 is a tissue-specific gene. The transcripts of tacer5 were found in leaf tissues and predominantly in stem tissues but were hardly detectable in root tissues. Stress inhibited the expression of tacer5. The expression was significantly decreased by salinity, low temperature, and 48-hour-darkness and also by exogenous phytohormone abscisic acid and salicylic acid, although not significantly. Exposure to polyethylene glycol either increased the expression or decreased it, depending on the duration of exposure. All the above factors also decreased the extent of wax deposited on wheat leaves. It is therefore concluded that transport of wax molecules is affected by environmental factors.