External Ba2+ block of Kv4.2 channels is enhanced in the closed-inactivated state

2013 ◽  
Vol 304 (4) ◽  
pp. C370-C381 ◽  
Author(s):  
Steven J. Kehl ◽  
David Fedida ◽  
Zhuren Wang
Keyword(s):  
Conformational Changes ◽  
Binding Sites ◽  
Direct Evidence ◽  
Cell Voltage ◽  
Hek293 Cells ◽  
Barium Ions ◽  
Pore Mouth ◽  
Dependent Inhibition ◽  
In Series ◽  
Outer Pore

The effect of external barium ions on rat Kv4.2 channels expressed in HEK293 cells was investigated using whole cell, voltage-clamp recordings to determine its mechanism of action as well as its usefulness as a tool to probe the permeation pathway. Ba2+ caused a concentration-dependent inhibition of current that was antagonized by increasing the external concentration of K+ ([K+]o), and the concentration and time dependence of the inhibition were well fitted by a model involving two binding sites aligned in series. Recovery from current inhibition was enhanced by increasing the intensity, duration, or frequency of depolarizing steps or by increasing [K+]o. These properties are consistent with the conclusion that Ba2+ is a permeant ion that, by virtue of a stable interaction with a deep pore site, is able to block conduction. This blocking action was subsequently exploited to gain insights into the pore configuration in different channel states. In addition to blocking one or more states populated by brief depolarizing pulses to 80 mV, Ba2+ blocked closed channels [the membrane voltage ( Vm) = −80 mV] and closed-inactivated channels ( Vm = −40 mV). Interestingly, the block of closed-inactivated channels was faster and more complete than for closed channels, which we interpret to mean that conformational changes underlying closed-state inactivation (CSI) enhance Ba2+ binding and that the outer pore mouth remains patent during CSI. This provides the first direct evidence that an inactivation process involving a constriction of the outer pore mouth does not account for CSI in Kv4.2.

scholarly journals The Blood-Brain Barrier Thyroxine Transporter Organic Anion-Transporting Polypeptide 1c1 Displays Atypical Transport Kinetics

Endocrinology ◽  
2009 ◽  
Vol 150 (11) ◽  
pp. 5153-5162 ◽  
Author(s):  
Daniel E. Westholm ◽  
David R. Salo ◽  
Kevin J. Viken ◽  
Jon N. Rumbley ◽  
Grant W. Anderson
Keyword(s):  
Binding Sites ◽  
Organic Anion ◽  
Transport Processes ◽  
Transport Kinetics ◽  
Brain Barrier ◽  
Hek293 Cells ◽  
Published Data ◽  
Organic Anion Transporting Polypeptide ◽  
Wide Range ◽  
Dependent Inhibition

Organic anion-transporting polypeptide (Oatp) 1c1 is a high-affinity T4 transporter expressed in brain barrier cells. Oatp1c1 transports a variety of additional ligands including the conjugated sterol estradiol 17β-glucuronide (E217βG). Intriguingly, published data suggest that E217βG inhibition of Oatp1c1-mediated T4 transport exhibits characteristics suggestive of atypical transport kinetics. To determine whether Oatp1c1 exhibits atypical transport kinetics, we first performed detailed T4 and E217βG uptake assays using Oatp1c1 stably transfected HEK293 cells and a wide range of T4 and E217βG concentrations (100 pm to 300 nm and 27 nm to 200 μm, respectively). Eadie-Hofstee plots derived from these detailed T4 and E217βG uptake experiments display a biphasic profile consistent with atypical transport kinetics. These data along with T4 and E217βG cis-inhibition dose-response measurements revealed shared high- and low-affinity Oatp1c1 binding sites for T4 and E217βG. T4 and E217βG recognized these Oatp1c1 binding sites with opposite preferences. In addition, sterols glucuronidated in the 17 or 21 position, exhibited preferential substrate-dependent inhibition of Oatp1c1 transport, inhibiting Oatp1c1-mediated E217βG transport more strongly than T4 transport. Together these data reveal that Oatp1c1-dependent substrate transport is a complex process involving substrate interaction with multiple binding sites and competition for binding with a variety of other substrates. A thorough understanding of atypical Oatp1c1 transport processes and substrate-dependent inhibition will allow better prediction of endo- and xenobiotic interactions with the Oatp transporter.

scholarly journals Monoclonal antibodies detect receptor-induced binding sites in Glu-plasminogen

Blood ◽  
2011 ◽  
Vol 118 (6) ◽  
pp. 1653-1662 ◽  
Author(s):  
Jaena Han ◽  
Nagyung Baik ◽  
Kee-Hwan Kim ◽  
Jian-Ming Yang ◽  
Gye Won Han ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Conformational Changes ◽  
Binding Sites ◽  
Direct Evidence ◽  
Immunoaffinity Chromatography ◽  
Tandem Mass ◽  
Linear Epitope ◽  
Cell Surfaces ◽  
Protease Domain ◽  
Reaction In Solution

Abstract When Glu-plasminogen binds to cells, its activation to plasmin is markedly enhanced compared with the reaction in solution, suggesting that Glu-plasminogen on cell surfaces adopts a conformation distinct from that in solution. However, direct evidence for such conformational changes has not been obtained. Therefore, we developed anti-plasminogen mAbs to test the hypothesis that Glu-plasminogen undergoes conformational changes on its interaction with cells. Six anti-plasminogen mAbs (recognizing 3 distinct epitopes) that preferentially recognized receptor-induced binding sites (RIBS) in Glu-plasminogen were obtained. The mAbs also preferentially recognized Glu-plasminogen bound to the C-terminal peptide of the plasminogen receptor, Plg-RKT, and to fibrin, plasmin-treated fibrinogen, and Matrigel. We used trypsin proteolysis, immunoaffinity chromatography, and tandem mass spectrometry and identified Glu-plasminogen sequences containing epitopes recognized by the anti-plasminogen-RIBS mAbs: a linear epitope within a domain linking kringles 1 and 2; a nonlinear epitope contained within the kringle 5 domain and the latent protease domain; and a nonlinear epitope contained within the N-terminal peptide of Glu-plasminogen and the latent protease domain. Our results identify neoepitopes latent in soluble Glu-plasminogen that become available when Glu-plasminogen binds to cells and demonstrate that binding of Glu-plasminogen to cells induces a conformational change in Glu-plasminogen distinct from that of Lys-Pg.

scholarly journals Ligand-Induced conformational changes in the lactose permease ofescherichia coli: Evidence for two binding sites

1994 ◽  
Vol 3 (12) ◽  
pp. 2294-2301 ◽  
Author(s):  
Jianhua Wu ◽  
Stathis Frillingos ◽  
John Voss ◽  
H. Ronald Kaback
Keyword(s):  
Conformational Changes ◽  
Binding Sites ◽  
Ofescherichia Coli ◽  
Lactose Permease

scholarly journals Human organic anion transporter MRP4 (ABCC4) is an efflux pump for the purine end metabolite urate with multiple allosteric substrate binding sites

2005 ◽  
Vol 288 (2) ◽  
pp. F327-F333 ◽  
Author(s):  
Rémon A. M. H. Van Aubel ◽  
Pascal H. E. Smeets ◽  
Jeroen J. M. W. van den Heuvel ◽  
Frans G. M. Russel
Keyword(s):  
Binding Sites ◽  
Efflux Pump ◽  
Substrate Binding ◽  
Organic Anion ◽  
Apical Cell ◽  
Cell Uptake ◽  
Hek293 Cells ◽  
Hill Coefficient ◽  
Anion Transporter ◽  
Substrate Binding Sites

The end product of human purine metabolism is urate, which is produced primarily in the liver and excreted by the kidney through a well-defined basolateral blood-to-cell uptake step. However, the apical cell-to-urine efflux mechanism is as yet unidentified. Here, we show that the renal apical organic anion efflux transporter human multidrug resistance protein 4 (MRP4), but not apical MRP2, mediates ATP-dependent urate transport via a positive cooperative mechanism ( Km of 1.5 ± 0.3 mM, Vmax of 47 ± 7 pmol·mg−1·min−1, and Hill coefficient of 1.7 ± 0.2). In HEK293 cells overexpressing MRP4, intracellular urate levels were lower than in control cells. Urate inhibited methotrexate transport (IC50 of 235 ± 8 μM) by MRP4, did not affect cAMP transport, whereas cGMP transport was stimulated. Urate shifted cGMP transport by MRP4 from positive cooperativity ( Km and Vmax value of 180 ± 20 μM and 58 ± 4 pmol·mg−1·min−1, respectively, Hill coefficient of 1.4 ± 0.1) to single binding site kinetics ( Km and Vmax value of 2.2 ± 0.9 mM and 280 ± 50 pmol·mg−1·min−1, respectively). Finally, MRP4 could transport urate simultaneously with cAMP or cGMP. We conclude that human MRP4 is a unidirectional efflux pump for urate with multiple allosteric substrate binding sites. We propose MRP4 as a candidate transporter for urinary urate excretion and suggest that MRP4 may also mediate hepatic export of urate into the circulation, because of its basolateral expression in the liver.

scholarly journals Inhibition of acanthamoeba actomyosin-II ATPase activity and mechanochemical function by specific monoclonal antibodies.

1984 ◽  
Vol 99 (3) ◽  
pp. 1024-1033 ◽  
Author(s):  
D P Kiehart ◽  
T D Pollard
Keyword(s):  
Monoclonal Antibodies ◽  
Atpase Activity ◽  
Conformational Changes ◽  
Binding Sites ◽  
Polyclonal Antibodies ◽  
Myosin Ii ◽  
Antigenic Site ◽  
Actin Binding ◽  
Filamentous Form ◽  
Antibody Effects

Monoclonal and polyclonal antibodies that bind to myosin-II were tested for their ability to inhibit myosin ATPase activity, actomyosin ATPase activity, and contraction of cytoplasmic extracts. Numerous antibodies specifically inhibit the actin activated Mg++-ATPase activity of myosin-II in a dose-dependent fashion, but none blocked the ATPase activity of myosin alone. Control antibodies that do not bind to myosin-II and several specific antibodies that do bind have no effect on the actomyosin-II ATPase activity. In most cases, the saturation of a single antigenic site on the myosin-II heavy chain is sufficient for maximal inhibition of function. Numerous monoclonal antibodies also block the contraction of gelled extracts of Acanthamoeba cytoplasm. No polyclonal antibodies tested inhibited ATPase activity or gel contraction. As expected, most antibodies that block actin-activated ATPase activity also block gel contraction. Exceptions were three antibodies M2.2, -15, and -17, that appear to uncouple the ATPase activity from gel contraction: they block gel contraction without influencing ATPase activity. The mechanisms of inhibition of myosin function depends on the location of the antibody-binding sites. Those inhibitory antibodies that bind to the myosin-II heads presumably block actin binding or essential conformational changes in the myosin heads. A subset of the antibodies that bind to the proximal end of the myosin-II tail inhibit actomyosin-II ATPase activity and gel contraction. Although this part of the molecule is presumably some distance from the ATP and actin-binding sites, these antibody effects suggest that structural domains in this region are directly involved with or coupled to catalysis and energy transduction. A subset of the antibodies that bind to the tip of the myosin-II tail appear to inhibit ATPase activity and contraction through their inhibition of filament formation. They provide strong evidence for a substantial enhancement of the ATPase activity of myosin molecules in filamentous form and suggest that the myosin filaments may be required for cell motility.

scholarly journals On the dynamics of some small structural motifs in rRNA upon ligand binding

2008 ◽  
Vol 73 (1) ◽  
pp. 41-53
Author(s):  
Aleksandra Rakic ◽  
Petar Mitrasinovic
Keyword(s):  
Molecular Dynamics ◽  
Conformational Changes ◽  
Binding Sites ◽  
De Novo ◽  
Reference Structure ◽  
Base Pairs ◽  
Rna Sequences ◽  
Conformational Model ◽  
Thermodynamic Variables ◽  
Dynamics Simulations

The present study characterizes using molecular dynamics simulations the behavior of the GAA (1186-1188) hairpin triloops with their closing c-g base pairs in large ribonucleoligand complexes (PDB IDs: 1njn, 1nwy, 1jzx). The relative energies of the motifs in the complexes with respect to that in the reference structure (unbound form of rRNA; PDB ID: 1njp) display the trends that agree with those of the conformational parameters reported in a previous study1 utilizing the de novo pseudotorsional (?,?) approach. The RNA regions around the actual RNA-ligand contacts, which experience the most substantial conformational changes upon formation of the complexes were identified. The thermodynamic parameters, based on a two-state conformational model of RNA sequences containing 15, 21 and 27 nucleotides in the immediate vicinity of the particular binding sites, were evaluated. From a more structural standpoint, the strain of a triloop, being far from the specific contacts and interacting primarily with other parts of the ribosome, was established as a structural feature which conforms to the trend of the average values of the thermodynamic variables corresponding to the three motifs defined by the 15-, 21- and 27-nucleotide sequences. From a more functional standpoint, RNA-ligand recognition is suggested to be presumably dictated by the types of ligands in the complexes.

scholarly journals Positive co-operative binding at two weak lysine-binding sites governs the Glu-plasminogen conformational change

1992 ◽  
Vol 285 (2) ◽  
pp. 419-425 ◽  
Author(s):  
U Christensen ◽  
L Mølgaard
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
Conformational Change ◽  
Conformational Changes ◽  
Binding Sites ◽  
High Specificity ◽  
Stopped Flow ◽  
Protein Fluorescence ◽  
Lysine Residues ◽  
Kinetics Of

The kinetics of a series of Glu-plasminogen ligand-binding processes were investigated at pH 7.8 and 25 degrees C (in 0.1 M-NaCl). The ligands include compounds analogous to C-terminal lysine residues and to normal lysine residues. Changes of the Glu-plasminogen protein fluorescence were measured in a stopped-flow instrument as a function of time after rapid mixing of Glu-plasminogen and ligand at various concentrations. Large positive fluorescence changes (approximately 10%) accompany the ligand-induced conformational changes of Glu-plasminogen resulting from binding at weak lysine-binding sites. Detailed studies of the concentration-dependencies of the equilibrium signals and the rate constants of the process induced by various ligands showed the conformational change to involve two sites in a concerted positive co-operative process with three steps: (i) binding of a ligand at a very weak lysine-binding site that preferentially, but not exclusively, binds C-terminal-type lysine ligands, (ii) the rate-determining actual-conformational-change step and (iii) binding of one more lysine ligand at a second weak lysine-binding site that then binds the ligand more tightly. Further, totally independent initial small negative fluorescence changes (approximately 2-4%) corresponding to binding at the strong lysine-binding site of kringle 1 [Sottrup-Jensen, Claeys, Zajdel, Petersen & Magnusson (1978) Prog. Chem. Fibrinolysis Thrombolysis 3, 191-209] were observed for the C-terminal-type ligands. The finding that the conformational change in Glu-plasminogen involves two weak lysine-binding sites indicates that the effect cannot be assigned to any single kringle and that the problem of whether kringle 4 or kringle 5 is responsible for the process resolves itself. Probably kringle 4 and 5 are both participating. The involvement of two lysine binding-sites further makes the high specificity of Glu-plasminogen effectors more conceivable.

scholarly journals Conformational epitopes at cadherin calcium-binding sites and p120-catenin phosphorylation regulate cell adhesion

2012 ◽  
Vol 23 (11) ◽  
pp. 2092-2108 ◽  
Author(s):  
Yuliya I. Petrova ◽  
MarthaJoy M. Spano ◽  
Barry M. Gumbiner
Keyword(s):  
Cell Surface ◽  
Conformational Changes ◽  
Binding Sites ◽  
Calcium Binding ◽  
Cell Types ◽  
P120 Catenin ◽  
Physiological Regulation ◽  
Conformational Epitopes ◽  
Calcium Binding Sites ◽  
E Cadherin

We investigated changes in cadherin structure at the cell surface that regulate its adhesive activity. Colo 205 cells are nonadhesive cells with a full but inactive complement of E-cadherin–catenin complexes at the cell surface, but they can be triggered to adhere and form monolayers. We were able to distinguish the inactive and active states of E-cadherin at the cell surface by using a special set of monoclonal antibodies (mAbs). Another set of mAbs binds E-cadherin and strongly activates adhesion. In other epithelial cell types these activating mAbs inhibit growth factor–induced down-regulation of adhesion and epithelial morphogenesis, indicating that these phenomena are also controlled by E-cadherin activity at the cell surface. Both types of mAbs recognize conformational epitopes at different interfaces between extracellular cadherin repeat domains (ECs), especially near calcium-binding sites. Activation also induces p120-catenin dephosphorylation, as well as changes in the cadherin cytoplasmic domain. Moreover, phospho-site mutations indicate that dephosphorylation of specific Ser/Thr residues in the N-terminal domain of p120-catenin mediate adhesion activation. Thus physiological regulation of the adhesive state of E-cadherin involves physical and/or conformational changes in the EC interface regions of the ectodomain at the cell surface that are mediated by catenin-associated changes across the membrane.

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