Homology and analogy in transmembrane channel design: lessons from synaptic membrane proteins.
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Amino Acid Sequence↗
Biomedical subjects
C M Becker
Publications and source records attributed to C M Becker.
Functional chloride channels by mammalian cell expression of rat glycine receptor subunit.
Cultured human cells were transfected with cloned rat glycine receptor (GlyR) 48 kd subunit cDNA. In these cells glycine elicited large chloride currents (up to 1.5 nA), which were blocked by nanomolar concentrations of strychnine. However, no corresponding high-affinity binding of [3H]strychnine was detected in membrane preparations of the transfected cells. Analysis by monoclonal antibodies specific for the 48 kd subunit revealed high expression levels of this membrane protein. After solubilization, the 48 kd subunit behaved as a macromolecular complex when analyzed by sucrose density centrifugation. Approximately 50% of the solubilized complex bound specifically to a 2-aminostrychnine affinity column, indicating the existence of low-affinity antagonist binding sites on most of the expressed GlyR protein. Thus, the 48 kd strychnine binding subunit efficiently assembles into high molecular weight complexes, resembling the native spinal cord GlyR. However, formation of functional receptor channels of high affinity for strychnine occurs with low efficiency.
Amino Acid Sequence↗
Primary cultures of mouse spinal cord express the neonatal isoform of the inhibitory glycine receptor.
Expression of the inhibitory glycine receptor complex was investigated in primary cultures of fetal mouse spinal cord using sensitive immunomethods. In these cells, glycine receptor is predominantly of the neonatal isoform characterized by a low affinity for the antagonist strychnine. It contains a ligand binding subunit that differs from that of the adult receptor in antigenic epitopes and apparent molecular weight. Whereas in vivo the neonatal receptor isoform is completely replaced by the adult isoform within 3 weeks after birth, this exchange of subtypes is not seen in culture. The increased expression of the cytoplasmic glycine receptor-associated polypeptide of 93 kd occurring after birth is also seen under culture conditions. Purification of glycine receptor from cultures yielded polypeptides of 49 kd and 93 kd, suggesting that the membrane-spanning core of the neonatal receptor may be a homooligomer composed of 49 kd subunits. About half of the 49 kd subunit is cleaved by trypsinization of the cultures, indicating a predominant cell surface localization of the receptor. Pulse-labeling experiments revealed the 49 kd subunit to be a metabolically stable glycoprotein (half-life approximately 2 days). After its synthesis, a transition time of 30-45 min is required for acquisition of a strychnine binding conformation.
Animals↗
Sensitive immunoassay shows selective association of peripheral and integral membrane proteins of the inhibitory glycine receptor complex.
The inhibitory glycine receptor of mammalian spinal cord is a ligand-gated chloride channel that, on affinity purification, contains two subunits of 48-kilodalton (kD) and 58-kD molecular mass in addition to an associated 93-kD protein. Ligand-binding 48-kD subunit and 93-kD protein were quantified in the CNS of the adult rat using a newly developed dot receptor assay (detection limit less than or equal to 1 fmol/assay) which employs monoclonal antibodies specific for glycine receptor polypeptides. The 93-kD protein was found to codistribute at a fixed stoichiometry with the 48-kD subunit throughout the CNS of the rat. Moreover, the 93-kD protein cofractionated with the ligand-binding subunit on solubilization and affinity chromatography or immunoprecipitation. However, both proteins were separated on sucrose gradient centrifugation of detergent extracts of spinal cord membranes in accord with earlier observations on purified receptor. These data prove that the 93-kD polypeptide is selectively associated with the membrane core of the strychnine-sensitive glycine receptor. The regional distribution of glycine receptor polypeptides was also determined in the CNS of the spastic rat mutant. In contrast to hereditary spasticity in mouse and cattle, no reduction of glycine receptors was found in the spastic rat.
Animals↗
Glycine receptor heterogeneity in rat spinal cord during postnatal development.
Two different isoforms of the inhibitory glycine receptor were identified during postnatal development of rat spinal cord. A neonatal form characterized by low strychnine binding affinity, altered antigenicity, and a ligand binding subunit differing in mol. wt (49 kd) from that of the adult receptor (48 kd) predominates at birth (70% of the total receptor protein). Separation from the adult form could be achieved by either use of a selective antibody or glycine gradient elution of 2-aminostrychnine affinity columns. Both isoforms co-purify with the mol. wt 93 kd peripheral membrane protein of the postsynaptic glycine receptor complex.
Animals↗
Fractionation of synaptophysin-containing vesicles from rat brain and cultured PC12 pheochromocytoma cells.
Synaptophysin is a transmembrane glycoprotein of neuroendocrine vesicles. Its content and distribution in subcellular fractions from cultured PC12 cells, rat brain and bovine adrenal medulla were determined by a sensitive dot immunoassay. Synaptophysin-containing fractions appeared as monodispersed populations similar to synaptic vesicles in density and size distribution. Membranes from synaptic vesicles contained approximately 100-times more synaptophysin than chromaffin granules. In conclusion, synaptophysin is located almost exclusively in vesicles of brain and PC12 cells which are distinct from dense core granules.
Adrenal Medulla↗
It may be interpretation, not experiment that is wrong. Interpreting non-competitive inhibition.
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Binding, Competitive↗
Bovine fibrinogen aggregates: electron microscopic observations of quasi-globular structures.
Electron microscopic observations of bovine fibrinogen preparations were made following negative staining with uranyl acetate and phosphotungstic acid. Preparations were first incubated at pH 6.6, gamma/2 0.3 and 0 degrees C to develop fibrinogen aggregates at concentrations approaching equilibrium levels. The fraction of fibrinogen present as aggregates, designated the product mass fraction (Pmf), was determined by gel filtration on Bio-Gel A-1.5m. Electron micrographs of preparations with significant Pmf revealed partially closed quasi-globular structures of variable size. Fibrinogen aggregates could be dissociated by incubating preparations at 2 mg/ml, 37 degrees C for 18h, with a resulting Pmf less than 0.01. In these dissociated systems, monomers were observed with no apparent aggregate forms. At intervals during the approach to equilibrium, the size distributions of aggregates present were estimated by Bio-Gel A-15m filtration. A thermodynamic analysis incorporating these and previous data led to the following model specifications. Fibrinogen aggregates develop by monomer addition, followed by periodic reactions among the constituent members of an aggregate. These intra-aggregate reactions produce a non-linear structure, most likely begin after the association of four monomers, and on the average then follow the addition of every other monomer. In this manner, quasiglobular aggregates containing up to about 28 constituent monomers may develop as equilibrium is approached.
Animals↗
The chloride channel blocking agent, t-butyl bicyclophosphorothionate, binds to the gamma-aminobutyric acid-benzodiazepine, but not to the glycine receptor in rodents.
The inhibitory neurotransmitters glycine and gamma-aminobutyric acid (GABA) both activate transmembrane chloride channels of similar physical characteristics. A common ion channel component has therefore been postulated for both the glycine and GABA receptor proteins. Different convulsant drugs as picrotoxin and t-butyl bicyclophosphorothionate (TBPS) have been reported as channel-blocking ligands of the GABA receptor. Here, we show that the distribution of [35S]TBPS binding sites parallels the binding of the GABA receptor ligand [3H]flunitrazepam, but not that of the glycine receptor antagonist [3H]strychnine. Binding was examined in membrane fractions from different regions of the rat CNS and of the mutant mouse spastic, an animal deficient in glycine receptors. Also, affinity purification of the glycine receptor on aminostrychnine-agarose resulted in almost complete removal of [35S]TPBS binding sites from the receptor preparation. It is concluded that TBPS selectively binds to the GABA, but not glycine, receptor chloride channel complex.
Animals↗
The Mr 93,000 polypeptide of the postsynaptic glycine receptor complex is a peripheral membrane protein.
The glycine receptor of mammalian spinal cord is an oligomeric membrane protein that, after affinity purification on aminostrychnine-agarose or immobilized antibody, contains three polypeptides of Mr 48,000, 58,000, and 93,000. Here, the association and the properties of the polypeptides of the rat glycine receptor were investigated. Upon phase partitioning in the nonionic detergent Triton X-114, the three receptor polypeptides behaved as a hydrophilic protein complex exhibiting phospholipid binding. Sucrose gradient centrifugation or gel filtration in the presence of dithiothreitol and Triton X-100 separated the Mr 93,000 polypeptide from the Mr 48,000 and 58,000 polypeptides, which harbor the antagonist binding site of the glycine receptor. Alkaline or dimethylmaleic acid anhydride treatment of crude synaptic membrane fractions resulted in extraction of the Mr 93,000 polypeptide. Lectin binding was observed for the Mr 48,000 and 58,000 glycine receptor subunits but not the Mr 93,000 polypeptide. These results indicate that the Mr 93,000 polypeptide is a peripheral membrane protein that is located at the cytoplasmic face of the postsynaptic glycine receptor complex.
Amino Acids↗
Structure and biology of central nervous system neurotransmitter receptors.
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Animals↗
Separation of human plasma fibrin stabilizing factor (factor XIII) from fibrinogen by fibrinogen polymer formation.
Human plasma fibrin stabilizing factor (factor XIII) may be separated from fibrinogen through reversible fibrinogen polymer formation at pH 6.6, gamma/2 0.3, 0 degrees C, and subsequent Bio-Gel A 1.5m filtration. Factor XIII activity is eluted after the monomer fibrinogen peak. Polymer fractions from eight preparations, processed in duplicate, contain a mean 0.002 units factor XIII per mg fibrinogen, or about 0.7% the factor XIII content of standard plasma. Factor XIII-free fibrinogen polymers are easily dissociated (greater than 98%) to the monomer form by incubation at 37 degrees C, 18 hours. The fibrinogen preparations utilized were devoid of plasma fibronectin; thus these studies also show that reversible human fibrinogen polymer formation occurs in its absence.
Chromatography, Gel↗
Nerve and muscle regeneration after axonal division in rats treated with isaxonine phosphate (isopropylamino-2-pyrimidine phosphate).
Isaxonine phosphate (isopropylamino-2-pyrimidine phosphate), a neurotrophic agent, was tested on Schwann cell and muscle metabolism as evidenced by radiophosphate uptake in the inorganic phosphate and organic-bound acid-soluble phosphate fractions. It had no demonstrable effect either on sciatic nerve Schwann cells above and below an experimental crush or on the corresponding gastrocnemius and soleus muscles in their denervated and reinnervated state (7th to 28th postoperative day). It also failed to influence the end results (150th postoperative day) at nerve and muscle level after surgical division of the nerve and repair by either microsuture or fibrinogen adhesive.
Animals↗
Dynamics of rural-urban migration in India: 1960-1981.
"This paper analyzes a multi-sectoral simulation model of the Indian economy designed to isolate the sources of Indian economic growth and urbanization since 1960. The model shares many common traits with other computable general equilibrium (CGE) simulation models, and its underlying framework is neoclassical. The model stresses spatial issues so that it can provide predictions on rural/urban labor demands, and hence on migration flows. The central issue we seek to evaluate is whether a neoclassical development paradigm can explain adequately the somewhat paradoxical patterns of urbanization and economic growth observed in India since 1960. Our conclusion is a qualified, affirmative response, based on the model's ability to replicate key macroeconomic variables."
Asia↗
The glycine receptor deficiency of the mutant mouse spastic: evidence for normal glycine receptor structure and localization.
Homozygotes of the mutant mouse spastic exhibit reduced binding of 3H-strychnine to homogenates from various regions of the CNS compared with unaffected littermates (White and Heller, 1982). Here we report evidence that the spastic mutation coincides with a reduced concentration and an unaltered structure of the glycine receptor in spinal cord. Scatchard analysis of 3H-strychnine binding revealed a single binding site with a Bmax of 267 +/- 62 fmol/mg protein for spastic and of 864 +/- 220 fmol/mg protein for control mice; no difference was found for the corresponding KD values. Also Ki values of glycine for 3H-strychnine binding and displacement of 3H-strychnine by beta-alanine and taurine were indistinguishable for both preparations. Photoaffinity labeling of synaptic membranes with 3H-strychnine identified an Mr = 48,000 polypeptide in both control and spastic mouse membranes. Tryptic digestion of these membranes produced radiolabeled peptide fragments of identical molecular weights, suggesting that the proteolytic cleavage sites around the antagonist binding site are conserved in the mutant glycine receptor protein. Glycine receptors from both control and mutant mice were purified by affinity chromatography on aminostrychnine agarose. SDS/PAGE revealed three polypeptides of Mr = 48,000, 58,000, and 93,000 in both receptor preparations. Monoclonal antibodies directed against different subunits of the glycine receptor were applied to an enzyme-linked immunosorbent assay. The same pattern of immunoreactivity was obtained for glycine receptor from spinal cord of spastic homozygotes, control mice, and rats, suggesting conservation of the antigenic epitopes in the mutant receptor.(ABSTRACT TRUNCATED AT 250 WORDS)
Affinity Labels↗
The vertebrate glycine receptor protein.
Glycine is a major inhibitory neurotransmitter in the central nervous system. The postsynaptic receptor for this amino acid is a transmembrane ion channel protein which, after affinity purification on an antagonist column, contains three polypeptides of Mr 48,000, 58,000 and 93,000. Biochemical and immunological data show that these polypeptides have different functional properties and topologies with respect to the postsynaptic membrane. A model summarizing the currently available structural data is presented.
Animals↗
Sutures or fibrin glue for divided rat nerves: Schwann cell and muscle metabolism.
Peripheral nerve anastomoses using either epiperineurial sutures or a fibrinogen adhesive technique have been compared in the rat sciatic nerve model. Evaluation of results was made using radiolabelling of the metabolically active acid-soluble phosphate fractions of both nerve and muscle. In none of the situations tested--traumatic degeneration and regeneration in the sciatic nerve proximal segment, Wallerian degeneration and regeneration in its distal segment, atrophy and regeneration of the fast gastrocnemius muscle, and atrophy and regeneration of the slow soleus muscle--was one repair method significantly superior to the other. A significant degree of cross-reinnervation was shown to take place after anastomosis, altering the characteristics of the regenerating muscles. Both repair methods were equally inferior to the spontaneous repair occurring after a simple nerve crush.
Animals↗
Alterations of the retina in chick embryos induced by systemic alpha-bungarotoxin application.
The application of alpha-bungarotoxin onto the chorio-allantoic membrane of chick embryos between the 11th and 18th day of incubation leads to alterations of retinal development. The most significant qualitative change is the appearance of retinal rosettes formed by receptor cells. These rosettes are infoldings of the receptor cell layer. Quantitatively, an enlargement in volume of the receptor and outer nuclear layer can be found together with a simultaneous decrease of the other retinal layers. The toxin seems to suspend the naturally occurring nerve cell death in the receptor cell population
Animals↗