Biomedical subjects

R Schoepfer

Publications and source records attributed to R Schoepfer.

At least 19 recordsLinked to original sources

PubMed2006

Subunit dependencies of N-methyl-D-aspartate (NMDA) receptor-induced alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor internalization.

N-Methyl-D-aspartate (NMDA) receptor (NMDAR) activity regulates the net number of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPAR) at the cell surface by modulating the balance between AMPAR membrane insertion and endocytosis. In this study, we addressed the role of NMDAR subtypes and of NMDAR-mediated Ca2+ influx in the NMDAR-induced endocytosis of GluR2-containing AMPARs in primary murine hippocampal neurons. We found that NMDAR activation enhanced the endocytosis of AMPARs containing the GluR2 splice variants with short, but not long, cytoplasmic tails. NMDA-induced GluR2 endocytosis was completely inhibited by pharmacological block of NR2B-containing NMDARs. In turn, preferential block of NR2A-containing NMDARs did not affect NMDA-induced AMPAR endocytosis, indicating that AMPAR internalization is controlled by a restricted set of NMDARs. The NMDA-induced GluR2 internalization was also observed in the absence of extracellular Na+ ions, suggesting that membrane depolarization is not a prerequisite for this effect. Furthermore, the activation of Ca2+-impermeable NMDARs containing the mutant NR1(N598R) subunit failed to enhance AMPAR endocytosis, indicating a requirement of Ca2+ influx directly through the NMDAR channels. In summary, our findings suggest that the NMDAR-induced selective internalization of short C-terminal GluR2-containing AMPARs requires a Ca2+ signal that originates from NMDAR channels and is processed in an NMDAR subtype-restricted manner.

Animals↗

PubMed2005

Chemically induced long-term potentiation increases the number of perforated and complex postsynaptic densities but does not alter dendritic spine volume in CA1 of adult mouse hippocampal slices.

Examination of the morphological correlates of long-term potentiation (LTP) in the hippocampus requires the analysis of both the presynaptic and postsynaptic elements. However, ultrastructural measurements of synapses and dendritic spines following LTP induced via tetanic stimulation presents the difficulty that not all synapses examined are necessarily activated. To overcome this limitation, and to ensure that a very large proportion of the synapses and spines examined have been potentiated, we induced LTP in acute hippocampal slices of adult mice by addition of tetraethylammonium (TEA) to a modified CSF containing an elevated concentration of Ca(2+) and no Mg(+). Quantitative electron microscope morphometric analyses and three-dimensional (3-D) reconstructions of both dendritic spines and postsynaptic densities (PSDs) in CA1 stratum radiatum were made on serial ultrathin sections. One hour after chemical LTP induction the proportion of macular (unperforated) synapses decreased (50%) whilst the number of synapses with simple perforated and complex PSDs (nonmacular) increased significantly (17%), without significant changes in volume and surface area of the PSD. In addition, the surface area of mushroom spines increased significantly (13%) whilst there were no volume differences in either mushroom or thin spines, or in surface area of thin spines. CA1 stratum radiatum contained multiple-synapse en passant axons as well as multiple-synapse spines, which were unaffected by chemical LTP. Our results suggest that chemical LTP induces active dendritic spine remodelling and correlates with a change in the weight and strength of synaptic transmission as shown by the increase in the proportion of nonmacular synapses.

Animals↗

PubMed2005

Phosphorylation state of postsynaptic density proteins.

The postsynaptic density (PSD) is an electron-dense structure located at the synaptic contacts between neurons. Its considerable complexity includes cytoskeletal and scaffold proteins, receptors, ion channels and signaling molecules, in line with the role of PSDs in signal transduction and processing. The phosphorylation state of components of the PSD is central to synaptic transmission and is known to play a role in synaptic plasticity, learning and memory. The presence of a range of kinases and phosphatases in the PSD defines potential key players in this context. However, the substrates that these enzymes target have not been fully identified to date. We analyzed the protein composition of purified PSD samples from adult mouse brains by strong cation exchange chromatography fractionation of a tryptic digest followed by nano-reverse phase liquid chromatography coupled with electrospray ionization-quadrupole time of flight tandem mass spectrometry. This led to the identification of 244 proteins. To gain an insight into the phosphoproteome of the PSD we then purified phosphorylated tryptic peptides by immobilized metal ion affinity chromatography. This approach for the specific enrichment of phosphopeptides resulted in the identification of 42 phosphoproteins in the PSD preparation, 39 of which are known PSD components. Here we present a total of 83 in vivo phosphorylation sites.

Animals↗

PubMed2001

Deletion of the alpha-synuclein locus in a subpopulation of C57BL/6J inbred mice.

BACKGROUND: The presynaptic protein alpha-synuclein is involved in a range of neurodegenerative diseases. Here we analyze potential compensatory mechanisms in alpha-synuclein null mutant mice. Furthermore, the findings reveal problems that may be associated with inbred mouse strains. RESULTS: Expression profiling by cDNA array technology in a transgenic mouse model revealed striking differences only in the expression level of alpha-synuclein. This was caused by a chromosomal deletion of the alpha-synuclein locus in the C57BL/6J inbred strain used for backcrossing. However, the deletion is only present in a subpopulation of C57BL/6J mice, namely animals from Harlan. No other genes are known to be affected by the deletion, which is estimated to be smaller than 2 cM. We propose to name this strain C57BL/6S. C57BL/6S animals appear phenotypically normal. They show no upregulation of beta-synuclein or gamma-synuclein, excluding a compensatory mechanism. Also, the expression of synphilin-1 was unaffected. CONCLUSIONS: The C57BL/6S strain should help in the understanding of the physiological function of alpha-synuclein and its involvement in synucleinopathies. Also, the findings exemplify unexpected complications that may arise during the study of transgenic models or inbred strains, in particular when combined with genome wide screening techniques.

Animals↗

PubMed2000

Single-channel analysis of an NMDA receptor possessing a mutation in the region of the glutamate binding site.

Recombinant NR1a/NR2A(T671A) N-methyl-D-aspartate (NMDA) receptor-channels, which carry a point mutation in the putative glutamate binding site that reduces glutamate potency by around 1000-fold, have been expressed in Xenopus laevis oocytes and their single-channel properties examined using patch-clamp recording techniques. Shut time distributions of channel activity were fitted with a mixture of five exponential components. The first three components in each distribution were considered to occur within a channel activation as they exhibited little or no dependence on agonist concentration. Bursts of single-channel openings were defined by a critical gap length with a mean of 5.65 +/- 0.37 ms. Shut intervals with a duration longer than this value were considered to occur between separate bursts of channel openings. Distributions of the lengths of bursts of openings were fitted with a mixture of four exponential components. The longest two components carried the majority of the charge transfer in the channel recordings and had means of 7.71 +/- 1.1 and 37.7 +/- 4.3 ms. The overall probability of a channel being open during a burst was high (mean 0.92 +/- 0.01). Brief concentration jumps (1 ms) of 10 mM glutamate were applied to outside-out patches so that a comparison between the macroscopic current relaxation and steady-state single-channel activity evoked by glutamate could be made. The decay of such macroscopic currents was fitted with a single exponential component with a mean of 32.0 +/- 3.53 ms. The good agreement between macroscopic current decay following brief agonist exposure and the value for the slowest component of the burst length distribution suggests that the bursts of openings that we identified in steady-state recordings represent individual activations of recombinant NR1a/NR2A(T671A) NMDA receptor-channels. A new way of displaying geometric distributions is suggested, and the utility of a modified definition of the 'probability of being open within a burst' is discussed. The single-channel data that we present in this paper support further the idea that the point mutation T671A in the NR2A NMDA receptor subunit affects mainly the ability of glutamate to remain bound to these channels.

Algorithms↗

PubMed2000

Molecular cloning and characterization of a novel ATP P2X receptor subtype from embryonic chick skeletal muscle.

We have cloned a new P2X ligand-gated ion channel receptor from embryonic chick skeletal muscle, which is tentatively named as chick P2X(8) (cP2X(8)) receptor. The cloned cDNA encodes a protein with 402 amino acids. Electrophysiological study of the recombinant cP2X(8) receptor expressed in Xenopus oocytes showed that 10 microm ATP induced a fast inward current followed by rapid and long lasting desensitization in medium containing 1.8 mm Ca(2+). In medium with 0. 3 mm Ca(2+) ATP induced a bi-phasic response as follows: a slower inward current succeeded the initial fast one. 2-Methylthio-ATP, alpha,beta-methylene-ATP, and adenosine 5'-O-(thio)triphosphate were potent agonists, whereas ADP was a very weak agonist. ATP-induced currents were blocked by 100 microm suramin and pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid. Northern blot analysis and reverse transcription-polymerase chain reaction showed that cP2X(8) RNA transcripts were mainly expressed in skeletal muscle, brain, and heart of Day 10 chick embryos. A moderate level of expression was also detected in gizzard and retina. Whole mount in situ hybridization showed that cP2X(8) RNA transcripts were expressed mainly in neurotube, notochord, and stomach in Day 3 embryos. In Day 4 and Day 6 embryos, the cP2X(8) RNA transcripts were highly expressed in the myotome and premuscle mass. The physiological role of this receptor in the establishment of the skeletal muscle innervation will be studied.

Amino Acid Sequence↗

PubMed1998

Properties of human glycine receptors containing the hyperekplexia mutation alpha1(K276E), expressed in Xenopus oocytes.

1. Inherited defects in human glycine receptors give rise to hyperekplexia (startle disease). We expressed human glycine receptors in Xenopus oocytes, in order to examine the pharmacological and single-channel properties of receptors that contain a mutation, alpha1(K276E), associated with an atypical form of hyperekplexia. 2. Equilibrium concentration-response curves showed that recombinant human alpha1(K276E)beta receptors had a 29-fold lower glycine sensitivity than wild-type alpha1beta receptors, and a greatly reduced Hill coefficient. The maximum response to glycine also appeared much reduced, whereas the equilibrium constant for the glycine receptor antagonist strychnine was unchanged. 3. Both wild-type and mutant channels opened to multiple conductance levels with similar main conductance levels (33 pS) and weighted mean conductances (41.5 versus 49.8 pS, respectively). 4. Channel openings were shorter for the alpha1(K276E)beta mutant than for the wild-type alpha1beta, with mean overall apparent open times of 0.82 and 6.85 ms, respectively. 5. The main effect of the alpha1(K276E) mutation is to impair the opening of the channel rather than the binding of glycine. This is shown by the results of fitting glycine dose-response curves with particular postulated mechanisms, the shorter open times of mutant channels, the properties of single-channel bursts, and the lack of an effect of the mutation on the strychnine-binding site.

Animals↗

PubMed1998

Identification of amino acid residues of the NR2A subunit that control glutamate potency in recombinant NR1/NR2A NMDA receptors.

The NMDA type of ligand-gated glutamate receptor requires the presence of both glutamate and glycine for gating. These receptors are hetero-oligomers of NR1 and NR2 subunits. Previously it was thought that the binding sites for glycine and glutamate were formed by residues on the NR1 subunit. Indeed, it has been shown that the effects of glycine are controlled by residues on the NR1 subunit, and a "Venus flytrap" model for the glycine binding site has been suggested by analogy with bacterial periplasmic amino acid binding proteins. By analysis of 10 mutant NMDA receptors, we now show that residues on the NR2A subunit control glutamate potency in recombinant NR1/NR2A receptors, without affecting glycine potency. Furthermore, we provide evidence that, at least for some mutated residues, the reduced potency of glutamate cannot be explained by alteration of gating but has to be caused primarily by impairing the binding of the agonist to the resting state of the receptor. One NR2A mutant, NR2A(T671A), had an EC50 for glutamate 1000-fold greater than wild type and a 255-fold reduced affinity for APV, yet it had single-channel openings very similar to those of wild type. Therefore we propose that the glutamate binding site is located on NR2 subunits and (taking our data together with previous work) is not on the NR1 subunit. Our data further imply that each NMDA receptor subunit possesses a binding site for an agonist (glutamate or glycine).

Amino Acid Sequence↗

PubMed1997

Recombinant nicotinic receptors, expressed in Xenopus oocytes, do not resemble native rat sympathetic ganglion receptors in single-channel behaviour.

1. In order to establish the subunit composition of neuronal nicotinic receptors in rat superior cervical ganglia (SCG), their single-channel properties were compared with those of recombinant receptors expressed in Xenopus oocytes, using outside-out excised patch recording. 2. The mean main conductance of SCG channels from adult and 1-day-old rats was 34.8 and 36.6 pS, respectively. Less frequent openings to lower conductances occurred both as isolated bursts and as events connected to the main level by direct transitions. There was considerable interpatch variability in the values of the lower conductances. 3. Nicotinic receptors from oocytes expressing alpha3beta4 and alpha4beta4 subunits had chord conductances lower than that of SCG neurones (22 pS for alpha3beta4 and 29 pS for alpha4beta4). 4. Prolonged recording from both native and recombinant channels was precluded by 'run-down', i.e. channel activity could be elicited for only a few minutes after excision. Nevertheless, SCG channel openings were clearly seen to occur as short bursts (slowest component, 38 ms), whereas recombinant channels opened in very prolonged bursts of activity, the major component being the slowest (480 ms). 5. Addition of the alpha5 subunit to the alpha3beta4 pair produced channels with a higher conductance than those observed after injection of the pair alone (24.9 vs. 22 pS), suggesting incorporation of alpha5 into the channel. Addition of the beta2 subunit did not change alpha3beta4 single-channel properties. In one out of fourteen alpha3alpha5beta4 patches, both ganglion-like, high conductance, short burst openings and recombinant-type, low conductance, slow burst openings were observed. 6. Channels produced by expression in Xenopus oocytes of neuronal nicotinic subunits present in rat SCG as a rule differ from native ganglion receptors in single-channel conductance and gross kinetics. While it is possible that an essential nicotinic subunit remains to be cloned, it is perhaps more likely that oocytes either cannot assemble neuronal nicotinic subunits efficiently into channels with the correct composition and stoichiometry, or that they produce post-translational channel modifications which differ from those of mammalian neurones.

Acetylcholine↗

PubMed1997

Regional, developmental and interspecies expression of the four NMDAR2 subunits, examined using monoclonal antibodies.

Mouse monoclonal antibodies were raised against bacterially expressed protein sequences of the NR2A, NR2B, NR2C and NR2D subunits of the rat NMDA receptor. From immunoblots of rat brain proteins, the apparent molecular weights of these subunits were 165, 170, 135 and 145 kDa, respectively. Proteins of similar masses were observed on immunoblots of specifically transfected HEK293 cells. Deglycosylation with endoglycosidase F reduced the mass of each endogenous NR2 subunit by approximately 10 kDa. In distribution studies, NR2A-immunoreactive protein (IRP) was located throughout the adult rat brain, NR2B-IRP was primarily in the forebrain, NR2C-IRP was predominantly in the cerebellum and NR2D-IRP was mainly found in the thalamus, midbrain and brainstem. Whereas NR2A- and NR2C-IRPs increased during rat brain post-natal development, NR2B- and NR2D-IRPs were abundant at birth and declined with age, especially in cerebellum. NR2-IRPs of mouse, rabbit, frog and human brain were of sizes similar to those of the corresponding rat subunits and were similarly distributed. In summary, NR2 subunits are large glycoproteins whose specific expression profiles in the brain are developmentally and regionally regulated and which are similarly expressed in a variety of species.

Aging↗

PubMed1996

Single-channel currents from recombinant NMDA NR1a/NR2D receptors expressed in Xenopus oocytes.

We have investigated the single-channel and whole-cell behaviour of recombinant N-methyl-D-aspartate (NMDA) receptors formed from NR1a and NR2D receptor subunits expressed in Xenopus oocytes. The EC50 for apparent steady-state activation of NR1a/NR2D receptors by glutamate was 450 nM, while extracellular MG2+ produced a voltage-dependent block of glutamate responses with an IC50 of 440 microM at -70 mV. At negative holding potentials glutamate-activated NR1a/NR2D single-channel currents, in 0.85 mM external Ca2+, had slope conductances of 35 pS for the main level, and 17 pS for the sublevel; direct transitions occurred between these two conductance levels. On average 35 pS events had mean open times of 1.01 +/- 0.04 ms, whereas the mean open times of 17 pS events were consistently longer (1.28 +/- 0.06 ms). In 5 mM external Ca2+ the larger conductance level was reduced to 20 pS whereas in Ca(2+)-free solutions it was increased to 50 pS. The frequency of transitions between the main and subconductance levels showed temporal asymmetry: 35-17 pS transitions were more frequent (61%) than 17-35 pS transitions. This asymmetry was not affected by alterations in the external Ca2+ concentration (up to 5 mM). In conclusion, the NR1a/NR2D channel is, like NR1a/NR2C, a 'low conductance' NMDA channel, but it can be distinguished from NR1a/NR2C channels on the basis of transition asymmetry and differences in the open times of its main and sub-conductance levels.

Animals↗

PubMed1996

Multiple structural elements determine subunit specificity of Mg2+ block in NMDA receptor channels.

In NMDA receptor channels, subtype-specific differences of Mg2+ block are determined by the NR2 subunits. Channels assembled from the NR1-NR2A or NR1-NR2B subunits are blocked more strongly than channels formed by the NR1-NR2C or NR1-NR2D subunits, predominantly reflecting a difference in voltage dependence. A determinant of Mg2+ block common to the NR2 subunits is located in the M2 domain (N-site or Q/R/N-site). However, subunit-specific differences of block suggested that additional structural elements exist. Chimeric NR2 subunits were constructed by replacing segments of the least sensitive NR2C subunit with homologous segments of the most sensitive NR2B subunit. Mutant NR2 subunits were coexpressed with wild-type NR1 in Xenopus oocytes, and Mg2+ block was quantified. Replacement of the entire M1-M4 region resulted in a chimera with a sensitivity of Mg2+ block similar to that of the NR2B wild type. Replacing smaller segments or introducing point mutations did not generate channels with Mg2+ block characteristic of NR2B wild type. However, combining in a single chimera three small segments (M1, M2-M3 linker, M4), each independently mediating an increase in Mg2+ block, produced channels close to NR2B wild type. Thus, differences in Mg2+ block as controlled by the NR2 subunits cannot be explained by a single structural determinant in addition to the N-site. Moreover, three elements of the NR2 subunit are the major determinants of subtype-specific differences of Mg2+ block in heteromeric NMDA receptor channels.

Amino Acid Sequence↗

PubMed1996

Phosphorylation of the predicted major intracellular domains of the rat and chick neuronal nicotinic acetylcholine receptor alpha 7 subunit by cAMP-dependent protein kinase.

The predicted major intracellular domains of the chick and rat neuronal nicotinic acetylcholine receptor alpha 7 subunits were expressed in E. coli as glutathione-S-transferase fusion proteins. These proteins were then purified to near homogeneity by chromatography on immobilized glutathione. The intracellular domains of the alpha 7 subunit from both species were phosphorylated to high stoichiometry by cAMP-dependent protein kinase, but not by protein kinase C, cGMP-dependent protein kinase, or calcium/calmodulin-dependent protein kinase. Phosphorylation occurred on serine residues only within an identical single tryptic peptide for both proteins. This conserved phosphorylation site was identified as Ser 342 utilizing site-directed mutagenesis. These results demonstrate that the intracellular domain of the alpha 7 subunit is a substrate of PKA, and suggest a role for protein phosphorylation in mediating cellular regulation upon neuronal AChRs containing this subunit.

Amino Acid Sequence↗

PubMed1995

Determination of NMDA NR1 subunit copy number in recombinant NMDA receptors.

Co-expression of wild-type and mutated NMDA NR1 (N598R) subunits in Xenopus oocytes has been used to determine the stoichiometry of the NMDA receptor-channel. When expressed together, wild-type NR2A and mutant NR1 (N598R) subunits produced channels with a main conductance of 2.6 pS and a sublevel of 1.2 pS. These conductances were clearly different from those obtained from wild-type NR1 and wild-type NR2A channels which gave characteristic 50 pS events with a 40 pS sublevel. When wild-type and mutant NR1 subunits were co-expressed together with NR2A subunits a different channel type with a main conductance of 15.2 pS and a sublevel of 11.4 pS was obtained, as well as the 'all wild-type' and 'all mutant' channels described above. These results indicate that there are likely to be two copies of the NR1 subunit in each NMDA receptor complex.

Animals↗

PubMed1995

A P2X purinoceptor cDNA conferring a novel pharmacological profile.

We have cloned P2X4, a member of the P2-purinoceptor family, which has a new pharmacological profile. Rat P2X4 is distantly related to P2X1, P2X2 and P2X3 and is expressed in brain, spinal cord, lung, thymus, bladder, adrenal, testis and vas deferens. This ligand gated ion channel is activated by ATP and analogs with the potency order of ATP > ATP gamma S > 2-methylthio ATP > ADP approximately alpha beta-methylene ATP. However, none of the currently used P2X purinoceptor antagonists suramin, reactive blue 2 and PPADS blocked ATP evoked currents; in contrast their application resulted in potentiation of the agonist response. Due to lack of any known antagonist for P2X4 it is unlikely that native P2X4 has previously been recognized as a P2X purinoceptor.

Adenosine Triphosphate↗

PubMed1994

A molecular determinant for submillisecond desensitization in glutamate receptors.

The decay of excitatory postsynaptic currents in central neurons mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) receptors is likely to be shaped either by receptor desensitization or by offset after removal of glutamate from the synaptic cleft. Native AMPA receptors show desensitization time constants of 1 to about 10 milliseconds, but the underlying molecular determinants of these large differences are unknown. Cloned AMPA receptors carrying the "flop" splice variants of glutamate receptor subtype C (GluR-C) and GluR-D are shown to have desensitization time constants of around 1 millisecond, whereas those with the "flip" variants are about four times slower. Cerebellar granule cells switch their expression of GluR-D splice variants from mostly flip forms in early stages to predominantly flop forms in the adult rat brain. These findings suggest that rapid desensitization of AMPA receptors can be regulated by the expression and alternative splicing of GluR-D gene transcripts.

Alternative Splicing↗

PubMed1994

Molecular biology of glutamate receptors.

The ligand-gated receptors for L-glutamate play a central role in acute neuronal degeneration. Recently cDNAs have been isolated for subunits of several glutamate receptor subtypes. By sequence homology all these subunits clearly belong to one large gene family. Several subfamilies exist and match roughly previously pharmacologically and electrophysiologically defined subtypes of glutamate receptors. Currently four genes (GluR A, B, C and D) are known that code for the AMPA subtypes of glutamate receptors. Recombinant expression of wild type and mutated sequences identified a critical residue in the putative TM2 channel-lining segment that controls Ca2+ ion permeability. The arginine (R) found in GluR B subunits at that position renders AMPA channels impermeable for Ca2+ ions, whereas glutamine (Q) containing GluR A, C and D subunits give rise to Ca2+ permeable channels. RNA editing converts the genomically encoded glutamine codon into the arginine codon found in GluR B cDNAs for the Q/R site. NMDA subtypes of glutamate receptors are formed after coexpression of the NR1 cDNA with a cDNA of the NR2 family. Depending on the member of the NR2 family used, NMDA receptors with different kinetical and pharmacological properties are generated. Common to all channels of these NMDA receptors is a high permeability for Ca2+ ions and a voltage dependent block by Mg2+ ions. All currently known NMDA receptor subunits have an asparagine at the Q/R homologous position. We found that this residue is critical for Mg2+ block and Ca2+ permeability of NMDA receptor channels.

Amino Acid Sequence↗

PubMed1993

Ethanol inhibits glutamate-induced currents in heteromeric NMDA receptor subtypes.

Maximal L-glutamate/glycine-evoked currents were inhibited by ethanol in Xenopus laevis oocytes expressing recombinant heteromeric NMDA receptors consisting of NR1-NR2A, NR1-NR2B, and NR1-NR2C subunit combinations. Concentration-dependent inhibition was observed at ethanol concentrations of > or = 50 mM both in Ca(2+)-containing and Ca(2+)-deficient, Ba(2+)-containing Mg(2+)-free media. The NR1-NR2C channels were slightly less sensitive to ethanol inhibition than the other heteromeric channels in Ca(2+)-deficient, Ba(2+)-containing medium. The inhibition was unaffected by the clamping-voltage and by a mutation [NR1-NR2A(N595Q)] that prevents the Mg(2+)-blockade of the channels, indicating that the mechanism of action of ethanol differs from that of Mg2+. The results are consistent with the hypothesis that the NMDA receptor subtypes can mediate many behavioural actions of ethanol.

Animals↗