scholarly journals Desensitization Contributes to the Synaptic Response of Gain-of-Function Mutants of the Muscle Nicotinic Receptor

2006 ◽  
Vol 128 (5) ◽  
pp. 615-627 ◽  
Author(s):  
Sergio Elenes ◽  
Ying Ni ◽  
Gisela D. Cymes ◽  
Claudio Grosman
Keyword(s):  
Nicotinic Receptor ◽  
Dissociation Rate ◽  
Gain Of Function ◽  
Naturally Occurring ◽  
Channel Opening ◽  
Speed Up ◽  
High Concentrations ◽  
Dissociation Rate Constants ◽  
Kinetics Of ◽  
Peak Value

Although the muscle nicotinic receptor (AChR) desensitizes almost completely in the steady presence of high concentrations of acetylcholine (ACh), it is well established that AChRs do not accumulate in desensitized states under normal physiological conditions of neurotransmitter release and clearance. Quantitative considerations in the framework of plausible kinetic schemes, however, lead us to predict that mutations that speed up channel opening, slow down channel closure, and/or slow down the dissociation of neurotransmitter (i.e., gain-of-function mutations) increase the extent to which AChRs desensitize upon ACh removal. In this paper, we confirm this prediction by applying high-frequency trains of brief (∼1 ms) ACh pulses to outside-out membrane patches expressing either lab-engineered or naturally occurring (disease-causing) gain-of-function mutants. Entry into desensitization was evident in our experiments as a frequency-dependent depression in the peak value of succesive macroscopic current responses, in a manner that is remarkably consistent with the theoretical expectation. We conclude that the comparatively small depression of the macroscopic currents observed upon repetitive stimulation of the wild-type AChR is due, not to desensitization being exceedingly slow but, rather, to the particular balance between gating, entry into desensitization, and ACh dissociation rate constants. Disruption of this fine balance by, for example, mutations can lead to enhanced desensitization even if the kinetics of entry into, and recovery from, desensitization themselves are not affected. It follows that accounting for the (usually overlooked) desensitization phenomenon is essential for the correct interpretation of mutagenesis-driven structure–function relationships and for the understanding of pathological synaptic transmission at the vertebrate neuromuscular junction.

scholarly journals Oversized galactosides as a probe for conformational dynamics in LacY

2018 ◽  
Vol 115 (16) ◽  
pp. 4146-4151 ◽  
Author(s):  
Irina Smirnova ◽  
Vladimir Kasho ◽  
Xiaoxu Jiang ◽  
Hong-Ming Chen ◽  
Stephen G. Withers ◽  
...  
Keyword(s):  
Binding Site ◽  
Conformational Dynamics ◽  
Dissociation Rate ◽  
Binding Kinetics ◽  
Lactose Permease ◽  
E Coli ◽  
Open Conformation ◽  
Dissociation Rate Constants ◽  
Kinetics Of ◽  
Micromolar Range

Binding kinetics of α-galactopyranoside homologs with fluorescent aglycones of different sizes and shapes were determined with the lactose permease (LacY) of Escherichia coli by FRET from Trp151 in the binding site of LacY to the fluorophores. Fast binding was observed with LacY stabilized in an outward-open conformation (kon = 4–20 μM−1·s−1), indicating unobstructed access to the binding site even for ligands that are much larger than lactose. Dissociation rate constants (koff) increase with the size of the aglycone so that Kd values also increase but remain in the micromolar range for each homolog. Phe27 (helix I) forms an apparent constriction in the pathway for sugar by protruding into the periplasmic cavity. However, replacement of Phe27 with a bulkier Trp does not create an obstacle in the pathway even for large ligands, since binding kinetics remain unchanged. High accessibility of the binding site is also observed in a LacY/nanobody complex with partially blocked periplasmic opening. Remarkably, E. coli expressing WT LacY catalyzes transport of α- or β-galactopyranosides with oversized aglycones such as bodipy or Aldol518, which may require an extra space within the occluded intermediate. The results confirm that LacY specificity is strictly directed toward the galactopyranoside ring and also clearly indicate that the opening on the periplasmic side is sufficiently wide to accommodate the large galactoside derivatives tested here. We conclude that the actual pathway for the substrate entering from the periplasmic side is wider than the pore diameter calculated in the periplasmic-open X-ray structures.

The Kinetic Activation Volumes for the Binding of Iodide to Cobalamin (Vitamin B12) Studied on a High Pressure Laser Temperature Jump Apparatus

1974 ◽  
Vol 52 (6) ◽  
pp. 910-914 ◽  
Author(s):  
Brian B. Hasinoff
Keyword(s):  
High Pressure ◽  
Metal Ion ◽  
Temperature Jump ◽  
Outer Sphere ◽  
Dissociation Rate ◽  
Vitamin B ◽  
Laser Temperature Jump ◽  
Dissociation Rate Constants ◽  
Kinetics Of ◽  
Activated Complex

The relaxation kinetics of the reaction of iodide with cobalamin (vitamin B12) were studied on a high pressure laser temperature jump apparatus in aqueous solution at 25° and ionic strength 0.2 M. Analysis of the pressure dependence of the formation and dissociation rate constants gave their respective volumes of activation to be: ΔVf* = 5.50 ± 0.8 cm3 mol−1 and ΔVd* = 11.5 ± 1.6 cm3 mol−1 The positive activation volume for formation of the complex, ΔVf*, after appropriate correction for the volume change due to formation of an outer sphere complex, is consistent with a dissociative type mechanism in which the metal ion – water bond is stretched in the activated complex.

scholarly journals CD80 (B7-1) Binds Both CD28 and CTLA-4 with a Low Affinity and Very Fast Kinetics

1997 ◽  
Vol 185 (3) ◽  
pp. 393-404 ◽  
Author(s):  
P.  Anton van der Merwe ◽  
Dale L. Bodian ◽  
Susan Daenke ◽  
Peter Linsley ◽  
Simon J. Davis
Keyword(s):  
T Cell ◽  
Cell Surface ◽  
Dissociation Rate ◽  
Fast Kinetics ◽  
Surface Molecules ◽  
Kd Values ◽  
Dissociation Rate Constants ◽  
Antigen Presenting ◽  
Kinetics Of ◽  
Rapid Binding

The structurally related T cell surface molecules CD28 and CTLA-4 interact with cell surface ligands CD80 (B7-1) and CD86 (B7-2) on antigen-presenting cells (APC) and modulate T cell antigen recognition. Preliminary reports have suggested that CD80 binds CTLA-4 and CD28 with affinities (Kd values ∼12 and ∼200 nM, respectively) that are high when compared with other molecular interactions that contribute to T cell–APC recognition. In the present study, we use surface plasmon resonance to measure the affinity and kinetics of CD80 binding to CD28 and CTLA-4. At 37°C, soluble recombinant CD80 bound to CTLA-4 and CD28 with Kd values of 0.42 and 4 μM, respectively. Kinetic analysis indicated that these low affinities were the result of very fast dissociation rate constants (koff); sCD80 dissociated from CD28 and CTLA-4 with koff values of ⩾1.6 and ⩾0.43 s−1, respectively. Such rapid binding kinetics have also been reported for the T cell adhesion molecule CD2 and may be necessary to accommodate dynamic T cell–APC contacts and to facilitate scanning of APC for antigen.

scholarly journals The kinetics of antibody binding to membrane antigens in solution and at the cell surface

1980 ◽  
Vol 187 (1) ◽  
pp. 1-20 ◽  
Author(s):  
D W Mason ◽  
A F Williams
Keyword(s):  
Cell Surface ◽  
Rate Constants ◽  
Equilibrium Constants ◽  
Surface Antigens ◽  
Dissociation Rate ◽  
Simple Relationship ◽  
Dissociation Kinetics ◽  
Cell Surface Antigens ◽  
Dissociation Rate Constants ◽  
Kinetics Of

The reaction kinetics of 125I-labelled mouse monoclonal antibodies binding to three cell-surface antigens of rat thymocytes (Thy-1.1, W3/25) were studied. The differences between bivalent and univalent interactions were determined by using antibody in the F(ab′)2 or Fab′ form and by using antigen in polymeric or monomeric forms. Association rate constants (k+1), dissociation rate constants (k-1) and equilibrium constants were determined. Also, the dissociation kinetics of rabbit antibodies against rat Thy-1 antigen were studied. The major findings were as follows. (i) With F(ab′)2 antibody there was no simple relationship between antigen density at the cell surface and extent of bivalent binding. Extensive univalent binding was observed unless the antibody had a high k-1 for the univalent interaction, in which case all binding was bivalent. (ii) k+1 values were similar for F(ab′)2 or Fab′ antibody, and for the different antibodies were in the range 0.8 × 10(5)–1.1 × 10(6) M-1.s-1. These differences were sufficient to affect the interpretation of serological assays with the different antibodies. (iii) Antibody bound bivalently dissociated much more slowly than that bound univalently. However, the k-1 values for the univalently bound antibody were sufficiently low in most cases that the lifetime of the univalent complex was similar to or greater than the time needed for the assay. Thus the results could be interpreted on the basis of irreversible reactions. The overall conclusion from the study is that for an understanding of the binding of antibody to cell-surface antigens the kinetics of the interaction are of major importance and theories based on equilibrium binding are inappropriate.

scholarly journals Laser Photolysis Investigations of Ligand Binding With Models of the Active Site of Respiratory Hemoproteins: Kinetic and Thermodynamic Aspects

1990 ◽  
Vol 10 (5-6) ◽  
pp. 297-318 ◽  
Author(s):  
D. Lavalette ◽  
C. Tetreau ◽  
M. Momenteau ◽  
J. Mispelter ◽  
J. M. Lhoste ◽  
...  
Keyword(s):  
Active Site ◽  
Structural Parameters ◽  
Dissociation Rate ◽  
Laser Photolysis ◽  
Concentration Jump ◽  
Linear Free Energy ◽  
Energy Relationships ◽  
Dissociation Rate Constants ◽  
Kinetics Of

During the past 15 years, laser photolysis has been the method of choice for probing the complex reaction kinetics of respiratory proteins. In an attempt to determine the structural parameters which govern their reactivity, synthetic heme model compounds capable of simulating particular aspects of the reactivity of the active site of hemoproteins have been successively proposed. Laser photolysis of heme compounds merely induces a reversible photodissociation of one ligand at a time. This is equivalent to performing a fast concentration jump "in situ" and provides a powerful, fast and "clean" chemical relaxation technique. To gather association and dissociation rate constants of various ligands (O2, CO, nitrogenous bases) special methods have been developed or adapted. The problem of comparing and classifying a large number of collected data has been greatly simplified by introducing a Linear Free Energy Relationships formalism. In the first part of this paper, some of the methods and concepts which have emerged from several years of investigations of heme proteins and heme models and which are of a sufficient generality to be useful in other fields of chemical kinetics are reviewed. In the second part of the paper we present the application of the preceding methods to a kinetic study of a series of heme models which were specifically designed to investigate the important problem of H-bonding as a stabilizing factor of the oxygenated heme model and hemoprotein complexes.

scholarly journals Resource uptake and the evolution of moderately efficient enzymes

2020 ◽  
Author(s):  
Florian Labourel ◽  
Etienne Rajon
Keyword(s):  
Kinetic Parameters ◽  
Mechanistic Model ◽  
Enzyme Concentration ◽  
Lower Efficiency ◽  
Intermediate Metabolites ◽  
Speed Up ◽  
High Concentrations ◽  
Kinetics Of ◽  
Competition For Resources ◽  
Joint Evolution

AbstractEnzymes speed up reactions that would otherwise be too slow to sustain the metabolism of self-replicators. Yet, most enzymes seem only moderately efficient, exhibiting kinetic parameters orders of magnitude lower than their expected physically achievable maxima. Here, we question how these parameters evolve using a mechanistic model where enzyme efficiency is a key component of individual competition for resources. We show that kinetic parameters are under strong directional selection only up to a point, above which enzymes appear to evolve under near-neutrality. A majority of kinetic parameters compiled elsewhere do spread onto this plateau. Nonetheless, using a population genetics model that includes genetic drift and mutational biases, we show that this is a very unlikely outcome of evolution on a common landscape, as even very moderate biases towards lower efficiency should prevent the occurrence of such a diversity. Instead, differences between species, and within a species between metabolic pathways and the reactions to perform, should be involved. Our results point to drift playing an important role, along with the kinetics of nutrient transporters, the tolerance to high concentrations of intermediate metabolites, and the reversibility of reactions. Enzyme concentration also shapes selection on kinetic parameters, suggesting that the joint evolution of concentration and efficiency, facilitated by the plateau, should matter. Interestingly, the position of an enzyme along the metabolic pathway is not key for its evolution, contrasting with the prediction of models assuming that fitness depends on a precise level of flux control, rather than on competitive abilities.

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