scholarly journals An Inactivation Stabilizer of the Na+ Channel Acts as an Opportunistic Pore Blocker Modulated by External Na+

2005 ◽  
Vol 125 (5) ◽  
pp. 465-481 ◽  
Author(s):  
Ya-Chin Yang ◽  
Chung-Chin Kuo
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Dissociation Constants ◽  
Channel Gating ◽  
Structural Motif ◽  
Na Channel ◽  
Channel Pore ◽  
Conduction Pathway ◽  
Gating Process ◽  
Pore Blocker

The Na+ channel is the primary target of anticonvulsants carbamazepine, phenytoin, and lamotrigine. These drugs modify Na+ channel gating as they have much higher binding affinity to the inactivated state than to the resting state of the channel. It has been proposed that these drugs bind to the Na+ channel pore with a common diphenyl structural motif. Diclofenac is a widely prescribed anti-inflammatory agent that has a similar diphenyl motif in its structure. In this study, we found that diclofenac modifies Na+ channel gating in a way similar to the foregoing anticonvulsants. The dissociation constants of diclofenac binding to the resting, activated, and inactivated Na+ channels are ∼880 μM, ∼88 μM, and ∼7 μM, respectively. The changing affinity well depicts the gradual shaping of a use-dependent receptor along the gating process. Most interestingly, diclofenac does not show the pore-blocking effect of carbamazepine on the Na+ channel when the external solution contains 150 mM Na+, but is turned into an effective Na+ channel pore blocker if the extracellular solution contains no Na+. In contrast, internal Na+ has only negligible effect on the functional consequences of diclofenac binding. Diclofenac thus acts as an “opportunistic” pore blocker modulated by external but not internal Na+, indicating that the diclofenac binding site is located at the junction of a widened part and an acutely narrowed part of the ion conduction pathway, and faces the extracellular rather than the intracellular solution. The diclofenac binding site thus is most likely located at the external pore mouth, and undergoes delicate conformational changes modulated by external Na+ along the gating process of the Na+ channel.

scholarly journals An integrated catch-and-hold mechanism activates nicotinic acetylcholine receptors

2012 ◽  
Vol 140 (1) ◽  
pp. 17-28 ◽  
Author(s):  
Snehal Jadey ◽  
Anthony Auerbach
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Nicotinic Acetylcholine Receptors ◽  
Single Channel ◽  
Equilibrium Constants ◽  
Protein Structures ◽  
Channel Gating ◽  
Structural Basis ◽  
Agonist Binding ◽  
Adult Type

In neuromuscular acetylcholine (ACh) receptor channels (AChRs), agonist molecules bind with a low affinity (LA) to two sites that can switch to high affinity (HA) and increase the probability of channel opening. We measured (by using single-channel kinetic analysis) the rate and equilibrium constants for LA binding and channel gating for several different agonists of adult-type mouse AChRs. Almost all of the variation in the equilibrium constants for LA binding was from differences in the association rate constants. These were consistently below the limit set by diffusion and were substantially different even though the agonists had similar sizes and the same charge. This suggests that binding to resting receptors is not by diffusion alone and, hence, that each binding site can undergo two conformational changes (“catch” and “hold”) that connect three different structures (apo-, LA-bound, and HA-bound). Analyses of ACh-binding protein structures suggest that this binding site, too, may adopt three discrete structures having different degrees of loop C displacement (“capping”). For the agonists we tested, the logarithms of the equilibrium constants for LA binding and LA↔HA gating were correlated. Although agonist binding and channel gating have long been considered to be separate processes in the activation of ligand-gated ion channels, this correlation implies that the catch-and-hold conformational changes are energetically linked and together comprise an integrated process having a common structural basis. We propose that loop C capping mainly reflects agonist binding, with its two stages corresponding to the formation of the LA and HA complexes. The catch-and-hold reaction coordinate is discussed in terms of preopening states and thermodynamic cycles of activation.

scholarly journals Compound-specific Na+channel pore conformational changes induced by local anaesthetics

2005 ◽  
Vol 564 (1) ◽  
pp. 21-31 ◽  
Author(s):  
Koji Fukuda ◽  
Tadashi Nakajima ◽  
Prakash C. Viswanathan ◽  
Jeffrey R. Balser
Keyword(s):  
Conformational Changes ◽  
Local Anaesthetics ◽  
Na Channel ◽  
Channel Pore

scholarly journals Electron-paramagnetic-resonance studies on nitrogenase of Klebsiella pneumoniae. Evidence for acetylene- and ethylene-nitrogenase transient complexes

1978 ◽  
Vol 173 (1) ◽  
pp. 277-290 ◽  
Author(s):  
D J Lowe ◽  
R R Eady ◽  
R N F Thorneley
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Klebsiella Pneumoniae ◽  
Binding Site ◽  
Dissociation Constants ◽  
Paramagnetic Resonance ◽  
Fe Protein ◽  
Low Concentrations ◽  
Direct Binding ◽  
Electron Paramagnetic ◽  
Single Binding Site

Klebsiella pneumoniae nitrogenase exhibited four new electron-paramagnetic-resonance signals during turnover at 10 degrees C, pH7.4, which were assigned to intermediates present in low concentrations in the steady state. 57Fe-substituted Mo–Fe protein showed that they arose from Fe–S clusters in the Mo–Fe protein of nitrogenase. The new signals are designated: Ic, g values at 4.67, 3.37 and approx. 2.0; VI, g values at 2.125, 2.000 and 2.000; VII, g values at 5.7 and 5.4; VIII, g values at 2.092, 1.974 and 1.933. The sharp axial signal VI arises from a Fe4S4 cluster at the −1 oxidation level. This signal was only detected in the presence of ethylene and provides the first evidence of an enzyme–product complex for nitrogenase. [13C]Acetylene and [13C]ethylene provided no evidence for direct binding of this substrate and product to the Fe–S clusters giving rise to these signals. The dependence of signal intensities on acetylene concentration indicated two types of binding site, with apparent dissociation constants K less than 16 micron and K approximately 13mM. A single binding site for ethylene (K=1.5mM) was detected. A scheme is proposed for the mechanism of reduction of acetylene to ethylene and inhibition of this reaction by CO.

scholarly journals Monoclonal antibodies identify residues 199–216 of the integrin α2 vWFA domain as a functionally important region within α2β1

2000 ◽  
Vol 350 (2) ◽  
pp. 485-493 ◽  
Author(s):  
Danny S. TUCKWELL ◽  
Lyndsay SMITH ◽  
Michelle KORDA ◽  
Janet A. ASKARI ◽  
Sentot SANTOSO ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Binding Site ◽  
Conformational Changes ◽  
Cation Binding ◽  
Inhibitory Antibody ◽  
Collagen Binding ◽  
Collagen Peptide ◽  
Binding Residue ◽  
Important Region ◽  
Domain Mapping

Integrin α2β1 is the major receptor for collagens in the human body, and the collagen-binding site on the α2 subunit von Willebrand factor A-type domain (vWFA domain) is now well defined. However, the biologically important conformational changes that are associated with collagen binding, and the means by which the vWFA domain is integrated into the whole integrin are not completely understood. We have raised monoclonal antibodies against recombinant α2 vWFA domain for use as probes of function. Three antibodies, JA202, JA215 and JA218, inhibited binding to collagen, collagen I C-propeptide and E-cadherin, demonstrating that their function is important for structurally diverse α2β1 ligands. Cross-blocking studies grouped the epitopes into two clusters: (I) JA202, the inhibitory antibody, Gi9, and a non-inhibitory antibody, JA208; (II) JA215 and JA218. Both clusters were sensitive to events at the collagen binding site, as binding of Gi9, JA202, JA215 and JA218 were inhibited by collagen peptide, JA208 binding was enhanced by collagen peptide, and binding of JA202 was decreased after mutagenesis of the cation-binding residue Thr221 to alanine. Binding of cluster I antibodies was inhibited by the anti-functional anti-β1 antibody Mab13, and binding of Gi9 and JA218 to α2β1 was inhibited by substituting Mn2+ for Mg2+, demonstrating that these antibodies were sensitive to changes initiated outside the vWFA domain. Mapping of epitopes showed that JA202 and Gi9 bound between residues 212–216, while JA208 bound between residues 199–216. We have therefore identified two epitope clusters with novel properties; i.e. they are intimately associated with the collagen-binding site, responsive to conformational changes at the collagen-binding site and sensitive to events initiated outside the vWFA domain.

scholarly journals Route, mechanism, and implications of proton import during Na+/K+ exchange by native Na+/K+-ATPase pumps

2014 ◽  
Vol 143 (4) ◽  
pp. 449-464 ◽  
Author(s):  
Natascia Vedovato ◽  
David C. Gadsby
Keyword(s):  
Binding Site ◽  
Single Molecule ◽  
Conformational Changes ◽  
Outward Current ◽  
Side Chain ◽  
Ion Binding ◽  
Inward Current ◽  
Na Release ◽  
K Transport ◽  
External K

A single Na+/K+-ATPase pumps three Na+ outwards and two K+ inwards by alternately exposing ion-binding sites to opposite sides of the membrane in a conformational sequence coupled to pump autophosphorylation from ATP and auto-dephosphorylation. The larger flow of Na+ than K+ generates outward current across the cell membrane. Less well understood is the ability of Na+/K+ pumps to generate an inward current of protons. Originally noted in pumps deprived of external K+ and Na+ ions, as inward current at negative membrane potentials that becomes amplified when external pH is lowered, this proton current is generally viewed as an artifact of those unnatural conditions. We demonstrate here that this inward current also flows at physiological K+ and Na+ concentrations. We show that protons exploit ready reversibility of conformational changes associated with extracellular Na+ release from phosphorylated Na+/K+ pumps. Reversal of a subset of these transitions allows an extracellular proton to bind an acidic side chain and to be subsequently released to the cytoplasm. This back-step of phosphorylated Na+/K+ pumps that enables proton import is not required for completion of the 3 Na+/2 K+ transport cycle. However, the back-step occurs readily during Na+/K+ transport when external K+ ion binding and occlusion are delayed, and it occurs more frequently when lowered extracellular pH raises the probability of protonation of the externally accessible carboxylate side chain. The proton route passes through the Na+-selective binding site III and is distinct from the principal pathway traversed by the majority of transported Na+ and K+ ions that passes through binding site II. The inferred occurrence of Na+/K+ exchange and H+ import during the same conformational cycle of a single molecule identifies the Na+/K+ pump as a hybrid transporter. Whether Na+/K+ pump–mediated proton inflow may have any physiological or pathophysiological significance remains to be clarified.

Nitrogenase iron-molybdenum cofactor binding site: Protein conformational changes associated with cofactor binding

Tetrahedron ◽  
1997 ◽  
Vol 53 (35) ◽  
pp. 11971-11984 ◽  
Author(s):  
Jon K. Magnuson ◽  
Timothy D. Paustian ◽  
Vinod K. Shah ◽  
Dennis R. Dean ◽  
Gary P. Roberts ◽  
...  
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Molybdenum Cofactor ◽  
Cofactor Binding ◽  
Protein Conformational Changes ◽  
Iron Molybdenum Cofactor

Overlap of IGF- and heparin-binding sites in rat IGF-binding protein-5

2000 ◽  
Vol 24 (1) ◽  
pp. 43-51 ◽  
Author(s):  
H Song ◽  
J Beattie ◽  
IW Campbell ◽  
GJ Allan
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Binding Protein ◽  
Binding Activity ◽  
Binding Domain ◽  
Site Directed Mutagenesis ◽  
Heparin Binding ◽  
Igf I ◽  
Heparin Binding Domain ◽  
Igf Binding

Using site-directed mutagenesis, we have undertaken a study of a potential IGF-binding site in the C-terminal domain of rat IGFBP-5, lying close to or within a previously described heparin-binding domain (residues 201-218) in this protein. After analysis of binding activity using three different methods - ligand blotting, solution phase equilibrium binding and biosensor measurement of real-time on- and off-rates - we report that the mutation of two highly conserved residues within this region (glycine 203 and glutamine 209) reduces the affinity of the binding protein for both IGF-I and IGF-II, while having no effect on heparin binding. In addition, we confirm that mutation of basic residues within the heparin-binding domain (R201L, K202E, K206Q and R214A) results in a protein that has attenuated heparin binding but shows only a small reduction in affinity for IGF-I and -II. Previous findings have described the reduction in affinity of IGFBP-5 for IGFs that occurs after complexation of the binding protein with heparin or other components of the extracellular matrix (ECM) and have postulated that such an interaction may result in conformational changes in protein structure, affecting subsequent IGF interaction. Our data suggesting potential overlap of heparin- and IGF-binding domains argue for a more direct effect of ECM modulation of the affinity of IGFBP-5 for ligand by partial occlusion of the IGF-binding site after interaction with ECM.

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.

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