Characterization of γ-Aminobutyric Acid Binding Sites on Crude Synaptic Membranes

[3H]GABA binding to crude synaptic membranes of rat brain was studied in an attempt to identify GABA binding to its synaptic receptor in the presence of Na+. Membrane vesicles prepared from crude synaptic membrane fractions were useful as a tool to differentiate synaptic GABA receptors from GABA uptake sites. The crude synaptic membranes treated with Triton X-100 [membranes (TX)] involved two classes of GABA binding sites (KD = 38.7 and 78.0 nM) in the absence of Na+, but the high-affinity sites disappeared in the presence of Na+ and a single class of GABA binding sites (KD = 75.0 nM) was detected. The failure to detect an active uptake of [3H]GABA into the vesicles prepared from membranes (TX) suggests that the [3H]GABA binding in the presence of Na+ was related to synaptic GABA receptors. It is probable that Na+ could mask the presence of the high-affinity class of GABA receptor.     

An Analysis of γ-Aminobutyrate Receptors and Uptake by Isolated Purkinje Cells

Abstract: An isolated fraction of Purkinje perikarya was prepared. This fraction had [³H]GABA receptor binding and [³H]muscimol binding analogous to that reported for heterogeneous cerebellar membranes. The finding of two binding components with each ligand suggests that these components do not represent two binding sites, one presynaptic and the other postsynaptic, since both are clearly seen on purified Purkinje cell bodies. Subcellular fractionation indicates that synaptic endings and plasma membranes are enriched in GABA receptors compared with intracellular organelles. Purkinje cell bodies were also found to possess a high-affinity transport system for GABA, which was sensitive to inhibition by diaminobutyric acid (DABA) but not by β-alanine. They showed no evidence of homoexchange (the movement of label without net transport). This supports our suggestion that homoexchange is an artifact of synaptic particle formation.     

Purification, Biochemical Characterization, Binding Activity, and Selectivity of a Glutamate Binding Protein from Bovine Brain

A glutamate binding protein was purified from bovine brain to apparent homogeneity. The procedure used for the purification of this protein involved extraction of a crude synaptic membrane fraction with Na-cholate, followed by solubilization of the binding protein from the membranes by Triton X-100, and, finally, affinity batch separation of the protein on L-glutamate-loaded glass fiber. The molecular characteristics of the purified protein were similar to those previously described for the glutamate binding protein from rat brain synaptic membranes and included the following: small Mr (14,000), acidic (pI = 4.7) protein with a single NH2-terminal amino acid (tyrosine), and significant absorption at wave-lengths greater than 300 nm. Complete amino acid analysis of the protein was not achieved, either because of destruction of some amino acids or of incomplete hydrolysis of the protein. The protein bound L-glutamate with high affinity (KD = 0.87 microM), exhibited one class of L-glutamate binding sites, and bound glutamate with a stoichiometry of 0.7 mol ligand/mol protein. The displacement of protein-bound L-glutamic acid by other neuroactive amino acids had characteristics similar to those observed for the displacement of L-glutamate from rat brain synaptic membrane or purified protein binding sites. Finally, the metal ligand formers KCN and NaN3 inhibited the activity of this protein just as they have been shown to do in rat brain synaptic membranes or the purified protein.     

CHARACTERIZATION OF THE BINDING OF [3H]MUSCIMOL, A POTENT γ-AMINOBUTYRIC ACID AGONIST, TO RAT BRAIN SYNAPTOSOMAL MEMBRANES USING A FILTRATION ASSAY

Abstract— The binding of [³H]muscimol, a potent GABA agonist, to crude synaptic membranes prepared from rat brain was studied using a filtration method to isolate membrane-bound ligand. Specific binding was found to be saturable and occurred to two binding sites of K _d5 5 and 30 n m . Binding was Na⁺-independent and enhanced by both freezing and Triton treatment. Regional and subcellular distribution studies and pharmacological characterization of specific [³H]muscimol binding are consistent with binding to the synaptic GABA receptor.     

The effect of triton X-100 on bovine brain synaptic membranes

Bovine brain synaptic membranes which were frozen and then extensively washed showed low affinity [³H]muscimol binding. These membranes contained GABA and calmodulin, apparently tightly bound within the membrane fraction. Membranes which were additionally treated with the detergent Triton X-100 showed high affinity [³H]muscimol binding. These membranes did not appear to contain GABA or calmodulin. Transmission electron microscopy studies demonstrated that the washed membrane fraction contained many synaptosomal and vesicular structures. Triton treatment led to the extensive rupture of these structures. These studies explain the well-reported findings of tightly bound GABA and calmodulin in brain membrane fractions, as being due to the entrapment of these compounds inside sealed membrane-bound structures which are still present after a freezethaw and extensive wash treatment, their complete removal requiring Triton-treatment to rupture the vesicles.     

The binding of 3H-Isoguvacine to mouse brain synaptic membranes

³H-Isoguvacine, a gamma aminobutyric acid (GABA) agonist, has been shown to bind to a mouse forebrain synaptic membrane preparation. The specific binding is displaceable by GABA, muscimol and bicuculline but not by picrotoxin or diaminobutyric acid. Kinetic data suggest two binding affinities. Highest levels of binding are observed in the cerebellum, cortex and hippocampus. It is suggested that isoguvacine binds to GABA binding sites and therefore represents a new ligand for measuring GABA receptor binding.     

An endogenous inhibitor of GABA receptor binding

An endogenous inhibitor of GABA receptor binding was prepared from synaptic membrane of rat brain with 0.05% Triton X-100. The endogenous inhibitor was competitive with GABA for GABA binding sites. The inhibition of GABA receptor binding by the endogenous inhibitor was blocked by the allosteric effect of diazepam. In the presence of diazepam, specific [³H]GABA binding was greater in a medium containing the endogenous inhibitor than in one containing an equal inhibitory potency of GABA, whereas there was no difference in the absence of diazepam. This indicated that the endogenous inhibitor was not GABA itself.     

牛小脑γ-氨基丁酸受体的生化特性

用放射性配基结合实验方法鉴定了牛小脑γ-氨基丁酸(GABA)受体的生化特性。高速离心制备的牛小脑突触膜与~3H-GABA有一饱和结合,K_D值为96nM,B_(max)为1.02pmol/mg蛋白,Hill系数为0.99。这一结合具有药理专一性和立体专一性,动力学实验测得结合速度常数为9.6×10~6M~(-1)min~(-1),解离速度常数为0.115min~(-1)。冻融、洗涤、超声波和TritonX-100处理突触膜,使~3H-GABA结合活性增加114倍。     

Increased γ-Aminobutyric Acid Receptor Function in the Cerebral Cortex of Myoclonic Calves with an Hereditary Deficit in Glycine/Strychnine Receptors

Inherited congenital myoclonus (ICM) of Poll Hereford cattle is a neurological disease in which there are severe alterations in spinal cord glycine-mediated neurotransmission. There is a specific and marked decrease, or defect, in glycine receptors and a significant increase in neuronal (synaptosomal) glycine uptake. Here we have examined the characteristics of the cerebral gamma-aminobutyric acid (GABA) receptor complex, and demonstrate that the malfunction of the spinal cord inhibitory system is accompanied by a change in the major inhibitory system in the cerebral cortex. In synaptic membrane preparations from ICM calves, both high-and low-affinity binding sites for the GABA agonist [3H]muscimol were found (KD = 9.3 +/- 1.5 and 227 +/- 41 nM, respectively), whereas only the high-affinity site was detectable in controls (KD = 14.0 +/- 3.1 nM). The density and affinity of benzodiazepine agonist binding sites labelled by [3H]diazepam were unchanged, but there was an increase in GABA-stimulated benzodiazepine binding. The affinity for t-[3H]butylbicyclo-o-benzoate, a ligand that binds to the GABA-activated chloride channel, was significantly increased in ICM brain membranes (KD = 148 +/- 14 nM) compared with controls (KD = 245 +/- 33 nM). Muscimol-stimulated 36Cl- uptake was 12% greater in microsacs prepared from ICM calf cerebral cortex, and the uptake was more sensitive to block by the GABA antagonist picrotoxin. The results show that the characteristics of the GABA receptor complex in ICM calf cortex differ from those in cortex from unaffected calves, a difference that is particularly apparent for the low-affinity, physiologically relevant GABA receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulation of benzodiazepine binding to rat brain membranes and solubilized receptor complex by avermectin B1a and gamma-aminobutyric acid

The binding of [3H]flunitrazepam to benzodiazepine receptors in synaptic membranes and a digitonin-solubilized receptor fraction of rat brain is increased by avermectin B1a and gamma-aminobutyric acid (GABA). The effects of avermectin B1a and GABA are both sensitive to inhibition by (+)-bicuculline. Avermectin B1a and GABA both decrease the Kd and increase the Bmax of [3H]flunitrazepam binding to membranes. Kinetic analysis of the binding of [3H]flunitrazepam to rat brain membranes indicates that avermectin B1a and GABA reduce the rate constants of both association and dissociation between the ligand and the receptor. These results suggest a similar mechanism of modulation of benzodiazepine binding by avermectin B1a and GABA. This modulation may involve in interaction among the receptors for benzodiazepine, GABA and avermectin B1a.     

Endogenous inhibitors of Na+-independent [3H]GABA binding to crude synaptic membranes

The characteristics of the Na⁺-independent high-affinity binding of [³H]GABA to various types of crude synaptic membranes (CSM) prepared from rat brain cortex were studied. In freshly prepared CSM the content of GABA was so high that the high-affinity [³H]GABA binding could not be determined. In contrast when the frozen-thawed CSM were incubated at 37° for 30 min with or without Triton X-100 or phospholipase C and then washed repeatedly, there was a virtual disappearance of GABA from the supernatant extracts and the binding constants of [³H]GABA to CSM could be determined. Two apparent populations of [³H]GABA binding sites, one with a low- and the other with a high-affinity constant, were detected. The ratio of the number of high- to low-affinity binding sites varies with the method used to prepare the membranes. The lowest value of this ratio was observed with membranes incubated at 37° for 30 min. However, when frozen-thawed CSM were treated with 0.05% Triton X-100 repeatedly, the ratio of the number of high- to low-affinity binding sites increased progressively. This increase in ratio is due to a selective increase in the number of the high-affinity sites without significant changes in the number of the low-affinity sites. The extent of the increase in the number of sites that bind [³H]GABA with high affinity after repeated Triton X-100 treatments was paralleled by a decrease of an endogenous protein which inhibits GABA binding. The reapplication of this endogenous material to membranes repeatedly treated with Triton X-100 reduces the number of high-affinity binding sites for [³H]GABA to values similar to those measured in membranes that were not treated with Triton X-100. The inhibitory preparation extracted from CSM incubated with Triton X-100 was shown to be free of GABA or phospholipids. The gel filtration chromatography reveals the presence of two molecular forms of the inhibitor; of these, the high-molecular-weight material fails to bind GABA, whereas the low-molecular-weight material appears to bind GABA. The high-molecular-weight endogenous inhibitor has been termed GABA modulin.     

A comparison of direct and indirect techniques using ferritin-conjugated ligands for the localization of concanavalin A binding sites on isolated hepatocyte plasma membranes

Concanavalin A binding sites have been localized on isolated plasma membranes both by a direct technique involving ferritin-concanavalin A and by an indirect technique in which membranes were treated successively with concanavalin A, rabbit anti-concanavalin A, and ferritin-conjugated sheep anti-rabbit F(ab')2. Binding studies showed that, at saturation, less than 25% of the concanavalin A binding sites were accessible to ferritin-concanavalin A. The decreased binding was apparently related to steric factors, since membranes saturated with the conjugated ligand were able to bind additional concanavalin A, and since the conjugated ligand, once bound to the membrane, caused the same inhibition of the membrane-bound enzyme 5'-nucleotidase as concanavalin A. Nonspecific binding sites accounted for 10% of the total binding of ferritin-concanavalin A and were localized mainly on the cytoplasmic side of the membrane, whereas specific sites were on the external side. The indirect technique, which was expected to increase the binding of ferritin-conjugate to the membrane, resulted in the binding of ferritin to less than 15% of the concanavalin A binding sites, and did not decrease the nonspecific binding.     

A modulating role of mercurials-sensitive sulfhydryl groups in synaptic GABA receptor binding

The specific binding of GABA (γ-aminobutyric acid) agonist ³H-muscimol, to synaptic membranes from the rat brain showed a significant increase, when the membranous preparations were treated with a low concentration (10^?4–10^?5M) of mercurial sulfhydryl reagents such as p-chloromercuribenzoate and mercuric chloride. This activation in GABA receptor binding was bicuculline-sensitive, and was partially restored by subsequent treatments with 10 mM cysteine, penicillamine, or mercaptoethanol. Scatchard analysis of the binding revealed that this activation was due to the increase in the affinity of both high and low affinity bindings sites but not in the B_max values. On the other hand, the treatment of synaptic membranes with hydrophilic sulfhydryl reagents such as N-ethylmaleimide and iodoacetate had no effect on the binding. These hydrophilic sulfhydryl reagents, however, induced an increase of the binding following the pretreatment of synaptic membranes with 0.01% Triton X-100 or 0.5 U/mg prot. of phospholipase A₂ (EC 3.1.1.4.). These results suggest that mercurials-sensitive sulfhydryl groups, which are normally masked by membrane lipids, may play a modulating role in GABA receptor binding at central synapses.     

Evidence of essential disulfide bonds in angiotensin II binding sites of rabbit hepatic membranes. Inactivation by dithiothreitol

Radiolabelled angiotensin II binds to a single class of high-affinity binding sites on purified rabbit hepatic membranes. The binding is specific, reversible and saturable. Displacement studies using angiotensin and various analogs of angiotensin II disclosed a structure-activity profile similar to that found in physiologically relevant angiotensin II receptor sites. Treatment of membranes with the reducing agent, dithiothreitol, cause a significant decrease in the affinity of angiotensin II binding sites for the native ligand. This effect is mimicked by a 15-fold higher concentration of the monosulfhydryl derivative, 2-mercaptoethanol. Kinetic studies also indicated that dithiothreitol increases the rate of dissociation of bound ligand from the membrane without significantly affecting the association rate. In contrast, treatment of membranes with the metal chelators, ethylenediaminetetracetic acid (EDTA) and ethyleneglycol bis(β-aminoethyl ether)-N,N′-tetracetic acid (EGTA), does not affect the binding of radiolabeled angiotensin II. Furthermore, dithiothreitol inhibited the binding of angiotensin II to a solubilized partially purified preparation of angiotensin II-binding protein from the same tissue and also increased the dissociation of bound angiotensin II. This indicates that the effect of the sulfhydryl reagents on the membrane binding sites is the result of a direct alteration of the binding sites rather than a gross modification of the structure of the membrane.     

Differential Solubilization of γ-Aminobutyric Acid Receptive Sites from Membranes of Mammalian Brain

Sodium-dependent (+Na) and sodium-independent (-Na) receptive sites for gamma-aminobutyric acid (GABA) residing in or on frozen synaptic plasma membranes (SPM) of bovine cerebral cortex were characterized as to binding constants, pharmacologic specificities, and sodium dependence. The SPM fraction was then treated with various concentrations of Triton X-100 resulting in the loss of pharmacologic specificity, binding characteristics, and sodium dependence associated with +Na GABA receptive sites in SPM. The resulting junctional complex preparation (JC), i.e., a fraction enriched in junctional complexes, possessed only the pharmacologic specificity and binding constants associated with -Na receptive sites whether assayed in the presence or absence of 100 mM-NaCl. This is probably due to the detergent dispersal or solubilization of the +Na GABA receptive site. The binding constants, KD and Bmax, for -Na GABA binding in SPM were 170 nM and 4.4 pmol/mg protein, while in JC they were 186 nM and 3.7 pmol/mg protein. Under repeated washing the KD was reduced to 60 +/- 6.9 nM and the Bmax was reduced to 2.5 +/- 0.5 pmol/mg protein in JC, probably owing to the removal of endogenous ligand or inhibitor, and not to inhibition by residual Triton X-100. Multiple extraction with 0.1% or 0.5% Triton X-100 did not alter the KD or Bmax values for the binding of [3H]GABA to JC. Sodium-independent GABA binding was lost from JC membranes with the use of sodium deoxycholate, probably through solubilization.     

The Benzodiazepine/GABA Receptor Complex: Molecular Size in Brain Synaptic Membranes and in Solution

Abstract: The molecular size of the benzodiazepine (BZ) receptor in the synaptic membrane of brain cortex (bovine or rat) was determined by an improved version of the radiation inactivation method to be 220,000. An identical size was found simultaneously for the associated γ-aminobutyric acid (GABA) receptor and for the component binding β-carboline esters. It is proposed that all three activities reside in a single protein or protein complex in the membrane. The size in solution, after extraction into Triton X-100 medium from exhaustively washed membranes, was estimated by sedimentation constant (9.4S) and by gel filtration (∼230,000 apparent MW), again with the BZ and GABA binding activities behaving identically. This size applies to the component that undergoes photoaffinity labelling by [³H]flunitrazepam in the membrane, and contains a 51,000 M_r polypeptide as the BZ-binding subunit. It is concluded that a protein complex or oligomer of 200,000–220,000 MW carries a class of BZ-binding sites and an associated class of GABA_A sites.     

Function of a calmodulin in postsynaptic densities. III. Calmodulin-binding proteins of the postsynaptic density

A method has been developed for binding calmodulin, radioiodinated by the lactoperoxidase method, to denaturing gels and has been used to attempt to identify the calmodulin-binding proteins of cerebral cortex postsynaptic densities (PSDs). Calmodulin primarily bound to the major 51,000 Mr protein in a saturatable manner; secondarily bound to the 60,000 Mr region, 140,000 Mr region, and 230,000 Mr protein; and bound in lesser amounts to a number of other proteins. The major 51,000 Mr calmodulin-binding protein is one of unknown identity. Binding of iodinated calmodulin to these proteins was blocked by EDTA, EGTA, chlorpromazine, and preincubation with unlabeled calmodulin. Calmodulin iodinated by the chloramine-T method, which inactivates calmodulin did not bind to the PSD but bound nonspecifically to histone. Calmodulin did not bind to proteins from a variety of sources for which calmodulin interactions have not been found. Except for three proteins, all of the proteins of synaptic membranes that bind calmodulin could be accounted for by proteins of the PSD which are a part of the synaptic membrane fraction. The major 51,000 M, protein and the corresponding iodinated calmodulin binding were greatly reduced in cerebellar PSDs and this difference between cerebral cortex and cerebellar PSDs is discussed in light of the possible function of calmodulin in synaptic excitatory responses.     

Microsomal Opiate Receptors: Characterization of Smooth Microsomal and Synaptic Membrane Opiate Receptors

In continuing studies on smooth microsomal and synaptic membranes from rat forebrain, we compared the binding properties of opiate receptors in these two discrete subcellular populations. Receptors in both preparations were saturable and stereospecific. Scatchard and Hill plots of [3H]naloxone binding to microsomes and synaptic membranes were similar to plots for crude membranes. Both synaptic membranes and smooth microsomes contained similar enrichments of low- and high-affinity [3H]naloxone binding sites. No change in the affinity of the receptors was observed. When [3H]D-ala2-D-leu5-enkephalin was used as ligand, microsomes possessed 60% fewer high-affinity sites than did synaptic membranes, and a large number of low-affinity sites. In competition binding experiments microsomal opiate receptors lacked the sensitivity to (guanyl-5'-yl)imidodiphosphate [Gpp(NH)p] shown by synaptic and crude membrane preparations. In this respect microsomal opiate receptors resembled membranes that were experimentally guanosine triphosphate (GTP)-uncoupled with N-ethylmaleimide (NEM). Agonist binding to microsomal and synaptic membrane opiate receptors was decreased by 100 mM NaCl. Like NEM-treated crude membranes, microsomal receptors were capable of differentiating agonist and antagonists in the presence of 100 mM NaCl. MnCl2 (50-100 microM) reversed the effects of 100 mM NaCl and 50 microM GTP on binding of the mu-specific agonist [3H]dihydromorphine in both membrane populations. Since microsomal receptors are unable to distinguish agonists from antagonists in the presence of Gpp(NH)p, they are a convenient source of guanine nucleotide-uncoupled opiate receptors.     

Solubilisation of the Benzodiazepine Receptor from Rat Cerebellum by the Detergent n-Octylpentaoxyethylene

The detergent n-octylpentaoxyethylene is one in the series of tenside detergents developed for membrane solubilisation. We have used this detergent to solubilise benzodiazepine receptors from rat cerebellum. The soluble receptor has an affinity (KD) for [3H]flunitrazepam of 1.8 nM +/- 0.2, which is not significantly different from that observed in synaptic membranes. Under optimal conditions (0.6% wt/vol), the number of soluble flunitrazepam binding sites (Bmax) of 0.35 pmol/mg protein suggests an apparent solubilisation of 40% of sites from the membrane. However, the absence of the characteristic facilitation of [3H]flunitrazepam binding by gamma-aminobutyric acid (GABA), cartazolate, and pentobarbital in this soluble receptor preparation suggests that such a preparation is unlikely to be a useful preparation for further studies on the molecular mechanisms underlying GABAA receptor function.     

Properties of NAD binding by synaptic membranes of rat brain

The binding of [14C]NAD to rat brain synaptic membranes is reversible and depends on incubation time, temperature and protein concentration in the reaction mixture. The value of the rate constant for [14C]NAD binding to the synaptic membranes at 24 degrees C (kl) is 1.1 X 10(-6) M-1 S-1, the rate constant for dissociation of the [14C]NAD-receptor complex (k-1) is 3.3 X 10(-3) S-1. The value of the constant for the ligand dissociation from this complex (Kd) is 3.0 nmole. Treatment of the experimental results in the Scatchard plots for the equilibrium binding of [14C]NAD to the synaptic membranes demonstrated that the receptor sites with high and low affinities for the ligand (Kd1 = 3.3 nmol, Kd2 = 14.4 nmole) and with binding capacities of 44 and 77 pmole of [14C]NAD, respectively. It was found that the synaptosomal membrane components which bind the labelled NAD have a protein nature. Data from [14C]NAD and [nicotinamide-3H]NAD binding suggest that brain synaptic membranes bind NAD at the nicotinamide and adenylic moieties.