Biomedical subjects

M A Raftery

Publications and source records attributed to M A Raftery.

At least 19 recordsLinked to original sources

PubMed2000

Roles of agonist-binding sites in nicotinic acetylcholine receptor function.

Under equilibrium conditions, the nicotinic acetylcholine receptor from Torpedo electroplax carries two high affinity-binding sites for agonists. It is generally assumed that these are the only agonist sites on the receptor and that their occupancy results in rapid channel activation followed by slower conformational transitions that lead to the high affinity equilibrium state. These slow transitions are thought to reflect the physiological process of desensitization. Here we show that preequilibration of the high affinity sites with saturating concentrations of carbamylcholine does not diminish the ion flux response to subsequent exposure to higher (activating) concentrations of this agonist. This finding has profound implications with respect to receptor function: (1) occupancy of the high affinity sites per se does not desensitize the receptor and (2) these sites cannot be directly involved in receptor activation. It is thus necessary to invoke the presence of additional binding sites in channel opening.

Animals↗

PubMed2000

Interaction of a semirigid agonist with Torpedo acetylcholine receptor.

The binding of the semirigid agonist [(3)H]arecolone methiodide to the Torpedo nicotinic acetylcholine receptor has been correlated with its functional properties measured both in flux studies with Torpedo membrane vesicles and by single-channel analysis after reconstitution in giant liposomes. Under both equilibrium and preequilibrium conditions, the binding of arecolone methiodide is similar to that of other agonists such as acetylcholine. At equilibrium, it binds to two sites per receptor with high affinity (K(d) = 99 +/- 12 nM), and studies of its dissociation kinetics suggest that each of these sites is made up of two subsites that are mutually exclusive at equilibrium. The kinetics of arecolone methiodide binding were monitored by the changes in the receptor intrinsic fluorescence, and the data are consistent with a model in which the initial binding event is followed by sequential conformational transitions of the receptor-ligand complex. In flux studies, arecolone methiodide was approximately 3-fold more potent (EC(50) = 31 +/- 5 microM) than acetylcholine but its maximum flux rate was 4-10-fold lower. This phenomenon has been studied further by single-channel analysis of Torpedo receptors reconstituted in giant liposomes. Whereas the flexible agonist carbamylcholine (5 microM) was shown to induce channels with conductances of 56 and 34 pS with approximately equal frequency, arecolone methiodide (2 microM) preferentially induced the channel of lower conductance. These results are interpreted in terms of a simple model in which the rigidity of arecolone methiodide restrains the conformation that the receptor-ligand complex can adopt, thus favoring the lower conductance state.

Animals↗

PubMed1999

Eserine and other tertiary amine interactions with Torpedo acetylcholine receptor postsynaptic membrane vesicles.

Interaction of the tertiary amines, arecolone, eserine (physostigmine), (+)-epibatidine, and (+/-)-epibatidine, with Torpedo nicotinic acetylcholine receptor-enriched membrane vesicles was investigated to characterize their action on the receptor, using stopped-flow thallium (I)-flux spectrofluorimetry. Arecolone, (+)-epibatidine, and (+/-)-epibatidine were agonists with activation constants of 390, 19, and 39 microM, respectively. Eserine was not an agonist but rather an antagonist for agonist-induced activation of the receptor with an inhibition constant of approximately 150 microM. The choice of the fluorescent dye used (entrapped within the membrane vesicles) was critical for interpretation of the effects of eserine. With 1,3,6,8-pyrene tetrasulfate (PTS), eserine appeared to act as an agonist. However, it was shown that such an effect was caused by rapid diffusion of the uncharged form of the amine across the membrane followed by direct interaction with PTS rather than eserine-induced cation transport. The use of a different fluorescent dye, 8-aminonaphthaline-1,3,6-trisulfate, with which eserine does not interact allowed demonstration of the action of eserine as an antagonist rather than as an agonist.

Amines↗

PubMed1997

Agonist binding to the Torpedo acetylcholine receptor. 1. Complexities revealed by dissociation kinetics.

Examination of the kinetics of dissociation of [3H]acetylcholine and [3H]suberyldicholine from the membrane-bound acetylcholine receptor from Torpedo californica has revealed complexities in the high-affinity binding of nicotinic agonists. Each agonist binds to two high-affinity sites per receptor with an equilibrium dissociation constant of approximately 15 nM. When dissociation of [3H]acetylcholine from the receptor complex was triggered by dilution, dissociation occurred as a monophasic process with an apparent rate of 0.023 +/- 0.010 s(-1). However, when micromolar concentrations of unlabeled agonists (acetylcholine, carbamylcholine or suberyldicholine) were included in the dilution buffer this rate increased about 5-fold. This accelerating effect occurred even when the two high-affinity sites were initially saturated with the radioligand. This suggested the presence of an additional site (or subsite) for agonist with affinity in the micromolar range. However, at concentrations of 0-20 microM, no additional sites for [3H]acetylcholine were detected at equilibrium. To explain these results, we propose that each high-affinity site is made up of two subsites, A and B, which are mutually exclusive at equilibrium. With [3H]acetylcholine initially occupying site A, occupancy of site B by unlabeled ligand reduces the affinity for site A and accelerates the dissociation of the radioligand. Studies of dissociation of [3H]suberyldicholine, a large bis-quaternary agonist, provide some clue as to the possible physical nature of these subsites. Whereas its dissociation rate was similar to that of [3H]acetylcholine (0.028 +/- 0.012 s(-1)), this rate was only marginally, if at all, affected by the presence of unlabeled ligands. These results, in addition to those presented in the accompanying manuscript, lead to the proposal that [3H]suberyldicholine is able to cross-link the two subsites or at least sterically occlude the second site.

Acetylcholine↗

PubMed1997

Agonist binding to the Torpedo acetylcholine receptor. 2. Complexities revealed by association kinetics.

The binding of suberyldicholine to membrane-bound Torpedo acetylcholine receptor has been monitored by fluorescence changes of covalently bound 5-iodoacetamidosalicylic acid (IAS). At equilibrium, suberyldicholine binds to two high-affinity binding sites (Kd approximately 20 nM). Kinetic experiments reveal that there is rapid formation of an initial complex (Kd approximately 2 microM) which undergoes sequential fast (k(app) approximately 1 s(-1)) and slow (k(app) approximately 0.05 s(-1)) conformational changes. These kinetics differ from those reported for other agonists [Blanchard, S. G., Dunn, S. M. J., & Raftery, M. A. (1982) Biochemistry 24, 6258-6264] in that, for suberyldicholine, there is no evidence for a second pathway involving the binding of an additional agonist molecule. These results, considered together with the observed dissociation kinetics (accompanying manuscript), suggest that each high-affinity site for acetylcholine is made up of two subsites, which suberyldicholine is able to bridge, thus occluding the binding of a second ligand. The kinetic mechanism for acetylcholine binding has been re-examined to accommodate the complexities of the [3H]-acetylcholine dissociation kinetics and the observation that, at equilibrium, no more than two occupied binding sites are detected [accompanying manuscript: Dunn, S. M. J., & Raftery, M. A. (1997) Biochemistry 36, 3846-3853]. It is suggested that, for each acetylcholine binding site, a second ligand is able to bind but that the ternary complex is transient since one of the two bound ligands again dissociates in the formation of the equilibrium mono-liganded complex. To further probe the physical nature of the two subsites, the binding of a series of bis-quaternary suberyldicholine analogues, (CH3)3N+CH2CH2OCO(CH2)n-COOCH2CH2N+(CH3)3, to IAS-labeled receptor preparations has been examined. Analogues in which n < 5 behave like acetylcholine, i.e., a second ligand binding pathway is observed, but longer ligands (n = 5-10) act like suberyldicholine and may be long enough to cross-link the sites.

Acetamides↗

PubMed1994

The nicotinic acetylcholine receptor: structure and autoimmune pathology.

The nicotinic acetylcholine receptors (AChR) are presently the best-characterized neurotransmitter receptors. They are pentamers of homologous or identical subunits, symmetrically arranged to form a transmembrane cation channel. The AChR subunits form a family of homologous proteins, derived from a common ancestor. An autoimmune response to muscle AChR causes the disease myasthenia gravis. This review summarizes recent developments in the understanding of the AChR structure and its molecular recognition by the immune system in myasthenia.

Amino Acid Sequence↗

PubMed1993

Cholinergic binding sites on the pentameric acetylcholine receptor of Torpedo californica.

The binding of agonists, antagonists, and the acetylcholinesterase inhibitor, eserine, to the nicotinic acetylcholine receptor from Torpedo californica has been monitored by the fluorescence changes of two extrinsic probes that have been covalently attached to the receptor protein. Although both probes, 5-(iodoacetamido)salicylic acid (IAS) and 4-[N-[(2-iodoacetoxy)ethyl]-N-methylamino]-7-nitrobenz-2-oxa-1,3-diaz ol e (IANBD) react with sulfhydryl groups, they do not react at the same location. The conditions for IAS labeling and competition studies have shown that, following reduction of the receptor, this fluorophore reacts with the same cysteines on each of the two alpha subunits that may be labeled by the alkylating agonist, [3H]bromoacetylcholine. The fluorescence of this probe is sensitive to the binding of agonists and competitive antagonists to two high-affinity sites on the receptor. IANBD does not react with the same cysteines as IAS, and its fluorescence is unchanged by the high-affinity binding of agonists and antagonists. The fluorescence of this probe is, however, specifically and saturably enhanced by the binding of agonists to distinct low-affinity sites. Heterogeneity in the NBD fluorescence changes induced by the bis-quaternary agonist, suberyldicholine, has indicated that the stoichiometry of low-affinity sites is also two per receptor. Stopped-flow studies of agonist binding to receptor preparations that had been doubly labeled by both fluorophores demonstrate that the conformational changes detected by IAS occur on slow time scales of seconds to minutes whereas a much faster conformational change is revealed by changes in NBD fluorescence.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗

PubMed1993

A high-affinity site for acetylcholine occurs close to the alpha-gamma subunit interface of Torpedo nicotinic acetylcholine receptor.

Affinity labeling techniques have been used to investigate the location of high-affinity binding sites for cholinergic agonists on the Torpedo acetylcholine receptor and the extent of overlap of these sites with those for long alpha-neurotoxins. Following reduction of the receptor by dithiothreitol, reaction with [3H]bromoacetylcholine leads to covalent incorporation into each of the two alpha subunits. At high concentrations of [3H]bromoacetylcholine (240 microM) and with prolonged incubation times (1-2 h), this labeling was not inhibited by either alpha-bungarotoxin or alpha-najatoxin. Following maximum labeling by [3H]bromoacetylcholine, no residual high binding sites for [125I]-alpha-bungarotoxin could be detected in the membrane-bound receptor, but 50% of the original sites were recovered by receptor solubilization. Since it has previously been reported that one of the two sites for alpha-bungarotoxin in the membrane-bound receptor is readily reversible but is converted to a high-affinity state by solubilization [Conti-Tronconi, B. M., Tang, F., Walgrave, S., & Gallagher, W. (1990) Biochemistry 29, 1046-1054], these results demonstrate that the covalently bound agonist inhibits the binding of alpha-bungarotoxin only to its higher affinity site in the membrane. When [3H]bromoacetylcholine labeling was carried out after reduction of the receptor by sodium borohydride rather than dithiothreitol, both alpha and gamma subunits of the receptor were labeled. Labeling of both subunits was completely inhibited if the receptor was first reduced with dithiothreitol and the alpha subunit sites were previously covalently labeled by unlabeled bromoacetylcholine.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗

PubMed1993

Photoaffinity labeling of Torpedo acetylcholine receptor at multiple sites.

The acetylcholine receptor from Torpedo californica electroplax was labeled with the photoaffinity reagent bis(3-azidopyridinium)decane perchlorate. All four receptor subunits (alpha, beta, gamma, and delta) were specifically labeled. In the presence of cholinergic agonists the gamma-, beta-, and delta-subunit labeling was decreased significantly, whereas labeling of the alpha subunit was minimally affected. Full occupancy of the two high-affinity sites involving the alpha subunits in the vicinity of alpha-Cys-192-Cys-193 by covalent reaction with bromoacetylcholine also caused a large decrease of gamma-subunit labeling by the photoaffinity reagent and lesser but significant decreases in beta- and delta-subunit labeling. No decrease in labeling of the alpha subunit was seen. Labeling of the alpha subunit could, however, be inhibited by high concentrations of the agonist carbamoylcholine. We conclude that the binding sites of high-affinity reside at interfaces of the alpha subunit and other subunits and that the alpha subunit also contributes to formation of a low-affinity site(s) for cholinergic compounds.

Acetylcholine↗

PubMed1993

Specific binding of ATP to extracellular sites on Torpedo acetylcholine receptor.

The beta- and delta-subunits of the nicotinic acetylcholine receptor from Torpedo californica were covalently photolabeled at the synaptic surface with the ATP photoaffinity analogue [alpha-32P]-8-azido-ATP. The specificity of labeling for nucleotide binding sites was demonstrated by the saturation of labeling with increasing concentration of 8-azido-ATP and the inhibition of photolabeling by ATP. Protection studies suggest that the binding sites for the photolabel are unique and are not associated with the cholinergic ligand binding sites.

Adenosine Triphosphate↗

PubMed1993

Monoclonal antibodies against synthetic sequences of the nicotinic receptor cross-react fully with the native receptor and reveal the transmembrane disposition of their epitopes.

Monoclonal antibodies (mAbs) were derived from mice immunized with synthetic peptide sequence regions of the alpha subunit of the nicotinic acetylcholine receptor from Torpedo electric tissue (TAChR). Sequence-specific mAbs were obtained against the following peptides: alpha 1-20, alpha 291-308, alpha 304-322, alpha 332-350, alpha 346-364, alpha 360-378, alpha 376-393, alpha 390-409, and alpha 420-437. The ability of mAbs to recognize native TAChR was quantitated by immunoprecipitation of TAChR solubilized in the nondenaturing detergent Triton X-100. mAbs against peptide alpha 304-322, alpha 332-350, and alpha 360-378 cross-reacted with most or all Triton-solubilized TAChR molecules and, in immunoelectron microscopy experiments, bound to the cytoplasmic surface of AChR-rich postsynaptic membrane fragments. Two mAbs specific for the sequence alpha 376-393, proposed to form an amphypathic alpha helix possibly involved in formation of the ion channel, recognized only approximately 35% of Triton-solubilized TAChR molecules and did not react with membrane-bound TAChR. All of these sequence-specific antibodies recognized SDS-denatured TAChR alpha subunit in Western blots. MAbs specific for the amino-terminal sequence region of the alpha subunit, alpha 1-20, and for the sequences alpha 291-308, alpha 346-364, and alpha 390-409 did not recognize native TAChR. A mAb directed against the carboxyl-terminal region, alpha 420-437, recognized with low apparent titer Triton-solubilized TAChR, not membrane-bound TAChR. In conclusion, a complex membrane protein, TAChR, contains several continuous sequence segments exposed on the TAChR surface, because different mAbs raised against certain synthetic sequences recognized most or all native TAChR molecules. By analogy, it should be possible for most proteins of known sequence to raise anti-peptide antibodies fully cross-reactive with the native cognate protein.

Amino Acid Sequence↗

PubMed1993

An assay for simultaneous multiple determinations of peptide binding to MHC class II molecules.

Binding of positively charged radiolabeled synthetic peptides to human major histocompatibility complex class II DR molecules, purified by affinity chromatography from lymphoblastoid B cell lines of different haplotypes, is rapidly, quantitatively, and specifically assayed by selective adsorption of the complexes between peptide and DR molecules onto DEAE-cellulose paper disks. This assay can be used as a revealing system of the ability of unlabeled test peptides to competitively inhibit the binding between the radiolabeled peptide and the DR molecules, thus measuring the binding of the competitor peptides, irrespective of their charge properties, to different DR molecules.

Amino Acid Sequence↗

PubMed1992

Scanning tunneling microscopy imaging of Torpedo acetylcholine receptor.

The synaptic surface of the acetylcholine receptor in membranes from Torpedo californica electric organ has been imaged by scanning tunneling microscopy. The molecule appears pentameric, with one major and four minor protrusions rising above the surface, and these protrusions encompass a large central cavity. The outer diameter of the molecule is 69 +/- 10 A, while the diameter of the cavity, measured at the widest complete contour line delimiting the opening, is 26 +/- 7 A. The images and dimensions obtained are consistent with the structure determined from hybrid density maps obtained by x-ray diffraction and electron microscopy. Thus, scanning tunneling microscopy can be used to obtain overall dimensions and low-resolution structural features of the surface of a membrane-embedded protein.

Animals↗

PubMed1988

Isolation and characterization of nicotinic acetylcholine receptor-like protein from fetal calf thymus.

A nicotinic acetylcholine receptor-like protein (AChR-LP) was isolated from fetal calf thymus by affinity chromatography using cobrotoxin-Sepharose after alkaline extraction and solubilization with Triton X-100. The AChR-LP had a specificity of 1.61 +/- 1.12 nmol of alpha-bungarotoxin binding sites per mg of protein. The isoelectric point, sedimentation coefficient and amino acid composition of the purified AChR-LP were very similar to those of muscle and electric organ AChRs. Upon SDS-polyacrylamide gel electrophoresis purified thymus AChR-LP preparations contained up to 6 polypeptide bands of molecular weights of 40,000, 43,000, 51,000, 56,000, 58,000, and 66,000, respectively. The peptides of 40,000, 51,000, 56,000, and 66,000 dalton cross-reacted with the four subunits of Torpedo californica and fetal calf muscle AChR.

Amino Acids↗

PubMed1987

Identification of subunits of acetylcholine receptor that interact with a cholesterol photoaffinity probe.

All four subunits of the acetylcholine receptor in membrane vesicles isolated from Torpedo californica have been labeled with [3H]cholesteryl diazoacetate. As this probe incorporates into lipid bilayers analogously to cholesterol, this result indicates that acetylcholine receptor interacts with cholesterol. This investigation also demonstrates that this probe is a useful reagent for studying the interaction of cholesterol with membrane proteins.

Affinity Labels↗

PubMed1986

Acetylcholine receptor dimers are stabilized by extracellular disulfide bonding.

Torpedo acetylcholine receptor (AcChR) exists predominantly as dimers, formed by two monomers held together by a disulfide bridge(s). The dimers are easily cleaved to monomers by reducing agents. 2-mercaptoethanesulfonic acid is shown to be a membrane-impermeant reducing agent which cleaves receptor dimers when it is present only on the outside of intact membrane vesicles. There is no increase in the extent of cleavage when 2-mercaptoethanesulfonic acid is also loaded inside the vesicles. Therefore the disulfide bond(s) involved in the dimerization of the Torpedo acetylcholine receptor is (are) formed by cysteine residues which are exposed on the extracellular side of the membrane.

Animals↗

PubMed1986

Nicotinic acetylcholine receptor contains multiple binding sites: evidence from binding of alpha-dendrotoxin.

We have studied the stoichiometry of the binding of the long alpha-neurotoxins from the venom of Dendroaspis viridis (alpha-dendrotoxin) and Naja naja siamensis (alpha-cobratoxin) to the membrane-bound acetylcholine receptor (AcChoR) from Torpedo californica electric organ. The number of toxin molecules bound to one AcChoR molecule was determined by simultaneous-quantitative gas-phase microsequencing of all the amino acid sequences present in AcChoR-alpha-neurotoxin complexes. This method permits the use of homogeneous (nonradiolabeled) preparations of native toxins to obtain molar ratios of neurotoxin-receptor complexes. The stoichiometry obtained for alpha-cobratoxin was 2.1 +/- 0.2 (n = 4), in agreement with the accepted view that alpha-cobratoxin, like alpha-bungarotoxin, binds to the two alpha subunits, which are constituent polypeptides of the AcChoR molecule. alpha-Dendrotoxin gave a stoichiometry of 4.1 +/- 0.5 (n = 12); therefore, the AcChoR molecule contains four binding sites for this alpha-neurotoxin, two of which are recognized by alpha-cobratoxin. In support of this contention we have also found that when the AcChoR is saturated with alpha-bungarotoxin, addition of alpha-dendrotoxin markedly accelerates the dissociation of the bound alpha-bungarotoxin, demonstrating that the occupancy of the additional two sites by the latter toxin influences and decreases the affinity of the former toxin for its two binding sites. The fact that the AcChoR molecule is a pseudosymmetric complex of five highly homologous peptides suggests the possibility that as many as five binding sites for cholinergic ligand could be present, one on each subunit.

Animals↗

PubMed1985

Structural and functional aspects of the nicotinic acetylcholine receptor.

The molecular structure of nicotinic acetylcholine receptors (AcChR) from different peripheral tissues and from different brain areas has been studied. AcChRs from Torpedo and Electrophorus electric organ and from piscine, avian, and mammalian muscle have been shown to be highly conserved proteins composed of four types of homologous subunits (alpha, beta, gamma, and delta) which associate in a stoichiometry alpha 2 beta 1 gamma 1 delta 1 to form a pseudosymmetric pentameric complex molecule. Genealogical analysis suggests that all the subunits of these AcChR derive from a common ancestral gene and that the divergence occurred very early in the evolution of the receptor. This shared ancestry and the very early divergence of the four subunits, as well as their highly conserved structures along the animal evolution, suggests that each of the subunits evolved to perform specific crucial roles in the function of the AcChR complex. In Torpedo the two alpha-subunits present in the AcChR molecule are glycosylated to a different extent. This may be the reason why the binding sites for cholinergic ligands that are located on the alpha-subunits are non-equivalent. The pseudosymmetric pentameric complex has all the properties of a physiological receptor with respect to known parameters leading to postsynaptic depolarization, and therefore contains the molecular structure/structures that constitute a cation selective channel for transport across the membrane. The complex also contains recognition sites for acetylcholine, cholinergic antagonists, polypeptide neurotoxins, small molecule neurotoxins, and local anesthetics. Therefore a multiplicity of receptor-ligand associations are possible. This leads to more complex models of such interactions than previously considered.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗