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A Maelicke

Publications and source records attributed to A Maelicke.

At least 73 records · Page 4Linked to original sources
PubMed1994

Diversity of nicotinic acetylcholine receptors in rat hippocampal neurons. II. The rundown and inward rectification of agonist-elicited whole-cell currents and identification of receptor subunits by in situ hybridization.

Our previous study demonstrated for the first time that nicotinic currents evoked in rat hippocampal neurons could be grouped into four categories (types IA, IB, II and III) according to their functional and pharmacological characteristics. In the second part of our continuing studies, the structural and functional diversity of nicotinic receptors expressed in hippocampal neurons was further explored. Type IA, the predominant and alpha-bungarotoxin-sensitive current, but not type II, the alpha-bungarotoxin-insensitive current, showed rundown in the peak amplitude during the whole-cell recording. The rundown of type IA currents could be prevented when the ATP-regenerating compound phosphocreatine, alone or in combination with ATP and creatine phosphokinase, was added to the internal recording solution. The addition to the internal solution of either the microfilament-stabilizing agent phalloidin (5 microM) or the microtubule-stabilizing agent taxol (50 microM) did not alter or prevent rundown in type IA currents. Type IA and type II currents showed inward rectification. The inward rectification of type IA currents was dependent on the presence of intracellular Mg++, whereas that of type II currents was independent of Mg++. When Mg++ was present in the internal pipette solution, the inward rectification of type IA currents was sustained throughout the recording time. However, when nominally Mg(++)-free internal solution was used, the inward rectification decreased with recording time in type IA currents, but not in type II currents, as a consequence of removal of intracellular Mg++. In situ hybridization demonstrated the presence of alpha 7-, alpha 4- and beta 2-nicotinic acetylcholine receptor subunit mRNAs in cultured hippocampal neurons. The distribution among the neurons of the mRNAs for alpha 7- and alpha 4-nicotinic acetylcholine receptor subunits, correlated with the frequency with which type IA and type II currents, respectively, could be evoked in these neurons. The present results provide evidence for 1) the presence of intracellular high-energy phosphate-dependent processes linked with the nicotinic acetylcholine receptor subserving type IA currents, 2) a requirement of intracellular Mg++ for the inward rectification of type IA currents and 3) a correlation between the distribution of nAChR subunits and the different probabilities of eliciting distinct types of nicotinic currents in hippocampal neurons.

Animals↗
PubMed1994

Physostigmine and galanthamine: probes for a novel binding site on the alpha 4 beta 2 subtype of neuronal nicotinic acetylcholine receptors stably expressed in fibroblast cells.

In the present study, we demonstrated that the chicken alpha 4 beta 2 neuronal nicotinic receptor stably expressed in transfected mouse fibroblasts (M10 cells) can be activated via the acetylcholine-binding site or via a site that is distinct from that for acetylcholine and recognizes physostigmine and galanthamine as agonists. In outside-out patches excised from dexamethasone-induced M10 cells, (+)-anatoxin-a, physostigmine and galanthamine (each at 1 microM) activated single channels with conductances of 18 and 30 pS. Dihydro-beta-erythroidine (1-30 nM), but not the nicotinic receptor-specific monoclonal antibody FK1, reduced the frequency of channels activated by anatoxin (1 microM). On the other hand, the frequency of channel activity induced by physostigmine (1 microM) was unaffected by dihydro-beta-erythroidine and was markedly decreased by FK1. In uninduced M10 cells and in dexamethasone-treated untransfected fibroblasts, we observed that physostigmine, galanthamine and nicotinic agonists did not evoke whole-cell or single-channel currents. Also, neither [3H]L-nicotine nor FK1 was able to bind to uninduced M10 cells. In dexamethasone-induced M10 cells, the nicotinic agonists acetylcholine, anatoxin, 1,1-dimethyl-4-phenylpiperazinium, (-)-nicotine, and cytisine (each at 100 microM) activated whole-cell currents that showed a marked inward rectification and were sensitive to blockade by dihydro-beta-erythroidine (100 nM). However, neither galanthamine nor physostigmine could evoke whole-cell currents in cells that were responsive to nicotinic agonists. Other effects of physostigmine and galanthamine on the nicotinic receptor that outweight the agonist properties of these compounds could account for their inability to evoke whole-cell currents.

Animals↗
PubMed1993

Photoaffinity labeling of Torpedo acetylcholine receptor by physostigmine.

The plant alkaloid physostigmine, an established anti-cholinesterase agent of the carbamate type, has recently been shown to bind to the nicotinic acetylcholine receptor from Torpedo marmorata electrocytes [Okonjo, K. O., Kuhlmann, J. & Maelicke, A. (1991) Eur. J. Biochem. 200, 671-677]. Pharmacological studies of physostigmine-induced ion flux into nicotinic-acetylcholine-receptor-rich membrane vesicles, indicated distinct binding sites for physostigmine and acetylcholine. As shown in this study by photoaffinity labeling with [phenyl-(n)-3H](-)physostigmine, the physostigmine-binding site is located within the same subunit (alpha polypeptide) of the receptor as the acetylcholine-binding site. Using a variety of proteolytic cleavage conditions for the purified alpha polypeptide, several [3H]physostigmine-labeled peptides were isolated and sequenced. From the radioactivity released in the course of the Edman degradations of the labeled peptides, it was found that the label was associated in all cases with Lys125. These results identify a novel ligand-binding site for the Torpedo nicotinic acetylcholine receptor that is different in location from binding sites identified previously for acetylcholine, its established agonists and antagonists, and direct channel blockers.

Affinity Labels↗
PubMed1993

Physostigmine and neuromuscular transmission.

Single channel studies carried out in cultured rat myoballs and cultured hippocampal neurons, and ion flux studies performed on Torpedo electrocyte membrane vesicles, showed that physostigmine (Phy), a well-established acetylcholinesterase inhibitor, interacts directly with nicotinic acetylcholine receptors (nAChR). Low concentrations (0.1 microM) of Phy activate the receptor integral channel, whereas higher concentrations blocked the channel in its opened state. In contrast to channel activation by acetylcholine (ACh) and classical cholinergic agonists, however, Phy was capable of activating the nAChR channel even when the ACh binding sites were blocked by competitive antagonists, such as alpha-neurotoxins and d-tubocurarine, or when the nAChR was desensitized by preincubation with high concentrations of ACh. The binding site at which Phy binds and activates the nAChR was mapped. It was located within the N-terminal extracellular region of the alpha-polypeptide, in close proximity to the binding site of the natural transmitter. These data identify a novel binding site at nAChRs from many species and tissues that may be involved in receptor regulatory processes.

Acetylcholine↗
PubMed1993

Biochemical characterization of a novel channel-activating site on nicotinic acetylcholine receptors.

We have studied the interaction of the reversible acetylcholine esterase inhibitor (-)physostigmine and several structurally related compounds with the nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata electric tissue by means of ligand-induced ion flux into nAChR-rich membrane vesicles, direct binding studies and photoaffinity labeling. (-)Physostigmine acts as a channel-activating ligand at low concentrations and as a direct channel blocker at elevated concentrations. Channel activation is not inhibited by desensitizing concentrations of ACh or ACh-competitive ligands (including alpha-bungarotoxin and D-tubocurarine) but is inhibited by antibody FK1 and several other compounds. From photoaffinity labeling using tritiated physostigmine and mapping of the epitope for the Phy-competitive antibody FK1, the binding site for physostigmine is located within the alpha-subunit of the Torpedo nAChR and is distinct from the acetylcholine binding site. Our data suggest a second pathway of nAChR channel activation that may function physiologically as an allosteric control of receptor activity.

Amino Acid Sequence↗
PubMed1993

A novel agonist binding site on nicotinic acetylcholine receptors.

This report provides evidence that physostigmine (Phy) and benzoquinonium (BZQ) are able to activate nicotinic acetylcholine receptors (nAChRs) through binding site(s) distinct from those of the natural transmitter, ACh. Such findings are in agreement with a second pathway of activation of nAChRs. Receptor activation may be modulated through the novel site, and, consequently, physiological processes involving nicotinic synapses could be controlled. Using patch clamp techniques, single channel currents activated by ACh and anatoxin were recorded from frog interosseal muscle fibers under cell-attached condition and outside-out patches excised from cultured rat hippocampal neurons. Whole cell nicotinic currents were also studied in the cultured neurons. In most of the neurons, nicotinic responses were blocked by the nicotinic antagonists methyllycaconitine (MLA) and alpha-bungarotoxin (alpha-BGT). Evaluation of the effects of Phy and BZQ on the muscle and on the alpha-BGT- and MLA-sensitive neuronal nAChRs demonstrated that both compounds were open channel blockers at these receptors. Furthermore, at low micromolar concentrations, Phy and BZQ activated the nAChRs of all preparations tested, such an effect being unexpectedly resistant to alpha-BGT or MLA. Thus, the nAChRs could be activated via two distinct binding sites: one for ACh and the other for Phy and BZQ. These findings and previous biochemical results led us to suggest that a putative endogenous ligand could bind to the new site and thereby regulate the activation of nAChRs in nicotinic synapses.

Animals↗
PubMed1993

Cloning of several genes coding for retinoic acid nuclear receptors in the mouse embryonal carcinoma cell line PCC7-MZ1.

Mouse embryonal carcinoma cell line PCC7-Mz1 can be induced by retinoic acid (RA) to differentiate into several well defined phenotypes of neuroectodermal origin (Lang, E. et al. (1989) J. Cell. Biol. 109, 2481-2493). Several subclones of the cell line (clonal variants) differ from each other in their developmental potential. To test whether these differences in cellular fate are due to somatic mutations in specific genes of these cells, we have cloned full length cDNAs coding for the alpha 1 and beta 2 isoforms, and partial length cDNAs coding for the alpha 2, beta 1 and beta 3 isoforms of the retinoic acid nuclear receptor (RAR). The cloned cDNAs did not differ in sequence from those of normal mouse cells. Using as probe the beta 2-RAR promoter region from mouse liver, we also checked for restriction fragment length polymorphism in the promoter regions of RA-inducible and RA-resistant cell variants. No alterations in this region of RAR genes was found in the clonal variants tested. The different patterns of derivatives produced by the variants upon exposure to RA therefore cannot be caused by somatic mutations in RAR genes of the tumor cell lines.

Amino Acid Sequence↗
PubMed1993

Identification and functional characterization of a new agonist site on nicotinic acetylcholine receptors of cultured hippocampal neurons.

Electrophysiological and biochemical techniques were used to demonstrate that alpha-bungarotoxin-, methyllycaconitine-sensitive neuronal nicotinic acetylcholine receptors (nAChRs) can be activated via a novel agonist site(s). The residue proposed to be essential to this site is the amino acid Lys-125 of the receptor alpha subunit. In outside-out patches excised from cultured hippocampal neurons, physostigmine (PHY) and 1-methyl-PHY activated single channels whose main conductances were 46 and 23 pS. This action was insensitive to DL-2-amino-5-phosphonovaleric acid, atropine, tetrodotoxin and competitive nicotinic antagonists, but blocked by benzoquinonium or FK1, a nAChR-specific antibody raised against rat muscle nAChR alpha subunits that binds to the novel site. Indirect immunofluorescence staining demonstrated that FK1 binds to the hippocampal neurons, as would be expected based on the high degree of homology among nAChR alpha subunits from diverse sources in the region surrounding Lys-125. PHY prevented the binding of FK1, thus supporting that FK1 is a specific probe for the PHY site. High-affinity sites (KD approximately 35 nM) for 1-methyl-PHY were identified in hippocampal neurons. Similar to PHY, benzoquinonium (0.1-10 microM) and galanthamine (1-10 microM) activated nicotinic single channels. The agonists benzoquinonium and PHY were also open-channel blockers at the neuronal nAChRs, whereas galanthamine was predominantly a desensitizing agent. In mouse fibroblasts transfected with cDNAs of alpha 4 and beta 2 neuronal nAChR subunits, PHY also activated single channels that were blocked by FK1. In these cells, dihydro-beta-erythroidine blocked single channels activated by (+)-anatoxin-a and did not affect those opened by PHY. Thus, the present results suggest that the novel agonist site located on the receptor alpha subunit is a common feature of neuronal nAChRs.

Amino Acid Sequence↗
PubMed1992

Expression of retinoic acid nuclear receptors in the mouse embryonal carcinoma cell line PCC7-Mz1.

Mouse embryonal carcinoma cell line PCC7-Mz1 can serve as a model of mammalian neural development [1989, J. Cell. Biol. 109, 2481-2493]. Upon exposure to all-trans retinoic acid (RA), Mz1 cells differentiate into a stable pattern of neurons, astroglia and fibroblasts whereas variants of the parental cell line either are restricted in their patterns of derivatives or do not respond at all to RA. Using gene probes specific for the alpha 1, alpha 2 and beta 2 isoforms of the retinoic acid nuclear receptor, we have studied by Northern blot analysis the expression of these transcription factors in uninduced and induced cells of clone Mz1 and in variants with different developmental potential. alpha 1-RAR is expressed constitutively in all variants independent of whether RA is present or not. Soon after addition of 10(-7) M RA, alpha 2-RAR is induced in RA-responsive cells reaching within a few hours a plateau level that remains unchanged throughout the developmental process. In contrast, the beta 2 isoform is expressed only transiently after RA-induction despite the continuous presence of RA. Other RAR isoforms are expressed only in trace amounts.

Animals↗
PubMed1992

Ligand binding to the membrane-bound acetylcholine receptor from Torpedo marmorata: a complete mathematical analysis.

We have studied by means of equilibrium binding and kinetic experiments the interaction of the membrane-bound nicotinic acetylcholine receptor (nACHR) from Torpedo marmorata with [3H]acetylcholine and the fluorescent agonist NBD-5-acylcholine. In agreement with previous studies by others, we observed the preexistence, in the absence of ligand, of an equilibrium between two states of the nAChR, one with high affinity and the other with low affinity for agonist. As additional requirements for a minimal reaction scheme, we recognized (i) the existence of two ligand-binding sites, each of which may exist in two conformational states when occupied, and (ii) ligand-induced transitions between these conformations. Employing a special form of the allosteric model which considers these requirements, we then developed a suitable algorithm in order to simultaneously fit the whole set of equilibrium binding and kinetic data obtained for the two ligands. In this way we determined for a minimal model of the mechanism of action of the nAChR the complete set of rate constants and KD values involved. With these values available, we were able to simulate the rise and fall in the concentrations of individual receptor-ligand complexes and conformations occurring in the course of excitatory events at the electrocyte synapse. The membrane environment of the nAChR plays a decisive role with respect to the rates of conformational change of the nAChR occurring in the course of ligand interaction. Thus, artificial changes in membrane structure and composition can speed up by several orders of magnitude the rate of conformational change ("desensitization"). A proper structure of the surrounding membrane hence is a prerequisite for the physiological function of the membrane-embedded nAChR.

Acetylcholine↗
PubMed1992

Synthesis and characterization of biotinylated and photoactivatable neuroleptics. Novel bifunctional probes for dopamine receptors.

We have synthesized and characterized a series of novel derivatives of established antagonists of the neurotransmitter dopamine, i.e. butyrophenones, hexahydrocarbolines and phenothiazines. All derivatives were biotinylated, some of them carried an additional (photoactivatable) azido group. In the case of butyrophenones, the structural modifications were introduced at the aliphatic keto group and/or the heterocyclic ring system, both modifications resulting in significant decreases in binding affinity to dopamine D2 and dopamine D1 receptor subtypes. Biotinylation of hexahydrocarbolines significantly increased their binding affinity to D1 receptors, with the affinity for D2 receptors increasing only slightly, or remaining approximately the same, as compared to the parent compound. As a consequence, the derivatized hexahydrocarbolines behaved as nonselective antagonists of dopamine. Biotinylation of phenothiazines increased their binding affinity to both main subtypes of dopamine receptors by at least one order of magnitude, resulting in binding affinities in the nM range. These derivatives bound to both D1 and D2 receptor subtypes. In three of the biotinylated derivatives the photoactivatable azido group was introduced. These compounds bound to synaptosomal membranes from bovine caudate nuclei with similar affinity and subtype specificity as the biotinylated derivatives, and photoaffinity labelling was shown to proceed under mild conditions and selectively. These novel bifunctional ligands may become useful tools in the purification and characterization of dopamine receptors including their visualization and localization in the central nervous system and in tissue culture.

Affinity Labels↗
PubMed1992

Nicotinic cholinoceptors in the rat pineal gland as analyzed by western blot, light- and electron microscopy.

The monoclonal antibody WF6, raised against purified Torpedo nicotinic acetylcholine receptor (nAChR) was used to study the distribution of cholinoceptors in the rat pineal gland by means of Western blot analysis, light- and electron microscopy. The immunoblot analysis using homogenized pineal gland revealed a labeled protein band of apparent molecular weight 40 kDa which was identified as alpha-subunit of a nAChR. In the light microscope, approximately one-fourth of the pinealocytes exhibited cytoplasmic immunoreactivity (IR) of varying density. In the electron microscope, IR was seen as patchy staining of cell membranes of pinealocyte somata and processes. Presynaptic IR material was not found. Distribution and intensity of the observed IR was not significantly different in pineal sections from ganglionectomized rats, nor were any alterations found that would relate to the animals' sex or to the time of killing (day vs night). Our results provide further evidence for the existence of cholinergic receptors in the mammalian pineal. They may be important for the understanding of the gland's regulation.

Animals↗
PubMed1992

Species- and subtype-specific recognition by antibody WF6 of a sequence segment forming an alpha-bungarotoxin binding site on the nicotinic acetylcholine receptor alpha subunit.

The monoclonal antibody WF6 competes with acetylcholine and alpha-bungarotoxin (alpha-BGT) for binding to the Torpedo nicotinic acetylcholine receptor (nAChR) alpha 1 subunit. Using synthetic peptides corresponding to the complete Torpedo nAChR alpha 1 subunit, we previously mapped a continuous epitope recognized by WF6, and the prototope for alpha-BGT, to the sequence segment alpha 1(181-200). Single amino acid substitution analogs have been used as an initial approach to determine the critical amino acids for WF6 and alpha-BGT binding. In the present study, we continue our analysis of the structural features of the WF6 epitope by comparing its cross-reactivity with synthetic peptides corresponding to the alpha 1 subunits from the muscle nAChRs of different species, the rat brain alpha 2, alpha 3, alpha 4 and alpha 5 nAChR subtypes, and the chick brain alpha-BGT binding protein subunits, alpha BGTBP alpha 1 and alpha BGTBP alpha 2. Our results indicate that WF6 is able to cross-react with the muscle alpha 1 subunits of different species by virtue of conservation of several critical amino acid residues between positions 190-198 of the alpha 1 subunit. These studies further define the essential structural features of the sequence segment alpha 1(181-200) required to form the epitope for WF6.

Amino Acid Sequence↗
PubMed1991

The refined crystal structure of alpha-cobratoxin from Naja naja siamensis at 2.4-A resolution.

The crystal structure of the "long" alpha-neurotoxin alpha-cobratoxin was refined to an R-factor of 19.5% using 3271 x-ray data to 2.4-A resolution. The polypeptide chain forms three loops, I, II, III, knotted together by four disulfide bridges, with the most prominent, loop II, containing another disulfide close to its lower tip. Loop I is stabilized by one beta-turn and two beta-sheet hydrogen bonds; loop II by eight beta-sheet hydrogen bonds, with the tip folded into two distorted right-handed helical turns stabilized by two alpha-helical and two beta-turn hydrogen bonds; and loop III by hydrophobic interactions and one beta-turn. Loop II and one strand of loop III form an antiparallel triple-pleated beta-sheet, and tight anchoring of the Asn63 side chain fixes the tail segment. In the crystal lattice, the alpha-cobratoxin molecules dimerize by beta-sheet formation between strands 53 and 57 of symmetry-related molecules. Because such interactions are found also in a cardiotoxin and alpha-bungarotoxin, this could be of importance for interaction with acetylcholine receptor.

Amino Acid Sequence↗
PubMed1991

A second pathway of activation of the Torpedo acetylcholine receptor channel.

We have studied the interaction of the reversible acetylcholine esterase inhibitor (-)physostigmine (D-eserine) with the nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata electric tissue by means of ligand-induced ion flux into nAChR-rich membrane vesicles and of equilibrium binding. We find that (-) physostigmine induces cation flux (and also binds to the receptor) even in the presence of saturating concentrations of antagonists of acetylcholine, such as D-tubocurarine, alpha-bungarotoxin or antibody WF6. The direct action on the acetylcholine receptor is not affected by removal of the methylcarbamate function from the drug and thus is not due to carbamylation of the receptor. Antibodies FK1 and benzoquinonium antagonize channel activation (and binding) of eserine, suggesting that the eserine binding site(s) is separate from, but adjacent to, the acetylcholine binding site at the receptor. In addition to the channel activating site(s) with an affinity of binding in the 50 microM range, there exists a further class of low-affinity (Kd approximately mM) sites from which eserine acts as a direct blocker of the acetylcholine-activated channel. Our results suggest the existence of a second pathway of activation of the nAChR channel.

Animals↗
PubMed1991

Alpha-bungarotoxin and the competing antibody WF6 interact with different amino acids within the same cholinergic subsite.

In the nicotinic acetylcholine receptors (AChRs), the sequence segment surrounding two invariant vicinal cysteinyl residues at positions 192 and 193 of the alpha subunit contains important structural component(s) of the binding site for acetylcholine and high molecular weight cholinergic antagonists, like snake alpha-neurotoxins. At least a second sequence region contributes to the formation of the cholinergic site. Studying the binding of alpha-bungarotoxin and three different monoclonal antibodies, able to compete with alpha-neurotoxins and cholinergic ligands, to a panel of synthetic peptides as representative structural elements of the AChR from Torpedo, we recently identified the sequence segments alpha 181-200 and alpha 55-74 as contributing to form the cholinergic site (Conti-Tronconi et al., 1990). As a first attempt to elucidate the structural requirements for ligand binding to the subsite formed by the sequence alpha 181-200, we have now studied the binding of alpha-bungarotoxin and of antibody WF6 to the synthetic peptide alpha 181-200, and to a panel of peptide analogues differing from the parental sequence alpha 181-200 by substitution of a single amino acid residue. CD spectral analysis of the synthetic peptide analogues indicated that they all have comparable structures in solution, and they can therefore be used to analyze the influence of single amino acid residues on ligand binding. Distinct clusters of amino acid residues, discontinuously positioned along the sequence 181-200, seem to serve as attachment points for the two ligands studied, and the residues necessary for binding of alpha-bungarotoxin are different from those crucial for binding of antibody WF6. In particular, residues at positions 188-190 (VYY) and 192-194 (CCP) were necessary for binding of alpha-bungarotoxin, while residues W187, T191, and Y198 and the three residues at positions 193-195 (CPD) were necessary for binding of WF6. Comparison of the CD spectra of the toxin/peptide complexes, and those obtained for the same peptides and alpha-bungarotoxin in solution, indicates that structural changes of the ligand(s) occur upon binding, with a net increase of the beta-structure component. The cholinergic binding site is therefore a complex surface area, formed by discontinuous clusters of amino acid residues from different sequence regions. Such complex structural arrangement is similar to the "discontinuous epitopes" observed by X-ray diffraction studies of antibody/antigen complexes [reviewed in Davies et al. (1988)]. Within this relatively large structure, cholinergic ligands bind with multiple points of attachment, and ligand-specific patterns of the attachment points exist.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗