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

BACKGROUND: The presynaptic protein α-synuclein is involved in a range of neurodegenerative diseases. Here we analyze potential compensatory mechanisms in α-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 α-synuclein. This was caused by a chromosomal deletion of the α-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 β-synuclein or γ-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 α-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

A P2X purinoceptor cDNA conferring a novel pharmacological profile

AbstractWe 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 > ATPγS > 2-methylthio ATP > ADP ≈ αβ-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

Absence of Whisker-Related Pattern Formation in Mice with NMDA Receptors Lacking Coincidence Detection Properties and Calcium Signaling

Precise refinement of synaptic connectivity is the result of activity-dependent mechanisms in which coincidence-dependent calcium signaling by NMDA receptors (NMDARs) under control of the voltage-dependent Mg2+ block might play a special role. In the developing rodent trigeminal system, the pattern of synaptic connections between whisker-specific inputs and their target cells in the brainstem is refined to form functionally and morphologically distinct units (barrelettes). To test the role of NMDA receptor signaling in this process, we introduced the N598R mutation into the native NR1 gene. This leads to the expression of functional NMDARs that are Mg2+ insensitive and Ca2+impermeable. Newborn mice expressing exclusively NR1 N598R-containing NMDARs do not show any whisker-related patterning in the brainstem, whereas the topographic projection of trigeminal afferents and gross brain morphology appear normal. Furthermore, the NR1 N598R mutation does not affect expression levels of NMDAR subunits and other important neurotransmitter receptors. Our results show that coincidence detection by, and/or Ca2+ permeability of, NMDARs is necessary for the development of somatotopic maps in the brainstem and suggest that highly specific signaling underlies synaptic refinement

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

The human medulloblastoma cell line TE671 expresses a muscle-like acetylcholine receptor Cloning of the α-subunit cDNA

AbstractNicotinic acetylcholine receptors (AChRs) from muscle bind α-bungarotoxin (αBgt) and are composed of four kinds of subunits, whereas AChRs from mammalian brains do not bind αBgt and are composed of two kinds of subunits. αBgt-binding proteins whose function is unknown are also found in brain. All these proteins belong to the same gene family. The human medulloblastoma cell line TE671 expresses a functional AChR which binds αBgt. Surprisingly, the AChR of this neuron-derived cell line has electrophysiological, immunological and biochemical properties different from neuronal AChRs and very similar to muscle AChRs. The TE671 AChR binds αBgt, but is different from αBgt-binding proteins in brain. Here we show that TE671 expresses the α-subunit mRNA coding for the muscle AChR, thereby proving that TE671 expresses a muscle-type AChR that is not expressed in adult brain. The isolated cDNA clones should prove useful for expression of large amounts of human muscle-type AChR α-subunit protein for studies of the autoimmune response to muscle AChRs in human myasthenia gravis

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 Ca2+-containing and Ca2+-deficient, Ba2+-containing Mg2+-free media. The NR1-NR2C channels were slightly less sensitive to ethanol inhibition than the other heteromeric channels in Ca(2+)-deficient, Ba2+-containing medium. The inhibition was unaffected by the clamping-voltage and by a mutation [NR1-NR2A(N595Q)] that prevents the Mg2+-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

The mechanism of magnesium block in NMDA receptors

The block of NMDA receptor channels by external magnesium (Mg2+) is believed to be of great physiological importance. The original model of a Mg2+ binding site deep inside the pore, near the cytoplasmic side of the channel has, however, become more and more questionable. In the experiments reported here, Mg2+ block was studied in inside-out patches and inside-out giant patches from Xenopus oocytes expressing recombinant NR1/NR2A receptors. The conductance of single NR1/NR2A receptor channels was rather weakly dependent on the concentration of permeant ions, and if only internal permeant ion concentration was changed the voltage for half-maximal block by Mg2+ shifted with the reversal potential. This suggests that NMDA receptor channels are strongly negatively charged. The charged spider venom Argiotoxin 636 (ATX) exhibited both external and internal block of NR1/NR2A receptors, with apparent electrical distances of almost 100% from both sides of the channel. This gives strong evidence against a considerable asymmetry of the channel. The electrical distance and voltage for half maximal block by Mg2+ was dependent on the monovalent ion species, especially on the cytoplasmic side of the membrane. This effect was independent of the reversal potential, suggesting a direct interaction between Mg2+ and other ions in the channel. A model is presented which explains the excessive voltage-dependence for block by Mg2+ and ATX by the interaction of monovalent ions with the blocking ion