scholarly journals A Picrotoxin-specific Conformational Change in the Glycine Receptor M2–M3 Loop

2005 ◽  
Vol 280 (43) ◽  
pp. 35836-35843 ◽  
Author(s):  
Rebecca Hawthorne ◽  
Joseph W. Lynch
Keyword(s):  
Conformational Change ◽  
Conformational Changes ◽  
Binding Sites ◽  
Glycine Receptor ◽  
Dissociation Rate ◽  
Open Probability ◽  
External Loop ◽  
Substituted Cysteine Accessibility ◽  
Specific Effects ◽  
Pore Lining

The external loop linking the M2 and M3 transmembrane domains is crucial for coupling agonist binding to channel gating in the glycine receptor chloride channel (GlyR). A substituted cysteine accessibility scan previously showed that glycine activation increased the surface accessibility of 6 contiguous residues (Arg271– Lys276) toward the N-terminal end of the homomeric α1 GlyR M2–M3 loop. In the present study we used a similar approach to determine whether the allosteric antagonist, picrotoxin, could impose conformational changes to this domain that cannot be induced by varying agonist concentrations alone. Picrotoxin slowed the reaction rate of a sulfhydryl-containing compound (MTSET) with A272C, S273C, and L274C. Before interpreting this as a picrotoxin-specific conformational change, it was necessary to eliminate the possibility of steric competition between picrotoxin and MTSET. Accordingly, we showed that picrotoxin and the structurally unrelated blocker, bilobalide, were both trapped in the R271C GlyR in the closed state and that a point mutation to the pore-lining Thr6′ residue abolished inhibition by both compounds. We also demonstrated that the picrotoxin dissociation rate was linearly related to the channel open probability. These observations constitute a strong case for picrotoxin binding in the pore. We thus conclude that the picrotoxin-specific effects on the M2–M3 loop are mediated allosterically. This suggests that the M2–M3 loop responds differently to the occupation of different binding sites.

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 Mechanical gating of the auditory transduction channel TMC1 involves the fourth and sixth transmembrane helices

2022 ◽  
Author(s):  
Nurunisa Akyuz ◽  
K. Domenica Karavitaki ◽  
Bifeng Pan ◽  
Panos I. Tamvakologos ◽  
Kelly P. Brock ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Hair Cells ◽  
Single Channel ◽  
Transmembrane Helices ◽  
Open Probability ◽  
Transmembrane Channel ◽  
Force Sensitivity ◽  
Electrophysiological Recordings ◽  
Auditory Transduction ◽  
Pore Lining

The transmembrane channel-like (TMC) 1 and 2 proteins play a central role in auditory transduction, forming ion channels that convert sound into electrical signals. However, the molecular mechanism of their gating remains unknown. Here, using predicted structural models as a guide, we probed the effects of twelve mutations on the mechanical gating of the transduction currents in native hair cells of Tmc1/2-null mice expressing virally introduced TMC1 variants. Whole-cell electrophysiological recordings revealed that mutations within the pore-lining transmembrane (TM) helices 4 and 6 modified gating, reducing the force sensitivity or shifting the open probability of the channels, or both. For some of the mutants, these changes were accompanied by a change in single-channel conductance. Our observations are in line with a model wherein conformational changes in the TM4 and TM6 helices are involved in the mechanical gating of the transduction channel.

scholarly journals Comparison of Taurine- and Glycine-induced Conformational Changes in the M2-M3 Domain of the Glycine Receptor

2004 ◽  
Vol 279 (19) ◽  
pp. 19559-19565 ◽  
Author(s):  
Nian-Lin R. Han ◽  
John D. Clements ◽  
Joseph W. Lynch
Keyword(s):  
Conformational Changes ◽  
Reaction Rate ◽  
Partial Agonist ◽  
Glycine Receptor ◽  
Reaction Rates ◽  
Structural Basis ◽  
Surface Accessibility ◽  
Partial Agonists ◽  
Substituted Cysteine Accessibility ◽  
Receptor Family

In the ionotropic glutamate receptor, the global conformational changes induced by partial agonists are smaller than those induced by full agonists. However, in the pentameric ligand-gated ion channel receptor family, the structural basis of partial agonism is not understood. This study investigated whether full and partial agonists induce different conformation changes in the glycine receptor chloride channel (GlyR). A substituted cysteine accessibility analysis demonstrated previously that glycine binding induced an increase in surface accessibility of all residues from Arg271to Lys276in the M2-M3 domain of the homomeric α1 GlyR. Here we compare the surface accessibility changes induced by the full agonist, glycine, and the partial agonist, taurine. In GlyRs incorporating the A272C, S273C, L274C, or P275C mutation, the reaction rate of the cysteine-specific compound, methanethiosulfonate ethyltrimethylammonium, depended on how strongly the receptors were activated but was agonist-independent. Reaction rates could not be compared in the R271C and K276C mutant GlyRs because methanethiosulfonate ethyltrimethylammonium did not modify the extremely small currents induced by saturating taurine or equivalent low glycine concentrations. The results indicate that bound taurine and glycine molecules impose identical conformational changes to the M2-M3 domain. We therefore conclude that the higher efficacy of glycine is due to an increased ability to stabilize a common activated configuration.

scholarly journals Conformational Variability of the Glycine Receptor M2 Domain in Response to Activation by Different Agonists

2007 ◽  
Vol 282 (49) ◽  
pp. 36057-36067 ◽  
Author(s):  
Stephan A. Pless ◽  
Mohammed I. Dibas ◽  
Henry A. Lester ◽  
Joseph W. Lynch
Keyword(s):  
Conformational Changes ◽  
Structural Changes ◽  
Glycine Receptor ◽  
Blue Shift ◽  
Receptor Activation ◽  
Emission Peak ◽  
Competitive Antagonist ◽  
Conformational Variability ◽  
Fluorescence Change ◽  
Pore Lining

Models describing the structural changes mediating Cys loop receptor activation generally give little attention to the possibility that different agonists may promote activation via distinct M2 pore-lining domain structural rearrangements. We investigated this question by comparing the effects of different ligands on the conformation of the external portion of the homomeric α1 glycine receptor M2 domain. Conformational flexibility was assessed by tethering a rhodamine fluorophore to cysteines introduced at the 19′ or 22′ positions and monitoring fluorescence and current changes during channel activation. During glycine activation, fluorescence of the label attached to R19′C increased by ∼20%, and the emission peak shifted to lower wavelengths, consistent with a more hydrophobic fluorophore environment. In contrast, ivermectin activated the receptors without producing a fluorescence change. Although taurine and β-alanine were weak partial agonists at the α1R19′C glycine receptor, they induced large fluorescence changes. Propofol, which drastically enhanced these currents, did not induce a glycine-like blue shift in the spectral emission peak. The inhibitors strychnine and picrotoxin elicited fluorescence and current changes as expected for a competitive antagonist and an open channel blocker, respectively. Glycine and taurine (or β-alanine) also produced an increase and a decrease, respectively, in the fluorescence of a label attached to the nearby L22′C residue. Thus, results from two separate labeled residues support the conclusion that the glycine receptor M2 domain responds with distinct conformational changes to activation by different agonists.

scholarly journals The long activations of α2 glycine channels can be described by a mechanism with reaction intermediates (“flip”)

2011 ◽  
Vol 137 (2) ◽  
pp. 197-216 ◽  
Author(s):  
Paraskevi Krashia ◽  
Remigijus Lape ◽  
Francesco Lodesani ◽  
David Colquhoun ◽  
Lucia G. Sivilotti
Keyword(s):  
Concentration Dependence ◽  
Binding Sites ◽  
Time Course ◽  
Single Channel ◽  
Glycine Receptor ◽  
Open Probability ◽  
Low Concentrations ◽  
Open Time ◽  
Single Channel Activity ◽  
Entire Sequence

The α2 glycine receptor (GlyR) subunit, abundant in embryonic neurons, is replaced by α1 in the adult nervous system. The single-channel activity of homomeric α2 channels differs from that of α1-containing GlyRs, as even at the lowest glycine concentration (20 µM), openings occurred in long (>300-ms) groups with high open probability (Popen; 0.96; cell-attached recordings, HEK-expressed channels). Shut-time intervals within groups of openings were dominated by short shuttings of 5–10 µs. The lack of concentration dependence in the groups of openings suggests that they represent single activations, separated by very long shut times at low concentrations. Several putative mechanisms were fitted by maximizing the likelihood of the entire sequence of open and shut times, with exact missed-events allowance (program hjcfit). Records obtained at several glycine concentrations were fitted simultaneously. The adequacy of the different schemes was judged by the accuracy with which they predicted not only single-channel data but also the time course and concentration dependence of macroscopic responses elicited by rapid glycine applications to outside-out patches. The data were adequately described only with schemes incorporating a reaction intermediate in the activation, and the best was a flip mechanism with two binding sites and one open state. Fits with this mechanism showed that for α2 channels, the opening rate constant is very fast, ∼130,000 s−1, much as for α1β GlyRs (the receptor in mature synapses), but the estimated true mean open time is 20 times longer (around 3 ms). The efficacy for the flipping step and the binding affinity were lower for α2 than for α1β channels, but the overall efficacies were similar. As we previously showed for α1 homomeric receptors, in α2 glycine channels, maximum Popen is achieved when fewer than all five of the putative binding sites in the pentamer are occupied by glycine.

Modulation of recombinant human alpha 1 glycine receptor by flavonoids and gingerols

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Ulrike Breitinger ◽  
Heinrich Sticht ◽  
Hans-Georg Breitinger
Keyword(s):  
Binding Sites ◽  
Glycine Receptor ◽  
Hek293 Cells ◽  
Plant Metabolites ◽  
Secondary Plant Metabolites ◽  
Cell Current ◽  
Modulatory Sites ◽  
Induced Currents ◽  
Pore Lining ◽  
Related Compounds

Abstract The inhibitory glycine receptor (GlyR) is a principal mediator of fast synaptic inhibition in mammalian spinal cord, brainstem, and higher brain centres. Flavonoids are secondary plant metabolites that exhibit many beneficial physiological effects, including modulatory action on neuronal receptors. Using whole-cell current recordings from recombinant human α1 GlyRs, expressed in HEK293 cells, we compared the flavonols kaempferol and quercetin, the flavanone naringenin, the flavones apigenin and nobiletin, the isoflavone genistein, and two gingerols, 6-gingerol and 8-gingerol for their modulation of receptor currents. All compounds were inhibitors of the GlyR with IC50 values ranging between 9.3 ± 2.6 µM (kaempferol) and 46.7 ± 6.5 µM (genistein), following a mixed mode of inhibition. Co-application of two inhibitors revealed distinct binding sites for flavonoids and gingerols. Pore-lining mutants T258A and T258S were strongly inhibited by quercetin and naringenin, but not by 6-gingerol, confirming the existence of distinct binding sites for flavonoids and gingerols. Apigenin, kaempferol, nobiletin, naringenin and 6-gingerol showed biphasic action, potentiating glycine-induced currents at low concentration of both, modulator and glycine, and inhibiting at higher concentrations. Identification of distinct modulatory sites for flavonoids and related compounds may present pharmacological target sites and aid the discovery of novel glycinergic drugs.

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 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.

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