Synaptosomal Membrane Preparations Research Articles

Mass spectrometric analysis of synaptosomal membrane preparations for the determination of brain receptors, transporters and channels.

The molecular composition of synaptic signal transduction machineries shapes synaptic neurotransmission. The repertoire of receptors, transporters and channels (RTCs) comprises major signaling events in the brain. RTCs are conventionally studied by candidate immunohistochemistry and biochemistry, which are low throughput with resolution greatly affected by available immunoreagents and membrane interference. Therefore, a comprehensive resource of synaptic brain RTCs is still lacking. In particular, studies on the detergent-soluble synaptosomal fraction, known to contain transporters and channels, are limited. We, therefore, performed sub-synaptosomal fractionation of rat cerebral cortex, followed by trypsin/chymotrypsin sequential digestion of a detergent-soluble synaptosomal fraction and a postsynaptic density preparation, stable-isotope tryptic peptide labeling and liquid chromatography mass spectrometry. Based on the current study, a total of 4784 synaptic proteins were submitted to the ProteomExchange database (PXD001948), including 274 receptors, 394 transporters/channels and 1377 transmembrane proteins. Function-based classification assigned 1781 proteins as probable drug targets with 834 directly linked to brain disorders. The analytical approach identified 499 RTCs that are not listed in the largest, curated database for synaptosomal proteins (SynProt). This is a threefold RTC increase over all other data collected to date. Taken together, we present a protein discovery resource that can serve as a benchmark for future molecular interrogation of synaptic connectivity.

CLC-3 Channels Modulate Excitatory Synaptic Transmission in Hippocampal Neurons

It is well established that ligand-gated chloride flux across the plasma membrane modulates neuronal excitability. We find that a voltage-dependent Cl(-) conductance increases neuronal excitability in immature rodents as well, enhancing the time course of NMDA receptor-mediated miniature excitatory postsynaptic potentials (mEPSPs). This Cl(-) conductance is activated by CaMKII, is electrophysiologically identical to the CaMKII-activated CLC-3 conductance in nonneuronal cells, and is absent in clc-3(-/-) mice. Systematically decreasing [Cl(-)](i) to mimic postnatal [Cl(-)](i) regulation progressively decreases the amplitude and decay time constant of spontaneous mEPSPs. This Cl(-)-dependent change in synaptic strength is absent in clc-3(-/-) mice. Using surface biotinylation, immunohistochemistry, electron microscopy, and coimmunoprecipitation studies, we find that CLC-3 channels are localized on the plasma membrane, at postsynaptic sites, and in association with NMDA receptors. This is the first demonstration that a voltage-dependent chloride conductance modulates neuronal excitability. By increasing postsynaptic potentials in a Cl(-) dependent fashion, CLC-3 channels regulate neuronal excitability postsynaptically in immature neurons.

Cholesterol is critical to the integrity of neuronal porosome/fusion pore

Secretion is one of the most fundamental cellular processes. Porosomes have been demonstrated as the universal secretory machinery in cells. Earlier studies determine the presence of a number of proteins in porosomes, among them the N- and P/Q-type calcium channels, actin, syntaxin-1, synaptotagmin-1, vimentin, the N-ethylmaleimide-sensitive factor (NSF), the chloride channel CLC-3, and the alpha subunit of the heterotrimeric GTP-binding protein G o. Studies demonstrate that t-SNAREs localize at the base of porosomes, and directly interact with calcium channels. In the present study, we demonstrate that Syntaxin-1 co-localizes with cholesterol in solubilized synaptosomal membrane preparations. Depletion of cholesterol, results in the dissociation of both Syntaxin-1 and N-type calcium channel from neuronal porosomes. Thus, cholesterol participates as an integral component of the neuronal porosome complex, and is required for its stability.

Pharmacological and neurochemical evidence for antidepressant-like effects of the herbal product Catuama

Catuama is a marketed herbal product currently used as a tonic, especially for the management of mental or physical fatigue. In the present study, we have shown pharmacological and neurochemical evidence for antidepressant-like actions of the product Catuama. Acute and chronic oral treatments with Catuama both resulted in a significant reduction of the immobility time in two models of depression in mice, the forced swimming and the tail suspension tests. Conversely, treatment with the same doses of Catuama did not significantly interfere with motor activity according to assessment in the open-field test. The antidepressant-like effects were comparable to those observed for classical antidepressant drugs. When assessed in vitro, Catuama inhibited, in a concentration-dependent manner, the synaptosomal uptake of noradrenaline and principally of serotonin and dopamine, in rat brain. Likewise, in vitro incubation of Catuama also resulted in a marked increase of the release of serotonin and dopamine in rat brain crude preparation of synaptosomal membranes. Finally, Catuama was found to be effective in interfering with the synaptosomal uptake of serotonin and dopamine following long-term oral treatment of rats. The present findings allow us to suggest that the herbal product Catuama might be useful for the clinical management of moderate and mild depressive states, alone or in association with current antidepressant drugs.

Differential postmortem delay effect on agonist-mediated phospholipase Cbeta activity in human cortical crude and synaptosomal brain membranes.

The phosphoinositide signal transduction system, and particularly, phospholipase Cbeta isozymes, are relevant in the etiopathogeny of human neuropsychiatric pathologies such as depression. Stimulation of phospholipase Cbeta activity by muscarinic receptors and G proteins was determined in crude and synaptosomal membrane preparations from nine postmortem human frontal cortices (postmortem delay range 8 to 50 h). Thus, the phospholipase Cbeta activity was determined by measuring the hydrolysis of exogenous [3H]-phosphatidylinositol 4,5-bisphosphate. There was a postmortem delay-mediated decrease in the PIP2 hydrolysis irrespective of the membrane preparation used (P < 0.05). Moreover, there were statistically significant differences for exponential decay curve parameters (K factor and Span) of PLCbeta activity induced by agonist-mediated activation between crude and synaptosomal membrane preparations. These results show that the postsynaptic component of the PLCbeta activity is more sensible to the postmortem delay effect.

A mechanism of the thearubigin fraction of black tea (camellia sinensis) extract protecting against the effect of tetanus toxin.

The aim of the present study was to elucidate the mechanism of the protective effect of black tea extract's thearubigin fraction against the action of tetanus toxin. The effects of thearubigin fraction extracted from a black tea infusion were examined for neuromuscular blocking action on tetanus toxin in mouse phrenic nerve-diaphragm preparations and on the binding of this toxin to the synaptosomal membrane preparations of rat cerebral cortices. The interaction between tetanus toxin and thearubigin fraction was also investigated. Tetanus toxin (4 micrograms/ml) abolished indirect twitches in mouse phrenic nerve-diaphragm preparations within 150 min. Thearubigin fraction mixed with tetanus toxin blocked the inhibitory effect of the toxin. Mixing iodinated toxin with thearubigin fraction inhibited the specific binding of [125I]tetanus toxin to the synaptosomal membrane preparation. The effects of thearubigin fraction were dose-dependent. The elution profile of [125I]tetanus toxin on Sephadex G-50 column chromatography was different from that of toxin mixed with thearubigin fraction. These findings indicate that thearubigin fraction protects against the action of tetanus toxin by binding with the toxin.

High-affinity [ 3H] kainic acid binding to brain membranes : a re-evaluation of ligand potency and selectivity

[ 3H]Kainic acid ([ 3H]KA) is a widely used tool for studying the KA class of excitatory amino acid receptors. [ 3H]KA of significantly higher specific activity has become available permitting use of radioligand concentrations below the dissociation constant (K D) of the high-affinity binding site. We employed low radioligand (0.05–0.2 nM) and receptor concentrations (0.01 nM) to gain new insights into the binding characteristics of the high-affinity KA binding site in a standard preparation of lyzed synaptosomal membranes from the cerebral cortex of male Sprague-Dawley rats. Under these conditions, KA binds to a single class of high-affinity sites with a K D of 1.0 ± 0.3 nM. The potencies of competing agents are considerably higher than published reports. Specifically, domoic acid, glutamate, and glutamine exhibit IC 50 values for displacing [ 3H]KA of 0.37 ± 0.02, 94 ± 13, and 1500 ± 500 nM, respectively. Domoate (1 μM) was tested against a panel of 32 central nervous system binding sites and found to be inactive at each, indicating this toxin displays considerable selectivity. This study illustrates the remarkable potency of domoic acid and underlines the importance of performing radioligand binding studies at concentrations of constituents that permit characterization of high-affinity interactions.

Voltage-dependent anion channel proteins in synaptosomes of the torpedo electric organ: immunolocalization, purification, and characterization.

In this study, we purified and characterized the voltage-dependent anion channel (VDAC) from the Torpedo electric organ. Using immunogold labeling, VDAC was colocalized with the voltage-gated Ca2+ channel in the synaptic plasma membrane. By immunoblot analysis, five protein bands in synaptosomes isolated from the Torpedo electric organ cross reacted with two monoclonal anti-VDAC antibody. No more than about 7 to 10% mitochondrial contains could be detected in any synaptosomal membrane preparation tested. This was estimated by comparing the specific activity in mitochondria and synaptosomes of succinate-cytochrome-c oxidoreductase and antimycin-insensitive NADH-cytochrome-c oxidoreductase activities; mitochondrial inner and outer membrane marker enzymes, respectively. [14C]DCCD (dicyclohexylcarbodiimide), which specifically label mitochondrial VDAC, labeled four 30-35 kDa protein bands that were found to interact with the anti-VDAC antibody. The distribution of the Torpedo VDAC protein bands was different among membranes isolated from various tissues. VDAC was purified from synaptosomes and a separation between two of the proteins was obtained. The two purified proteins were characterized by their single channel activity and partial amino acid sequences. Upon reconstitution into a planar lipid bilayer, the purified VDACs showed voltage-dependent channel activity with properties similar to those of purified mitochondrial VDAC. Amino acid sequence of four peptides, derived from VDAC band II, exhibited high homology to sequences present in human VDACI (98%), VDAC2 (91.8%), and VDAC3 (90%), while another peptide, derived from VDAC band III, showed lower homology to either VDAC1 (88.4%) or VDAC2 (79%). Two more peptides show high homology to the sequence present in mouse brain VDAC3 (100 and 78%). In addition, we demonstrate the translocation of ATP into synaptosomes, which is inhibited by DCCD and by the anion transport inhibitor DIDS. The possible function of VDAC in the synaptic plasma membrane is discussed.

5-HPETE Is a Potent Inhibitor of Neuronal Na+, K+-ATPase Activity

The effects of 1 uM concentrations of arachidonic acid hydroperoxide (HPETES) products of 5-, 12- and 15-lipoxygenase on Na+, K+-ATPase activity were investigated in synaptosomal membrane preparations from rat cerebral cortex. 5-HPETE inhibited Na+, K+-ATPase activity by up to 67 %. In contrast, 12-HPETE and 15-HPETE did not inhibit Na+, K+-ATPase activity. In addition, neither 5-HETE or LTA4inhibited Na+, K+-ATPase activity. Dose-response studies indicated that 5-HPETE was a potent (IC25= 10−8M) inhibitor of Na+, K+-ATPase activity. These findings indicate that 5-HPETE inhibits Na+, K+-ATPase activity by a mechanism that is dependent on the hydroperoxide position and independent of further metabolism by 5-lipoxygenase. It is proposed that 5-HPETE production by 5-lipoxygenase and subsequent inhibition of neuronal Na+, K+-ATPase activity may be a mechansim for modulating synaptic transmission.

Preparation of synaptosomal plasma membranes by subcellular fractionation.

Preparation of synaptosomal plasma membranes by subcellular fractionation.

SNAP-25 and synaptotagmin involvement in the final Ca(2+)-dependent triggering of neurotransmitter exocytosis.

In neurons, depolarization induces Ca2+ influx leading to fusion of synaptic vesicles docked at the active zone for neurotransmitter release. While a number of proteins have now been identified and postulated to participate in the assembly and subsequent disengagement of a vesicle docking complex for fusion, the mechanism that ultimately triggers neuroexocytosis remains elusive. Using a cell-free, lysed synaptosomal membrane preparation, we show that Ca2+ alone is sufficient to trigger secretion of glutamate and furthermore that Ca(2+)-signaled exocytosis is effectively blocked by antibodies and peptides to SNAP-25, a key constituent of the vesicle docking complex. In addition, Ca2+ inhibits the ability of synaptotagmin, a synaptic vesicle protein proposed as a calcium sensor and triggering device, to associate with this docking complex. These results support a model in which Ca(2+)-dependent triggering of neurotransmission at central synapses acts after ATP-dependent potentiation of the docking-fusion complex for membrane fusion.

Perturbation of artificial and biological membranes by organic compounds of aliphatic, alicyclic and aromatic structure

Aliphatic, alicyclic and aromatic hydrocarbons interact with biological membranes. Until now little has been known about their mode of interaction with the membrane bilayer and membrane integral proteins with toxic effects to cells. The lipid theory hypothesis explains the toxic effects by the organic solvent-induced disorder in the lipid bilayer, which indirectly affects the function of membrane-embedded proteins. The extent of bilayer perturbations is ascribed to the solvent accumulation in the bilayer, which is related only to the lipophilicity of the molecule, independent of the chemical structure. In this study the fluidizing effects of aliphatic, alicyclic and aromatic hydrocarbons were compared. Membrane fluidity changes were estimated from the pyrene excimer formation, using pyrene and pyrene derivatives to label specifically the localization of solvent molecules in the transverse plane of the bilayer. Liposomal, microsomal and synaptosomal membrane preparations were evaluated because proteins and cholesterol, as natural membrane components, increase the bilayer order and reduce the organic solvent membrane/buffer partition. In the concentration range investigated, only the aromatic solvents disorder the lipid bilayer, with the greatest perturbation in the centre of the bilayer. These results are related to structural properties of the organic solvents investigated.

Membrane receptors for estrogen, progesterone, and testosterone in the rat brain: fantasy or reality.

1. There are numerous circumstantial evidence supporting the concept that steroid hormones control cellular function by means other than the nuclear receptor steroid binding mechanism. It is the intent of this report to present evidence indicating that steroids bind to specific sites in neuronal membranes. 2. Some of the criteria to define steroid membrane receptors using steroid-BSA conjugates that can be radioiodinated to desired specific activity have been fulfilled for each of the three sex steroids using crude synaptosomal membrane preparations (P2 fractions) from the CNS of female and male rats. Ligand binding for each of the three steroids indicate high-affinity and high-capacity sites with distinct brain selectivity and stereospecificity. For example, 17 beta-E-6-[125I]BSA binds hypothalamic P2 fractions (HYP-P2) with an estimated Kd of about 3 +/- 0.7 nM (X +/- SE; n = 3), whereas the cerebellum P2 (CB-P2) fractions bind the ligand with a Kd of 34 +/- 7 nM and, a Bmax of 3 and 42 pmol/mg protein, respectively. Estrogen and testosterone binding fit best a one-single site, while progesterone binding sites can be best represented by a two-binding site, one high-affinity (Kd = 1-2 nM) and one low affinity (Kd = 62 nM), in CB-P2 fractions from intact adult female rat brain. Kinetics studies for T-3-[125I]BSA indicate that the estimated Kd of 30 +/- 2 nM for the olfactory bulb P2 fractions (OB-P2) from male rats is in good agreement with Kd values computed from Scatchard-derived data using the LIGAND algorithm. 3. 17 beta-E-6-[125I]BSA binding sites are stereospecific and appears to be present as early as 5 days of age in both the OB- and the CB-P2 fractions without changes during development. In contrast, P-6-[125I]BSA binding sites are practically absent during days 5 and 12 and appear by day 22. 4. Finally, membrane receptor molecules for estrogen and progesterone have been isolated and purified by affinity chromatography and characterized by PAGE and Western blot. Microsequencing of one of the membrane estrogen binding proteins indicates that the high-affinity site corresponds to the OSCP subunit of the proton ATP synthase. 5. It remains to be determined if P and T also bind to this complex enzyme or if they bind to other subunits of the family of proton ATPases. Overall the data indicate that steroid hormones conjugated to BSA are important tools to study the "reality of membrane steroid receptors."

Thyroid hormones modulate both adenosine transport and adenosine A1 receptors in rat brain

Adenosine transport and adenosine A1 receptors in rat brain are subjected to regulation by thyroid hormone levels. The studies were carried out with brain stem synaptosomal preparations from rat brain in euthyroid and various hypothyroid situations. The maximum velocity of the nitrobenzylthioinosine (NBTI)-sensitive adenosine transport was 3.3 +/- 0.3 pmol.mg protein-1.s-1 in euthyroid rats. The transport in 1-wk thyroidectomized rats was decreased by 45.8% with respect to controls. No changes were found in the affinity of euthyroid and hypothyroid rats, with the Michaelis-Menten constant values equal to 1.9 +/- 0.9 and 2.0 +/- 0.5 microM, respectively. The transporter number measured by NBTI binding also decreased; the maximum binding capacity (Bmax) was 112.9 +/- 21.9 and 31.3 +/- 4.1 fmol/mg protein for euthyroid and hypothyroid rats, respectively. The adenosine A1 receptors were measured in synaptosomal membrane preparations in the presence of 100 microM guanosine-5'-O-3-thiotriphosphate for cylopenthyl-1,3-dipropylxanthine 8-[dipropyl 2,3-3H(N)] ([3H]DPCPX) binding. In euthyroid rats, the Bmax value was 227.6 +/- 27.6 fmol/mg protein, a significant decrease of 23% was obtained in 1-wk hypothyroid rats. In all other thyroid situations studied, adenosine transport capacity, adenosine transporter number, and adenosine A1 receptor number were restored to control levels.

Characterization of binding sites of a memory-enhancing peptide AVP(4–8) in rat cortical synaptosomal membranes

The pentapeptide pGlu-Asn-Cyt-Pro-Arg [AVP(4–8), termed ZNC(C)PR in this article] has been found as a metabolite of arginine-vasopressin (AVP) in rat brain and has been shown to have potent memory-enhancing activity and to yield a series of biochemical events in brain tissues. [ 35S]ZNC(C)PR was chemically synthesized with high specific activity (232 Ci/mmol), and specific binding sites for ZNC(C)PR were located in synaptosomal membrane preparations of rat brain. We identified a specific binding site for ZNC(C)PR with a K d of 3.12 n M and B max of 31 fmol/mg protein in anterior cortical synaptosomal membranes in the presence of 5m M Ni 2+. A comparison of synthetic analogues of ZNC(C)PR competing with [ 35S]ZNC(C)PR for binding to anterior cortical receptor are presented. ZDC(C)PR, a 2-aspartyl substitute of the 2-asparaginyl residue in ZNC(C)PR, had the most potent competition, but AVP did not show significant ability to compete for the receptor with ZNC(C)PR.

Interconvertible kinetic states of t-butylbicycloorthobenzoate binding sites of the gamma-aminobutyric acidA ionophores.

The kinetics of t-[3H]butylbicycloorthobenzoate (TBOB) binding to the convulsant sites of the gamma-aminobutyric acidA (GABAA) receptor-ionophore complex were examined in synaptosomal membrane preparations of rat brain. On and off rates of TBOB binding were accelerated by 1 microM GABA and decelerated by 1 microM bicuculline methochloride, a GABAA antagonist. The presence of GABA and bicuculline methochloride created rapid and slow phases of dissociation, respectively. The three groups of rate constants distinguished for the dissociation of 4 nM and 30 nM [3H]TBOB represent multi-affinity states of the convulsant sites depending on the presence of GABA or bicuculline methochloride. Apparent association rate constants do not obey the equation k(app) = k(off) + k(on) [TBOB] without assuming interconvertibility of the kinetic states during binding. Avermectin B1a (AVM B1a), a chloride channel opening agent, accelerated the association and dissociation of TBOB and resulted in a biphasic effect on TBOB binding, i.e., enhancement at low concentrations (EC50 7.8 nM) followed by displacement at high concentrations (IC50 6.3 microM) of AVM B1a. AVM B1a resulted in similar biphasic effects on t-[35S]butylbicyclophosphorothionate binding. DIDS, an isothiocyanatostilbene derivative with irreversible anion channel blocking effect, selectively inhibited basal [3H]TBOB binding (IC50 125 microM DIDS) leaving the enhancement by AVM B1a unaffected.

Anandamide, an endogenous cannabimimetic eicosanoid, binds to the cloned human cannabinoid receptor and stimulates receptor-mediated signal transduction.

Arachidonylethanolamide (anandamide), a candidate endogenous cannabinoid ligand, has recently been isolated from porcine brain and displayed cannabinoid-like binding activity to synaptosomal membrane preparations and mimicked cannabinoid-induced inhibition of the twitch response in isolated murine vas deferens. In this study, anandamide and several congeners were evaluated as cannabinoid agonists by examining their ability to bind to the cloned cannabinoid receptor, inhibit forskolin-stimulated cAMP accumulation, inhibit N-type calcium channels, and stimulate one or more functional second messenger responses. Synthetic anandamide, and all but one congener, competed for [3H]CP55,940 binding to plasma membranes prepared from L cells expressing the rat cannabinoid receptor. The ability of anandamide to activate receptor-mediated signal transduction was evaluated in Chinese hamster ovary (CHO) cells expressing the human cannabinoid receptor (HCR, termed CHO-HCR cells) and compared to control CHO cells expressing the muscarinic m5 receptor (CHOm5 cells). Anandamide inhibited forskolin-stimulated cAMP accumulation in CHO-HCR cells, but not in CHOm5 cells, and this response was blocked with pertussis toxin. N-type calcium channels were inhibited by anandamide and several active congeners in N18 neuroblastoma cells. Anandamide stimulated arachidonic acid and intracellular calcium release in both CHOm5 and CHO-HCR cells and had no effect on the release of inositol phosphates or phosphatidylethanol, generated after activation of phospholipase C and D, respectively. Anandamide appears to exhibit the essential criteria required to be classified as a cannabinoid/anandamide receptor agonist and shares similar nonreceptor effects on arachidonic acid and intracellular calcium release as other cannabinoid agonists.

A specific membrane binding protein for progesterone in rat brain: sex differences and induction by estrogen.

Progesterone conjugated to bovine serum albumin (BSA) was used as a probe to study sex differences and the effects of hormonal status on binding of progesterone to crude synaptosomal membrane preparations (P2) derived from the mediobasal hypothalamic-anterior hypothalamic-preoptic area or the corpus striatum. Binding of 125I-labeled BSA linked to progesterone at the 11 position of the steroid (P-11-BSA) was decreased by competition with unlabeled P-11-BSA or P-3-BSA (in which progesterone is bound to BSA at the 3 position). P-3-BSA displayed higher affinity than P-11-BSA. Hypothalamic and striatal preparations from adult females show high specific binding (60-80%) to the progesterone-BSA conjugate. Specific binding was reduced more than 80% 14 days after ovariectomy. Estrogen treatment (10 micrograms per rat for 4 days) of 14-day ovariectomized rats restored specific binding to levels equivalent to intact females. In contrast, adult males displayed drastically reduced or no specific binding in either tissue. No specific binding was detected after orchidectomy. Estrogen treatment of orchidectomized animals induced specific binding sites similar to those in intact females. Additionally, an affinity probe was developed by linking primary amines on the P-3-BSA conjugate to agarose activated aldehydes in an AminoLink column. A digitoxin-solubilized fraction from female rat P2 cerebellum preparations yielded a single major band after affinity purification with an estimated molecular mass of 40-50 kDa in an SDS/PAGE system after silver stain. These results show a reversible sex difference in the specific binding of progesterone to synaptosomal membrane sites in the central nervous system of male and female rats which is dependent on estrogen.

Modification of benzodiazepine receptors supports the distinctive role of histidine residues

The effect of selective protein modifying reagents was examined on benzodiazepine (BZ) receptors in synaptosomal membrane preparations of rat whole brain and cerebellum. The potency of diethyl pyrocarbonate, a histidine modifying reagent, to inactivate BZ receptor binding, correlated with the rank order of agonist-inverse agonist efficacies of BZ ligands, the binding of the partial inverse agonist [ 3H]Ro 15–4513 was inactivated least. Diethyl pyrocarbonate slightly enhanced the displacing potency of Ro 15–4513 and enhanced its binding in low concentrations (1–2 mM). Diazepam-sensitive and -insensitive components of [ 3H]Ro 15–4513 binding were separated in cerebellum. Diethyl pyrocarbonate inactivated the diazepam-sensitive component with a potency (IC 50 = 1.8 mM) similar to that on the binding of other benzodiazepines, while the diazepam-insensitive component was resistant to diethyl pyrocarbonate. Tetranitromethane and 2,3-butanedione (diacetyl), reagents specific for tyrosine and arginine residues respectively, exerted concentration-dependent partial inactivation of [ 3H]Ro 15–4513 binding. The diazepam-insensitive component of cerebellar Ro 15–4513 binding was more sensitive to inactivation by diacetyl but less sensitive to inactivation by tetranitromethane. These findings are consistent with a distinctive role of histidine-101 in α 1, α 2, α 3 and α 5 subunits of the γ-aminobutyric acid A receptor complex and the His is replaced by an arginine residue in the α 6 subunit of the diazepam-insensitive cerebellar benzodiazepine receptors. The only other point of the protein sequence where histidine residues conserved in α 1, α 2, α 3 and α 5 subunits are replaced in α 6 is tyrosine-214 but this residue does not appear to contribute to benzodiazepine binding.

Characterization of EGTA-washed synaptosomal membrane with emphasis on its calmodulin-binding proteins. Demonstration of possible reconstitution with added calcium/calmodulin

Endogenous calmodulin (CaM) in the EGTA-washed cerebral-cortical synaptosomal membrane (SM) preparation was estimated below 3 μg/ml protein by the semiquantitative immunoblot analysis (Natsukari, N., Ohta, H. and Fujita, M. (1989) J. Immunol. Methods 125, 159–166). Membrane-bound CaM was immunoelectron-microscopically demonstrated in EGTA-washed, non-treated (control), and Ca 2+-treated cerebral-cortical synaptosomal membranes (SM) as well as for the SM enriched with added CaM. The density of CaM increased in the above order. CaM-dependent adenylate cyclase and CaM-dependent protein kinase II (CaM-kinase II) activities were restored, whereas the phosphodiesterase (PDE) activity was not affected by exogenous CaM over all the Ca 2+ concentrations tested. Adenylate cyclase at p Ca 6.2 was synergistically activated either by GTP and CaM or by CaM and β-adrenergic agonist, (±)-isoproterenol, reflecting the intactness of signal transduction pathway in the SM. Also demonstrated were the presence of protein kinase A, CaM-kinase II, and their endogenous substrates in the SM. Based on 32P-autoradiography and 125I-CaM overlay data certain CaM-binding proteins such as CaM-kinase II and synapsin I were identified on SDS-PAGE. Ca 2+-dependent and -independent CaMBPs were distinguished by 125I-CaM gel overlay with and without Ca 2+. The former had bigger molecular size (≧ 49 kDa) than the latter (≦ 34 kDa). Yield of Ca 2+-dependent CaMBPs was not affected by Ca 2+ concentration during preparation of the SM while that of Ca 2+-independent CaMBI's was reduced by exposure to 100 μM Ca 2+. In contrast with the CaMBPs of brain SM, those of enterocyte and erythrocyte plasma membranes especially, microvillous membrane of the enterocyte, showed quite distinct CaMBP profiles. The present findings suggested that the EGTA-washed SM preparation made a useful system for studying the role of CaM in the brain SM.