Biomedical subjects

H R Arias

Publications and source records attributed to H R Arias.

25 records · Page 2Linked to original sources

PubMed1993

Transverse localization of the quinacrine binding site on the Torpedo acetylcholine receptor.

We demonstrated previously that a phencyclidine-displaceable quinacrine binding site exists at the lipid-protein interface of the Torpedo acetylcholine receptor (AcChR) (Valenzuela, C. F., Kerr, J. A., and Johnson, D. A. (1992) J. Biol. Chem. 267, 8238-8244). In this manuscript, we assess (1) the transverse position of this site in the lipid bilayer by examining the ability of a series of paramagnetic n-doxyl stearates (n-SALs) and iodide to quench receptor-bound quinacrine and membrane-partitioned octadecyl rhodamine B (C18-Rho) fluorescence and (2) the stoichiometry of histrionicotoxin- or phencyclidine-displaceable quinacrine binding. Initial experiments established what fraction of the n-doxyl stearates partitioned into the membranes and that the n-doxyl stearates do not interfere with quinacrine binding to the receptor at the concentrations used in the quenching studies. The n-doxyl stearate quenching experiments indicated relatively small (< 2) differences between the n-doxyl stearates to quench receptor-bound quinacrine fluorescence, with a rank order of 7-SAL > or = 5-SAL > 12-SAL > 16-SAL. This contrasts with the n-doxyl stearate quenching of the membrane-partitioned C18-Rho which showed as much as an 8.6-fold difference between the various isomers with a rank order of quenching efficiencies of 5-SAL > 7-SAL > 12-SAL > or = 16-SAL. Iodide quenching measurements indicated significant solute accessibility to membrane-partitioned C18-Rho but not to receptor-bound quinacrine. The ratios of the bimolecular quenching rate constants for free to bound quinacrine and for free rhodamine B to membrane-partitioned C18-Rho were 53.4 and 6.6, respectively. Direct titration of quinacrine into suspensions of a high concentration of AcChR-associated membranes yielded an upper limit to the binding stoichiometry of 1.4 HTX- or PCP-displaceable quinacrine binding sites/AcChR functional units. The results suggest that there is a single phencyclidine- or histrionicotoxin-displaceable quinacrine binding site located at or somewhat below the level of the C5-C7 in the phospholipid acyl chains at the lipid-protein interface.

Animals↗

PubMed1990

Association of spin-labeled local anesthetics at the hydrophobic surface of acetylcholine receptor in native membranes from Torpedo marmorata.

The interactions between a series of spin-labeled local anesthetic analogues and the nicotinic acetylcholine receptor (AChR) have been investigated by means of electron spin resonance (ESR) and fluorescence spectroscopy. The paramagnetic local anesthetic analogues quenched the intrinsic tryptophan fluorescence of AChR-rich membranes in an agonist-dependent manner, demonstrating a direct interaction with the AChR. The quenching efficiency was greater for the benzocaine than for the thioprocaine analogue. The protein was found to restrict directly the molecular motion of the spin-labeled analogues, as seen by the appearance of a highly anisotropic component in the ESR spectrum. The relative affinity of the population of local anesthetic probes which interacts directly with the integral protein of the AChR-rich membranes was calculated on the basis of relative association constants, Kr, determined by ESR. By comparison with the relative association constant for spin-labeled phospholipid, Kro, it was possible to differentiate between local anesthetic analogues interacting with high (Kr/Kro greater than 2), intermediate (Kr/Kro = 1.6-1.9), and low (Kr/Kro less than or equal to 1.3) specificity and to calculate the fraction of protein-associated probe in each case. Differences were observed in the presence of agonist (0.1 mM carbamylcholine) with some, but not all, of the spin-labeled derivatives. The role of the protonatable diethylammonium group in the specificity of the interaction of the procaine and thioprocaine analogues was investigated. Only in the uncharged form, or in the charged form at high ionic strength, was there a preferential association of these two local anesthetic analogues.(ABSTRACT TRUNCATED AT 250 WORDS)

Allosteric Regulation↗

PubMed1990

Effect of local anaesthetics on steroid-nicotinic acetylcholine receptor interactions in native membranes of Torpedo marmorata electric organ.

Interactions between steroids and the nicotinic acetylcholine receptor (AChR) have been studied in native membrane vesicles from Torpedo marmorata electric organ by electron spin resonance (ESR) and fluorescence techniques. ESR spectra of spin-labelled cholestane (CSL) revealed that this steroid probe was incorporated into the AChR-rich membrane vesicles in regions which were to a certain extent enriched preferentially in the steroid, both in the presence and in the absence of local anaesthetics. Since the nitroxide group present in CSL is also a paramagnetic quencher of the intrinsic protein fluorescence, this property was used to characterize the AChR-steroid interactions. The quenching induced by CSL was sensitive both to AChR concentration and to the action of cholinergic agonists. In competition experiments, the ability of CSL to quench the AChR intrinsic fluorescence was markedly inhibited by benzocaine, tetracaine and QX-222 (a quaternary trimethylammonium derivative of lidocaine), and was totally inhibited by procaine. The effectiveness of local anaesthetics in inhibiting CSL-induced quenching followed the order: procaine much greater than benzocaine approximately greater than tetracaine greater than QX-222. This inhibition effect was shown not to be charge-dependent. The data can be interpreted in terms of a model requiring specific association sites for local anaesthetics on the hydrophobic surface of the AChR which at least partially overlap with those for steroids.

Anesthetics, Local↗

PubMed1987

Composition of lipids in elasmobranch electric organ and acetylcholine receptor membranes.

The composition of phospholipids from electric organ and from membranes enriched in acetylcholine receptors (AChRs) is analyzed in three elasmobranch fish (Torpedo marmorata, Torpedo californica, and Discopyge tschudii). Irrespective of their purity, AChR-containing membranes are similar to electric organ in lipid and fatty acid composition. The following characteristics are common to the three species: (a) Choline, ethanolamine, and serine glycerophospholipids account for 80-90% of the phospholipids. (b) Their major fatty acid constituents are monoenes, saturates, and long-chain (n-3) polyenes (especially docosahexaenoate). (c) A large proportion of the ethanolamine glycerophospholipids (30-50%) is made up by plasmenylethanolamine, which contains fewer polyenes than phosphatidylethanolamine per mole of lipid. (d) Polyphosphoinositides represent 20-30% of the inositides of electric organ. (e) Phosphatidylinositol and phosphatidate have large proportions of 20- and 22-carbon polyenes. (f) Diphosphatidylglycerol and triacylglycerols are rich in oleate but also contain long-chain polyenes. (g) Sphingomyelin has monoenes and saturates ranging from 14 to 26 carbons. Species-related variations are observed (a) in the ratios between some phospholipid classes and subclasses and (b) in the relative abundance of the major polyunsaturated acyl chains of phospholipids. Despite these differences, the average unsaturation and length of fatty acids in major phospholipid classes are similar for the three species.

Animals↗

PubMed1987

Lipid metabolism in electroplax.

The in vivo labeling of electrocyte lipids is followed after injection of radioactive glycerol and two fatty acids, oleate and arachidonate, into the electric organ of an elasmobranch (Discopyge tschudii). De novo synthesis of lipids and acyl-exchange reactions are operative in the electrocyte. The three precursors are preferentially incorporated into phosphatidylcholine, phosphatidylinositol, and triacylglycerols. The highest specific activities are attained by triacylglycerols and polyphosphoinositides. Electrocyte stacks from electric organ show an efficient and continuous esterification of oleate and arachidonate into lipids after several hours of incubation. Except for an apparently more active labeling of triacylglycerols, which is attributed to the larger availability of free fatty acid precursors under the in vitro experimental conditions, the pattern of lipid labeling is similar to that attained in vivo. 32P-labeled lipids are also steadily produced in electrocyte stacks (24 h of incubation with [32P]phosphate) using glucose as the sole exogenous source of energy. Polyphosphoinositides are the lipids preferentially labeled. The ability to sustain the labeling of lipids under in vitro conditions renders isolated electrocyte stacks an interesting model for future research on lipid involvement in cholinergic function.

Animals↗

PubMedDate not supplied

Agonist-induced displacement of quinacrine from its binding site on the nicotinic acetylcholine receptor: plausible agonist membrane partitioning mechanism.

It was previously demonstrated that high concentrations of cholinergic agonists such as acetylcholine (ACh), carbamylcholine (CCh), suberyldicholine (SubCh) and spin-labelled acetylcholine (SL-ACh) displaced quinacrine from its high-affinity binding site located at the lipid-protein interface of the nicotinic acetylcholine receptor (AChR) (Anas, H. R. and Johnson, D. A. (1995) Biochemistry, 34, 1589-1595). In order to account for the agonist self-inhibitory binding site which overlaps, at least partially, with the quinacrine binding site, we determined the partition coefficient (Kp) of these agonists relative to the local anaesthetic tetracaine in AChR native membranes from Torpedo californica electric organ by examining (1) the ability of tetracaine and SL-ACh to quench membrane-partitioned 1-pyrenedecanoic acid (C10-Py) monomer fluorescence, and (2) the ability of ACh, CCh and SubCh to induce an increase in the excimer/monomer ratio of C10-Py-labelled AChR membrane fluorescence. To further assess the differences in agonist accessibility to the quinacrine binding site, we calculated the agonist concentration in the lipid membrane (CM) at an external agonist concentration high enough to inhibit 50% of quinacrine binding (IC50), which in turn was obtained by agonist back titration of AChR-bound quinacrine. Initial experiments established that high agonist concentrations do not affect either transmembrane proton concentration equilibria (pH) of AChR membrane suspension or AChR-bound quinacrine fluorescence spectra. The agonist membrane partitioning experiments indicated relatively small (< or = 20) Kp values relative to tetracaine. These values follow the order: SL-ACh>SubCh>>CCh-ACh. A direct correlation was observed between Kp and the apparent inhibition constant (Ki) for agonists to displace AChR-bound quinacrine. Particularly, agonist with high KpS such as SL-ACh and SubCh showed low Ki values, and this relationship was opposite for CCh and ACh. The calculated CM values indicated significant (between 7 and 54 mM) agonist accessibility to lipid membrane. By themselves, these results support the conjecture that agonist self-inhibition seems to be mediated by the quinacrine binding site via a membrane approach mechanism. The existence of an agonist self-inhibitory binding site, not located in the channel lumen would indicate an allosteric mechanism of ion channel inhibition; however, we can not discard that the process of agonist self-inhibition can also be mediated by a steric blockage of the ion channel.

Acetylcholine↗

PubMedDate not supplied

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.

Affinity Labels↗