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Biomedical subjects

A Auerbach

Publications and source records attributed to A Auerbach.

At least 55 records · Page 3Linked to original sources
PubMed1995

Activation of recombinant mouse acetylcholine receptors by acetylcholine, carbamylcholine and tetramethylammonium.

1. The kinetic properties of cloned mouse embryonic nicotinic acetylcholine receptors (AChRs) expressed in HEK 293 cells or Xenopus oocytes were examined using high concentrations of acetylcholine (ACh), carbamylcholine (CCh), or tetramethylammonium (TMA). The rate constants of agonist binding and channel gating were estimated by fitting kinetic models to idealized open and closed intervals over a range of agonist concentrations. 2. Once doubly liganded, TMA-activated receptors open at approximately 3000 s-1. The equilibrium binding constants for TMA are 525 and 12,800 microM. Doubly liganded CCh-activated receptors open at approximately 11,500 s-1; the equilibrium binding constants for this agonist are 14 and 570 microM. If we assume that doubly liganded, ACh-activated receptors open at 60,000 s-1, then the equilibrium binding constants for ACh are 20 and > 650 microM, similar to those for CCh. For all three agonists the higher affinity site both binds and releases agonists more slowly than does the lower affinity site. 3. ACh and CCh bind to the two sites equally rapidly, at approximately 2 x 10(7) and 4 x 10(7) M-1 s-1 at the first and second binding sites, respectively. Compared with ACh, the TMA association rate is approximately 100 times slower at the first binding site, and approximately 30 times slower at the second binding site. These results indicate that at both binding sites the association rate of TMA is not limited by diffusional or steric factors. 4. All three agonists dissociate from the receptor binding sites at similar rates. The dissociation rate for all agonists was approximately 40 times slower at the first binding site than at the second. These results suggest that the interaction of the quarternary amine moiety with the receptor determines the rate of release of the agonist, and that the nature of this interaction is quite different at the two binding sites. 5. Although the channel opening rates for the three agonists varied approximately 20-fold, the channel closing rates were not strongly agonist dependent, and varied less than 3-fold. We speculate that the ester moiety of the agonist promotes both rapid binding and fast opening of the ligand receptors, and that interactions of the quarternary amine moiety of the agonist with the receptor determine the channel closing rate constant.

Acetylcholine↗
PubMed1995

Mutation of the acetylcholine receptor alpha subunit causes a slow-channel myasthenic syndrome by enhancing agonist binding affinity.

In five members of a family and another unrelated person affected by a slow-channel congenital myasthenic syndrome (SCCMS), molecular genetic analysis of acetylcholine receptor (AChR) subunit genes revealed a heterozygous G to A mutation at nucleotide 457 of the alpha subunit, converting codon 153 from glycine to serine (alpha G153S). Electrophysiologic analysis of SCCMS end plates revealed prolonged decay of miniature end plate currents and prolonged activation episodes of single AChR channels. Engineered mutant AChR expressed in HEK fibroblasts exhibited prolonged activation episodes strikingly similar to those observed at the SCCMS end plates. Single-channel kinetic analysis of engineered alpha G153S AChR revealed a markedly decreased rate of ACh dissociation, which causes the mutant AChR to open repeatedly during ACh occupancy. In addition, ACh binding measurements combined with the kinetic analysis indicated increased desensitization of the mutant AChR. Thus, ACh binding affinity can dictate the time course of the synaptic response, and alpha G153 contributes to the low binding affinity for ACh needed to speed the decay of the synaptic response.

Acetylcholine↗
PubMed1995

Activation kinetics of recombinant mouse nicotinic acetylcholine receptors: mutations of alpha-subunit tyrosine 190 affect both binding and gating.

Affinity labeling and mutagenesis studies have demonstrated that the conserved tyrosine Y190 of the acetylcholine receptor (AChR) alpha-subunit is a key determinant of the agonist binding site. Here we describe the binding and gating kinetics of embryonic mouse AChRs with mutations at Y190. In Y190F the dissociation constant for ACh binding to closed channels was reduced approximately 35-fold at the first binding site and only approximately 2-fold at the second site. At both binding sites the association and dissociation rate constants were decreased by the mutation. Compared with wildtype AChRs, doubly-liganded alpha Y190F receptors open 400 times more slowly but close only 2 times more rapidly. Considering the overall activation reaction (vacant-closed to fully occupied-open), there is an increase of approximately 6.4 kcal/mol caused by the Y-to-F mutation, of which at least 2.1 and 0.3 kcal/mol comes from altered agonist binding to the first and second binding sites, respectively. The closing rate constant of alpha Y190F receptors was the same with ACh, carbamoylcholine, or tetramethylammonium as the agonist. This rate constant was approximately 3 times faster in ACh-activated S, W, and T mutants. The equilibrium dissociation constant for channel block by ACh was approximately 2-fold lower in alpha Y190F receptors compared with in wildtype receptors, suggesting that there are changes in the pore region of the receptor as a consequence of the mutation. The activation reaction is discussed with regard to energy provided by agonist-receptor binding contacts, and by the intrinsic folding energy of the receptor.

Acetylcholine↗
PubMed1995

A large-scale gene-trap screen for insertional mutations in developmentally regulated genes in mice.

We have used a gene-trap vector and mouse embryonic stem (ES) cells to screen for insertional mutations in genes developmentally regulated at 8.5 days of embryogenesis (dpc). From 38,730 cell lines with vector insertions, 393 clonal integrations had disrupted active transcription units, as assayed by beta-galactosidase reporter gene expression. From these lines, 290 clones were recovered and injected into blastocysts to assay for reporter gene expression in 8.5-dpc chimeric mouse embryos. Of these, 279 clones provided a sufficient number of chimeric embryos for analysis. Thirty-six (13%) showed restricted patterns of reporter-gene expression, 88 (32%) showed widespread expression and 155 (55%) failed to show detectable levels of expression. Further analysis showed that approximately one-third of the clones that did not express detectable levels of the reporter gene at 8.5 dpc displayed reporter gene activity at 12.5 dpc. Thus, a large proportion of the genes that are expressed in ES cells are either temporally or spatially regulated during embryogenesis. These results indicate that gene-trap mutageneses in embryonic stem cells provide an effective approach for isolating mutations in a large number of developmentally regulated genes.

Animals↗
PubMed1995

Kinetic properties of NMDA receptors in embryonic Xenopus spinal neurons.

1. The kinetic properties of embryonic Xenopus spinal cord neuron N-methyl-D-aspartate (NMDA)-activated receptors (NMDAR) were examined at the single-channel level. These receptors have a main conductance state of 50 pS and make occasional sojourns to a subconductance level of approximately 40 pS. The open channel lifetime is 1.3 ms at -80 mV and in 1 mM Ca(2+)-, Mg(2+)-free solution. Extracellular Mg2+ blocks the channel at a rate of 2.9 x 10(8) M-1 s-1 and with a Kd of approximately 20 microM at -80 mV. In patches with only one channel active, the closed interval duration distribution requires at least four exponentials to be fit. The time constant of one closed interval component decreases with increasing NMDA concentration. Kinetic modeling indicates that these NMDA receptors open at a rate of 230 s-1 and close at a rate of 167 s-1, that in the absence of desensitization the maximum probability of being open is approximately 0.5, and that this probability is half-maximal at approximately 150 microM NMDA.

Animals↗
PubMed1994

Essential role of Mash-2 in extraembryonic development.

The outer layer of the blastocyst, or trophectoderm, is the first cell lineage to differentiate in the mouse embryo, but little is known about the genetic control of its development. Lineage-specific transcription factors may be important in lineage specification, and the product of the Mash-2 gene fulfils the criteria for such a factor. Mash-2 is a mammalian member of the achaete-scute family which encodes basic-helix-loop-helix transcription factors and is strongly expressed in the extraembryonic trophoblast lineage. Mash-2 transcripts are found in the female germ line and in the embryo throughout preimplantation development, but are highly expressed later only in the ectoplacental cone, the chorion and their derivatives in the placenta. Mash-2 transcripts are not found in primary and secondary giant cells, yolk sac or allantois at any post-implantation stage, and are present only transiently and at low levels in the embryo during gastrulation. To analyse the role of Mash-2 in development, we have used gene targeting to generate mice having no Mash-2 function. We report here that Mash-2-/- embryos die from placental failure at 10 days postcoitum. In mutant placentas, spongiotrophoblast cells and their precursors are absent and chorionic ectoderm is reduced. We have rescued this placental mutant phenotype by constructing chimaeras with tetraploid wild-type embryos which contribute almost exclusively to extraembryonic tissues. Mash-2-/- embryos developed normally and adult Mash-2-/- mice were viable, demonstrating that Mash-2 has no major role in the embryo itself. Mash-2 is the first transcription factor shown to play a critical part in the development of the mammalian trophoblast lineage.

Animals↗
PubMed1994

Dominant-negative and targeted null mutations in the endothelial receptor tyrosine kinase, tek, reveal a critical role in vasculogenesis of the embryo.

The receptor tyrosine kinases (RTKs) expressed on the surface of endothelial cells are likely to play key roles in initiating the program of endothelial cell growth during development and subsequent vascularization during wound healing and tumorigenesis. Expression of the Tek RTK during mouse development is restricted primarily to endothelial cells and their progenitors, the angioblasts, suggesting that Tek is a key participant in vasculogenesis. To investigate the role that Tek plays within the endothelial cell lineage, we have disrupted the Tek signaling pathway using two different genetic approaches. First, we constructed transgenic mice expressing a dominant-negative form of the Tek receptor. Second, we created a null allele of the tek gene by homologous recombination in embryonic stem (ES) cells. Transgenic mice expressing dominant-negative alleles of Tek or homozygous for a null allele of the tek locus both died in utero with similar defects in the integrity of their endothelium. By crossing transgenic mice that express the lacZ reporter gene under the transcriptional control of the endothelial cell-specific tek promoter, we found that the extraembryonic and embryonic vasculature was patterned correctly. However, homozygous tek embryos had approximately 30% and 75% fewer endothelial cells at day 8.5 and 9.0, respectively. Homozygous null embryos also displayed abnormalities in heart development, consistent with the conclusion that Tek is necessary for endocardial/myocardial interactions during development. On the basis of the analysis of mice carrying either dominant-negative or null mutations of the tek gene, these observations demonstrate that the Tek signaling pathway plays a critical role in the differentiation, proliferation, and survival of endothelial cells in the mouse embryo.

Animals↗
PubMed1993

Mammalian achaete-scute homolog 1 is required for the early development of olfactory and autonomic neurons.

The mouse Mash-1 gene, like its Drosophila homologs of the achaete-scute complex (AS-C), encodes a transcription factor expressed in neural precursors. We created a null allele of this gene by homologous recombination in embryonic stem cells. Mice homozygous for the mutation die at birth with apparent breathing and feeding defects. The brain and spinal cord of the mutants appear normal, but their olfactory epithelium and sympathetic, parasympathetic, and enteric ganglia are severely affected. In the olfactory epithelium, neuronal progenitors die at an early stage, whereas the nonneuronal supporting cells are present. In sympathetic ganglia, the mutation arrests the development of neuronal precursors, preventing the generation of sympathetic neurons, but does not affect glial precursor cells. These observations suggest that Mash-1, like its Drosophila homologs of the AS-C, controls a basic operation in development of neuronal progenitors in distinct neural lineages.

Adrenal Medulla↗
PubMed1993

The nature of cation-pi binding: interactions between tetramethylammonium ion and benzene in aqueous solution.

A combined quantum mechanical and molecular mechanical Monte Carlo simulation method was used to determine the free energy of binding between tetramethylammonium ion (TMA+) and benzene in water. The computed free energy as a function of distance (the potential of mean force) has two minima that represent contact and solvent-separated complexes. These species are separated by a broad barrier of about 3 kJ/mol. The results are in good accord with experimental data and suggest that TMA+ binds to benzene more favorably than to chloride ion, with an association constant of about 0.8 M-1.

Benzene↗