Noncompetitive Binding Sites Research Articles

The high-affinity quinacrine binding site is located at a non-annular lipid domain of the nicotinic acetylcholine receptor

This work deals with the localization of the high-affinity non-competitive quinacrine binding site on the muscle-type nicotinic acetylcholine receptor (AChR). Specifically, quantitative steady-state fluorescence spectroscopy is used to determine whether quinacrine binds to a site located at either the annular or the non-annular lipid domain. For this purpose, we measure the ability of spin-labelled phosphatidylcholine (SL-PC) to quench AChR-bound quinacrine, AChR-bound ethidium and membrane-partitioned 7-(9-anthroyloxy)stearate (7-AS) fluorescence. Additionally, we compare the accessibility of SL-PC which is considered to bind only to the annular lipid domain of the AChR with the accessibility of two non-annular domain-sensing lipids such as 5-doxylstearate (5-SAL) and spin-labelled androstane (ASL). Initial experiments using 7-AS established the experimental conditions for maximum SL-PC membrane partitioning. The non-specific quenching elicited by increasing turbidity of the sample after addition of SL-PC is corrected by means of parallel experiments with unlabelled egg yolk phosphatidylcholine. After correction, the SL-PC quenching experiments show the following order in quenching efficiency: 7-AS>quinacrine≫ethidium. The relative intrinsic sensitivity of quinacrine to TEMPO paramagnetic quenching in acetonitrile is considered to be approximately two times higher than that for 7-AS. Thus, SL-PC was found to be more accessible (about 5-fold) to the membrane-partitioned 7-AS than to the quinacrine locus. In addition, SL-PC was virtually not accessible to the high-affinity non-luminal binding site for ethidium. The relative capacity of SL-PC, 5-SAL, and ASL to quench AChR-bound quinacrine fluorescence indicated that the spin-labelled lipid accessibility to the quinacrine binding site follows the order: 5-SAL>ASL≫SL-PC. Examination of the effect of high concentrations of 5-SAL, of its unlabelled parent stearate, and of SL-PC on ethidium and quinacrine binding showed that: (a) both fatty acids displace quinacrine, but not ethidium, from its high-affinity binding site, however (b) 5-SAL was found to be more effective than stearate to displace quinacrine from its locus, whereas (c) SL-PC competes neither for the ethidium locus nor for the quinacrine binding site. The results suggest that the high-affinity binding site for quinacrine is located at a non-annular lipid domain of the AChR. This particular area has been considered to be located at the intramolecular interfaces of the five AChR subunits and/or at the interstices of the transmembrane domains.

Preclinical Evidence for a Role of Glutamatergic Systems in Opioid Tolerance and Dependence

Opioid tolerance and physical dependence are undesirable consequences of chronic opioid use or misuse. Evidence from rodents, using a variety of modes of drug coadministration, reveals that drugs with glutamatergic antagonist activity at the competitive, noncompetitive, or glycine binding sites of the NMDA receptor complex or inhibitors of certain forms of nitric oxide synthase (NOS) can attenuate the development of morphine tolerance and in some cases reverse established tolerance or dependence. Some of these drugs modulate tolerance and dependence without affecting morphine's analgesic effects, suggesting that they prevent neuronal plasticity associated with adaptive changes mediated by the NMDA/NO cascade. Drugs that have a favorable preclinical safety margin are providing leads for new drugs for clinical evaluation.

Agonist self-inhibitory binding site of the nicotinic acetylcholine receptor.

A major focus of current research on the nicotinic acetylcholine receptor (AChR) has been to understand the molecular mechanism of ion channel inhibition. In particular, we put special emphasis on the description of the localization of the agonist self-inhibitory binding site. Binding of agonist in the millimolar concentration range to this particular site produces inhibition of the ion flux activity previously elicited by the same agonist at micromolar concentrations. Due to the similitude in the pharmacological and electrophysiological behavior in inhibiting the ion channel of both high agonist concentrations and noncompetitive antagonists, we first describe the localization of noncompetitive inhibitor binding sites on the AChR. There is a great body of experimental evidence for the existence and location of luminal high-affinity noncompetitive inhibitor binding sites. In this regard, the most simple mechanism to describe the action of noncompetitive inhibitors which bind to luminal sites and, by its semblance, the agonist self-inhibition itself, is based on the assumption that these compounds enter the open channel, bind to different rings within the M2 transmembrane domain of the receptor, and block cation flux by occluding the receptor pore. However, the existence of high-affinity nonluminal noncompetitive inhibitor binding sites is not consistent with the open-channel-blocking mechanism. Instead, the presence of the quinacrine locus at the lipid-protein (alpha M1) interface approximately 7 A from the lipid-water interface and the ethidium domain located approximately 46 A from the membrane surface in the wall of the vestibule open the possibility for the regulation of cation permeation by an allosteric process. Additionally, the observed (at least partially) overlapping between the quinacrine and the agonist self-inhibitory binding site also suggests an allosteric process for agonist self-inhibition. For this alternative mechanism, cholinergic agonist molecules first need to be partitioned into (or to be adsorbed onto) the lipid membrane to further interact with its binding site located at the lipid-protein interface.

Luminal and non-luminal non-competitive inhibitor binding sites on the nicotinic acetylcholine receptor.

The nicotinic acetylcholine receptor presents two very well differentiated domains for ligand binding that account for different cholinergic properties. In the hydrophilic extracellular region of the alpha subunit exist the binding sites for agonists such as the neurotransmitter acetylcholine, which upon binding trigger the channel opening, and for competitive antagonists such as d-tubocurarine, which compete for the former inhibiting its pharmacological action. For non-competitive inhibitors, a population of low-affinity binding sites have been found at the lipid-protein interface of the nicotinic acetylcholine receptor. In addition, at the M2 transmembrane domain, several high-affinity binding sites have been found for non-competitive inhibitors such as chlorpromazine, triphenylmethylphosphonium, the local anaesthetic QX-222 and the hydrophobic probe trifluoromethyl-iodophenyldiazirine. They are known as luminal binding sites. Although the local anaesthetic meproadifen seems to be located between the hydrophobic domains M2-M3, this locus is considered to form part of the channel mouth, thus this site can also be called a luminal binding site. In contraposition, experimental evidences support the hypothesis of the existence of other high-affinity binding sites for non-competitive inhibitors located not at the channel lumen, but at non-luminal binding domains. Among them, we can quote the binding site for quinacrine, which is located at the lipid-protein interface of the alpha M1 domain, and the binding site for ethidium, which is believed to interact with the wall of the vestibule very far away from both the lumen channel and the lipid membrane surface. The aim of this review is to discuss these recent findings relative to both structurally and functionally relevant aspects of non-competitive inhibitors of the nicotinic acetylcholine receptor. We will put special emphasis on the description of the localization of molecules with non-competitive antagonist properties that bind with high-affinity to luminal and non-luminal domains. The information described herein was principally obtained by means of methods such as photolabelling and site-directed mutagenesis in combination with patch-clamp. Our laboratory has contributed with data obtained by using biophysical approaches such as paramagnetic electron spin resonance and quantitative fluorescence spectroscopy.

Effects of Troglitazone (CS-045) on insulin secretion in isolated rat pancreatic islets and HIT cells: an insulinotropic mechanism distinct from glibenclamide

In order to elucidate the direct effects of (+/-)-5-[4-(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl-methoxy) benzyl]-2,4-thiazolidinedione (Troglitazone), a newly-developed oral hypoglycaemic agent, on pancreatic beta-cell function, in vitro investigation of isolated rat pancreatic islets and a hamster beta-cell line (HIT cell) were performed. Troglitazone stimulates both glucose, and glibenclamide-induced insulin release at a concentration of 10(-6) mol/l in these cells but, conversely, inhibits insulin secretion at 10(-4) mol/l. Glucose uptake in HIT cells is similarly enhanced by 10(-6) mol/l Troglitazone, but is reduced in the presence of 10(-4) mol/l Troglitazone. However, a quantitative immunoblot analysis with a specific antibody for GLUT 2 glucose transporter revealed no significant change in GLUT 2 protein in HIT cells with 10(-6) mol/l Troglitazone. Specific binding of [3H]-glibenclamide to beta-cell membranes is replaced by Troglitazone in a non-competitive manner, but 10(-6) mol/l Troglitazone failed to eliminate ATP-sensitive K++ channel activity. These results suggest that Troglitazone has a putative non-competitive binding site at, or in the vicinity of, the sulphonylurea receptor in rat pancreatic islets and HIT cells and that the dual effect of Troglitazone on insulin secretory capacity is mediated through the modulation of glucose transport activity, possibly due to the modification of intrinsic activity in glucose transporter in pancreatic beta cells by this novel agent.

The effects of drugs on the incorporation of a conformationally-sensitive, hydrophobic probe into the ion channel of the nicotinic acetylcholine receptor

The pattern of incorporation of the hydrophobic photolabel 3-( trifluoromethyl)-3-(m-[ 125 I] iodophenyl)diazirine([ 125 I] TID) into the nicotinic acetylcholine receptor (AChR) is a sensitive measure of AChR conformation (resting state or desensitized). We determined the ability of tetracaine, dibucaine, procaine, lidocaine, chlorpromazine or phencyclidine to inhibit [ 125I]TID photolabeling of the AChR as a function of drug concentration, both as a measure of the ability of these drugs to desensitize the AChR, and to characterize the [ 125I]TID binding site. To localize the site(s) of drug action, experiments were performed in the absence and presence of saturating concentrations of α-bungarotoxin (BgTx), to block drug binding to the agonist binding site. On the basis of the concentration dependence of their effects, which was not altered by the presence of BgTx, tetracaine and dibucaine appeared to block [ 125I]TID incorporation competitively, suggesting that the high-affinity[ 125I]TID binding site is the non-competitive blocker binding site presumed to exist in the interior of the AChR ion channel. Procaine, chlorpromazine, lidocaine and phencyclidine blocked [ 125I]TID incorporation at lower concentrations in the absence of BgTx than in its presence, suggesting that these drugs block incorporation by inducing desensitization when bound to their high-affinity non-competitive blocker binding sites and that BgTx countered the drug effect by allosterically stabilizing the resting state.

Paradoxical effects of 2‐amino‐4‐phosphonobutanoic acid and other 2‐amino‐ω‐phosphonoalkanoic acid on the n‐methyl‐d‐aspartate receptor ion channel

AbstractAn extensively washed membrane preparation was used to measure the noncompetitive binding site of the N‐methyl‐D‐aspartate (NMDA) receptor complex with [3H]‐1‐[1‐(2‐thienyl) cyclo‐hexyl] piperidine ([3H]‐TCP). Addition of glutamic acid (Glu) or Glu + glycine (Gly) increased the binding of [3H]‐TCP, reflecting that the NMDA receptor ion channel was opened by these compounds. [3H]‐TCP binding was also increased by various 2‐amino‐ω‐phosphonoalkanoic (AP) compounds, of which 2‐amino‐4‐phosphonobutanoic acid (AP4) was the most effective. The effects of AP compounds were also observed in the presence of Gul. However, in the presence of Gly, or in the presence of Glu + Gly, 2‐amino‐5‐phosphonopentanoic acid (AP5) and 2‐amino‐7‐phosphonoheptanoic acid (AP7) inhibited the binding. The enhancing effect of AP4 was also inhibited by these compounds. Our conclusion is that AP compounds exert complex effects: they bind to and effect the NMDA receptor complex ion channel not only via the competitive binding site but may also interact with the potentiating Gly binding site. © Wiley‐Liss, Inc.

Synthesis and activity of spiroisoindolines as novel noncompetitive NMDA antagonists

A series of novel spiroisoindolines was designed and synthesized as potential noncompetitive NMDA antagonists. Affinities of these compounds for the noncompetitive NMDA binding site were determined using [ 3H]TCP and found to possess IC 50s ranging from 0.065 to 17μM. In vivo testing of 2′-methylspiro-[4,5,6,7-tetrahydrobenzothiophene-4,1′-(1,3-dihydroisoindole)] ( 43) showed it to antagonize NMDA-induced convulsions, to be neuroprotective in a gerbil model of ischemia, and not to generalize to MK-801 in a drug discrimination paradigm.

Quinacrine binds to the lipid-protein interface of the Torpedo acetylcholine receptor: a fluorescence study.

It has been argued both that there is a high affinity noncompetitive inhibitor binding site in the lumen of the acetylcholine receptor and that this lumen exists on the central axis of the receptor. Such a site would be expected to be 20-40 A from the membrane lipids. We tested whether, in fact, quinacrine, a potent fluorescent noncompetitive inhibitor, binds to such a site. We measured quenching of receptor-bound quinacrine fluorescence by fluorescence dipolar energy transfer to lipid probes, 5-(N-dodecanoylamino)eosin and N-(3-sulfopropyl)-4-(p-didecylaminostyryl)pyridinium, or by collision with paramagnetic lipid probes 2,2,6,6-tetramethylpiperidine-1-oxyl and 3-doxyl-17 beta-hydroxy-5 alpha-androstane (spin-labeled androstane). Initial control experiments established that in the presence of carbamylcholine, quinacrine binds to a phencyclidine-sensitive site on the Torpedo receptor with a Kd equal to 0.14 microM and with a quantum yield of 0.18. Fluorescence energy transfer from receptor-bound quinacrine had a magnitude consistent with quinacrine being less than 10 A from the lipid fluorescent probes. 2,2,6,6-Tetramethylpiperidine-1-oxyl and spin-labeled androstane were two to five times more effective at quenching receptor-bound quinacrine fluorescence than the fluorescence from membrane-partitioned 5-(dodecanoylamino)fluorescein. These results suggest that the quinacrine binding site is too close to the lipid domain to be in the lumen of the receptor, and therefore it is probably located on the outer surface of the membrane-spanning domain of the acetylcholine receptor.

Acetylcholine receptor inhibition by d-tubocurarine involves both a competitive and a noncompetitive binding site as determined by stopped-flow measurements of receptor-controlled ion flux in membrane vesicles

The issue of whether d-tubocurarine, the classical acetylcholine receptor inhibitor, inhibits the receptor by a competitive or noncompetitive mechanism has long been controversial. d-Tubocurarine, in this study, has been found to be both a competitive (KC = 120 nM) and a noncompetitive (KNC = 4 microM) inhibitor of receptor-mediated ion flux at zero transmembrane voltage in membrane vesicles prepared from Electrophorus electricus electroplax. A spectrophotometric stopped-flow method, based on fluorescence quenching of entrapped anthracene-1,5-disulfonic acid by Cs+, was used to measure both the rate coefficient of ion flux prior to receptor inactivation (desensitization) and the rate coefficient of the rapid inactivation process. Inhibition by d-tubocurarine of the initial rate of ion flux decreased with increasing acetylcholine concentration, consistent with competitive inhibition, but the inhibition by micromolar concentrations of d-tubocurarine could not be overcome with saturating concentrations of acetylcholine, consistent with noncompetitive inhibition. A minimum mechanism is proposed in which d-tubocurarine competes for one of the two acetylcholine activating sites and also binds to a noncompetitive site. The present data do not distinguish between one or two competitive sites, although one successfully accounts for all of the data. By variation of the acetylcholine concentration, the two types of sites could be studied in isolation. Binding of d-tubocurarine to the noncompetitive site does not change the rate of rapid receptor inactivation, whereas binding of d-tubocurarine to the competitive site decreases the rate of rapid inactivation by displacing acetylcholine, in agreement with the observation that d-tubocurarine does not inactivate (desensitize) the E. electricus receptor by itself.(ABSTRACT TRUNCATED AT 250 WORDS)

Parathyroid Hormone Bindingin Vivoto Renal, Hepatic, and Skeletal Tissues of the Rat Using a Radioautographic Approach*

We have examined in vivo binding of bovine (b) PTH-(1-84) and the analog [Nle8,18, Tyr34]bPTH-(1-34) amide to hepatic, skeletal, and renal rat tissues. Bioactive 125I-labeled bPTH-(1-84) or the 125I-labeled bPTH-(1-34) analog was injected alone into experimental animals or with either unlabeled PTH or unlabeled unrelated hormone into control animals. Corresponding tissues from experimental and control animals were then processed for light and electron microscope radioautography and analyzed quantitatively and qualitatively. Binding of both PTH forms occurred on hepatocytes and sinusoidal cells in liver, and hepatocyte binding was clearly specific and competitive. Intact PTH-(1-84), but not the amino-terminal fragment, bound to Kupffer cells, indicating a sequence-specific interaction. In bone, specific competitive PTH binding was seen over osteoblasts, but not osteoclasts. Skeletal PTH binding was also seen over connective tissue mononuclear cells, and sinusoidal endothelial cells. In kidney, specific competitive PTH binding was seen over glomerular podocytes, and over the antiluminal surface of cells of the proximal tubule, the thick ascending limb of Henle's loop, and the distal tubule. Noncompetitive binding was seen on the luminal surface of proximal convoluted tubule cells. We have, therefore, distinguished specific competitive binding sites, probably related to hormone action, from noncompetitive binding sites, presumably associated with hormone metabolism, on discrete cell types or cell regions within several tissues. Our approach provides morphological correlates to the biochemical interaction of PTH with its target tissues and should enhance our understanding of the relationship of target cell structure and function.

Molecular pharmacology of the nicotinic acetylcholine receptor

The functional transitions of the nicotinic acetylcholine receptor are regulated by acetylcholine binding sites and noncompetitive inhibitor binding sites. Amino acid residues contributing to these sites are being located in the sequences of the receptor subunits. The relative proximity on the α-subunit of portions of the acetylcholine binding site and of a noncompetitive inhibitor site might be a structural basis for the interaction of these sites and suggests a model for channel gating. In this review, Arthur Karlin, Peter Kao and Mario DiPaolo will focus on these functional sites.

On the Regulation of Phosphoenolpyruvate Carboxylase Activity from Maize Leaves by L-malate. Effect of pH

The effect of L-malate as reversible inhibitor of phosphoenolpyruvate carboxylase purified from maize leaves was studied between pH 7 and 8. The malate concentration required to reach fifty per cent inhibition was pH dependent being higher at pH 8. Complex inhibition kinetics with upwardly curved Dixon plots were obtained. Hill plots showed cooperative effects giving coefficients (n(H)) between 2.0 and 3.9. The K(i) values were 0.8 and 10 mM at pH 7 and 8 respectively. In addition, at lower pH the type of inhibition was mainly competitive with respect to phosphoenolpyruvate while at pH 8 it was non-competitive. The presence of glucose-6-phosphate in the assay medium reduced the competitive inhibition by malate without modifying the non-competitive effect, changing the Hill coefficient values to one at pH 8.0. Moreover, the dissociation constants for phosphoenolpyruvate (plus or minus MgCl(2)) and malate in the presence of glucose-6-phosphate were calculated at pH 7.0, 7.5, and 7.9 from the protection afforded by these compounds against chemical modification of phosphoenolpyruvate carboxylase by phenylglyoxal. The possible existence of competitive as well as non-competitive malate binding sites in phosphoenolpyruvate carboxylase and the physiological meaning of this interaction are discussed.

Side-effect reduction by use of drugs that bind to separate but equivalent binding sites.

If two drugs cause the same molecular effect by binding to separate noncompetitive binding sites, therapy with a mixture of the two drugs can provide a given desired effect with a lower level of side effects than therapy with either drug alone.

Surface-associated host proteins on virulent Treponema pallidum.

A surface coat of host serum proteins was detected on virulent Treponema pallidum by sodium dodecyl sulfate-gel electrophoresis. The loosely associated serum proteins could be removed by repeated washings in a protein-free medium. Washed T. pallidum retained the ability to readsorb numerous host proteins from rabbit serum as well as iodinated rabbit or human albumin. In addition, various avidly associated host serum proteins including albumin, alpha(2)-macroglobulin, transferrin, ceruloplasmin, immunoglobulin G, immunoglobulin M, and C3 were identified on the outer envelope of washed treponemes by an immunoadsorbent technique with protein A-bearing staphylococcus. Hyaluronidase treatment did not remove the avidly associated host proteins from the surface of washed treponemes, whereas trypsin treatment resulted in decreased levels of agglutination. Electrophoretic patterns of trypsin-treated treponemes showed that treponemal proteins as well as adsorbed host proteins were released concurrently by protease digestion. Reacquisition studies involving alpha(2)-macroglobulin and transferrin suggested the presence of noncompetitive binding sites for serum proteins on the treponemal outer envelope. Finally, differences among the T. pallidum preparations from individual rabbits with respect to incorporation of [(35)S]methionine, extent of agglutination with antisera, and length of time required for removal of avidly associated host proteins by trypsin treatment indicated biological variability among the treponemal populations.

Reversible inhibition of glucose transport in human erythrocytes by a series of pyridine derivatives.

A series of pyridine derivatives was chosen on the basis of structural similarity to glucose and to the hydrogen bonding capacity of this sugar. These compounds were found to reversibly inhibit glucose transport in a systematic fashion. The inhibition data can be rationalized by assuming that both competitive and noncompetitive binding sites exist on the cell membrane. The competitive site appears to have a much lower dissociation constant than the noncompetitive site for most derivatives. The effects of changes in substituents on the pyridine ring closely match, both in ring position and in the magnitude of the change in binding constant, those reported for derivatives of glucose. The stimulation of fluorodinitrobenzene inactivation of glucose transport by 2-amino-5-chloropyridine is in accord with this analysis. The strongest inhibitors bind about 100-fold more tightly than glucose itself. This series of pyridine compounds would appear to present a useful starting point for the development of site-specific irreversible inhibitors.