Rat Brain Synaptic Plasma Membranes Research Articles

125I]margatoxin, an extraordinarily high affinity ligand for voltage-gated potassium channels in mammalian brain.

Monoiodotyrosine margatoxin ([125I]MgTX) specifically and reversibly labels a maximum of 0.8 pmol of sites/mg of protein in purified rat brain synaptic plasma membrane vesicles with a dissociation constant of 0.1 pM under equilibrium binding conditions. This Kd value was confirmed by kinetic experiments (Kd of 0.07 pM), competition assays employing native margatoxin (MgTX) (Ki of 0.15 pM), and receptor saturation studies (Kd of 0.18 pM). Thus, this toxin represents the highest affinity, reversible radioligand for any membrane-bound receptor or ion channel described to date. [125I]MgTX binding in this system is modulated by charybdotoxin (Ki of 5 pM), kaliotoxin (Ki of 1.5 pM), and the agitoxins I and II (Ki's of 0.1 and 0.3 pM, respectively), in a noncompetitive manner. Moreover, alpha-dendrotoxin displayed a Ki value of 0.5 pM. Iberiotoxin was without any effect, suggesting that the receptor site is likely to be associated with a voltage-gated K+ channel complex. [125I]MgTX binding is inhibited by cations that are established blockers of voltage-dependent K+ channels (Ba2+, Ca2+, Cs+). The monovalent cations Na+ and K+ stimulate binding at low concentrations before producing complete inhibition as their concentrations are increased. Stimulation of binding results from an allosteric interaction that decreases Kd, whereas inhibition is due to an ionic strength effect. Affinity labeling of the binding site in rat brain synaptic plasma membranes employing [125I]MgTX and the bifunctional cross-linking reagent, disuccinimidyl suberate, causes specific and covalent incorporation of toxin into a glycoprotein of an apparent molecular weight (M(r)) of 74,000. Deglycosylation studies reveal an M(r) for the core polypeptide of the MgTX receptor of 63,000.(ABSTRACT TRUNCATED AT 250 WORDS)

Nitrous oxide and xenon enhance phospholipid-N-methylation in rat brain synaptic plasma membranes

Halothane and isoflurane increase the rate of phospholipid methylation (PLM) in rat brain synaptosomal membranes, a process linked to the coupling of neuronal excitation to neurotransmitter release. In contrast, synaptic plasma membrane (SPM) Ca 2+ ATPase (PMCA) pumping is reduced by exposure to halothane, isoflurane, xenon and nitrous oxide (N 2O). To examine further the relationship between PLM, PMCA and anesthetic action, we investigated the effect of clinically relevant concentrations of two less potent anesthetic gases, N 2O and xenon, on PLM in SPM. Biochemical assays were performed on SPM exposed to 1.3 MAC of N 2O (2 atm), 1.3 MAC of xenon (1.23 atm) or an equivalent pressure of helium for control. N 2O or xenon exposure increased PLM to 115% or 113%, respectively, of helium control (p < 0.02). Similar exposures to N 2O or xenon depressed PMCA activity to 78% and 85% of control (p < 0.05). Observations that PLM and PMCA are both altered by a wide variety of inhalation anesthetic agents at clinically relevant partial pressures lend support to a possible involvement and interaction of these processes in anesthetic action.

Diminished brain synaptic plasma membrane ca 2+-atpase activity in spontaneously hypertensive rats: Association with reduced anesthetic requirements

We have recently reported that plasma membrane Ca 2+-ATPase ( PMCA) pumping activity in rat brain synaptic plasma membranes (SPM) was reduced by in vitro or prior in vivo exposure to inhalation anesthetics (IA). In addition, rats with streptozocin-induced diabetes were found to have diminished brain synaptic PMCA pumping and a decrease in the partial pressures of several IA required to prevent movement in response to stimulation, defined as the minimum effective dose or MED. Diminished PMCA activity in erythrocytes of spontaneously hypertensive rats (SHR) has been noted. Because PMCA is ubiquitous, it seemed possible that PMCA pumping might be decreased in the brain of SHR and perhaps associated with decreased IA requirement. Eighteen SHR and 18 control, normotensive Wistar-Kyoto rats (WKY) were studied. PMCA activity was assessed by measurement of Ca 2+ uptake into synaptic plasma membrane vesicles prepared from cerebrum and diencephalon-mesencephalon (D-M) in WKY and SHR. Ca 2+ pumping was significantly less in SHR than in WKY, 85% of control in the cerebrum and 90% in the D-M (p < 0.01). The MEDs for halothane, isoflurane and desflurane were also lower in SHR than in WKY, 91%, 90% and 89%, respectively, of control (p < 0.05). Thus, an animal model of primary hypertension (SHR) manifested diminished brain synaptic PMCA activity and reduced MED for several volatile anesthetics. These findings provide further evidence for a role for PMCA in anesthetic action.

Reduced anesthetic requirements, diminished brain plasma membrane Ca(2+)-ATPase pumping, and enhanced brain synaptic plasma membrane phospholipid methylation in diabetic rats: effects of insulin.

We have recently reported that streptozocin (STZ)-induced diabetes in rats was associated with i) reduced Ca2+ pumping by rat brain synaptic plasma membrane Ca(2+)-ATPase (PMCA) and ii) a substantial reduction in the partial pressures of halothane and xenon required to prevent movement in response to stimulation (minimum effective dose or MED). MED for both agents correlated well with the degree of hemoglobin glycation and with PMCA activity. We now report that MEDs for isoflurane, enflurane, and desflurane were also substantially reduced in STZ-diabetic rats, compared with placebo-injected controls. In addition, we examined the effect of insulin treatment, begun 2 weeks after induction of diabetes and continued for 3 more weeks, on isoflurane MED and on brain synaptic PMCA and phospholipid-N-methyltransferase I (PLMT I), another enzyme altered by inhalation anesthetics (IA). Partial treatment of diabetes, as indicated by decreased glycated hemoglobin (GHb) compared to untreated diabetic rats, was associated with an isoflurane MED of 1.05 vol%, intermediate between a control mean of 1.57 vol% and an untreated diabetic mean of 0.82 vol% (p < 0.01), with a trend toward normalization of both PMCA and PLMT I activity. We also examined isoflurane MED and PMCA activity in the cerebrum and diencephalon-mesencephalon (D-M) of control and diabetic rats 2 and 12 weeks after induction of diabetes. Isoflurane MED was substantially reduced in diabetic rats from both treatment periods. Cerebral and D-M PMCA activities were each reduced to about 90% of control values 2 weeks after STZ induction. At 12 weeks, cerebral PMCA pumping in SPM from diabetic rats did not differ from control values, but PMCA pumping in SPM from the D-M was reduced to about 85% of control levels. Good correlation (r = 0.89, p < 0.01) was found between isoflurane MED and GHb in all treatment groups. These findings provide further evidence for an important role for PMCA in IA action. They also suggest that anesthetic effects on the calcium pump at specific anatomic sites may be of major importance in producing anesthesia.

The asymmetric distribution of phosphatidylcholine in rat brain synaptic plasma membranes

The distribution of phosphatidylcholine between inner and outer monolayers of rat brain synaptic plasma membrane was investigated by means of a phosphatidylcholine specific exchange protein. About 70% of the total membranal phosphatidylcholine was in the outer leaflet, 33% of which was exposed and readily exchanged in intact synaptosomes while the remainder was exchangeable following osmotic shock. Permeabilization of the synaptic plasma membranes by overnight incubation in buffer or by saponin (<0.08%) exposed an additional 30% of phosphatidylcholine to exchange, presumably from the inner cytoplasmic leaflet. Phosphatidylcholine is therefore asymmetrically distributed in the synaptosomal plasma membrane, as it is in other plasma membranes.

Substrate specificity of neutral phospholipase D from rat brain studied by selective labeling of endogenous synaptic membrane phospholipids in vitro.

We have designed a novel approach for studying the specificity of neutral phospholipase D from rat brain synaptic plasma membranes for endogenous phospholipid substrates in native membranes. A procedure was established that provides synaptic membranes labeled in selected phospholipids. This labeling procedure exploits the presence of endogenous acyl-coenzyme A synthetase and acyl-coenzyme A:lysophospholipid acyltransferase in synaptosomes for acylating various lysophospholipid acceptors with radioactive fatty acid. With [3H]arachidonate for acylation and optimal concentrations of the respective lysophospholipids, membranes were labeled in either of the following phospholipids: phosphatidylcholine (93% of total label in phospholipids), 1-O-alkyl-phosphatidylcholine (87%), phosphatidylinositol (90%), phosphatidylethanolamine (85%), phosphatidylethanolamine-plasmalogen (81%) or phosphatidylserine (59%). These membranes were employed to study the substrate specificity of the neutral, oleate-activated rat brain phospholipase D. This phospholipase exhibited almost absolute specificity for the choline-phospholipids phosphatidylcholine and 1-O-alkyl-phosphatidylcholine: 0.34% of the former labeled substrate were transphosphatidylated to phosphatidylpropanol during the assay and 0.28% of the latter. Activity toward other phospholipids was barely detectable and could largely be accounted for by utilization of residual labeled phosphatidylcholine present in those preparations. The phospholipase D exhibited some preference for fatty acids in the C-2 position of phosphatidylcholine in the following order: 2-oleoyl-phosphatidylcholine (0.67% of this labeled phosphatidylcholine were converted to phosphatidylpropanol), 2-myristoyl-phosphatidylcholine (0.60%), 2-palmitoyl-phosphatidylcholine (0.46%) and 2-arachidonoyl-phosphatidylcholine (0.34%). The present approach of labeling membrane phospholipids in vitro could be useful in studies of phospholipase specificity as an alternative to the use of sonicated vesicles or mixed detergent-phospholipid micellar systems.

Immunologic identification of [formula omitted] exchange protein in rat brain synaptic plasma membrane

Polyclonal antibodies raised against partially purified dog cardiac Na Ca exchanger react with cardiac sarcolemmal proteins of 160, 120 and 70 kDa on SDS-PAGE. Using the same specific antiserum, we detected three prominent immunoreactive bands of about 150, 120 and 70 kDa on immunoblots with rat forebrain synaptic plasma membrane proteins. These data indicate that the Na Ca exchange protein in rat brain synaptic plasma membrane is structurally and antigenically similar to the exchange protein in dog cardiac sarcolemma.

Effects of acromelic acid A on the binding of [3H]‐kainic acid and [3H]‐AMPA to rat brain synaptic plasma membranes

1. The ability of acromelic acid A to inhibit [3H]-kainic acid and [3H]-(RS)-alpha-amino-3-hydroxy-5-methyloxazole-4-propionic acid ([3H]-AMPA) binding to rat brain synaptic plasma membranes was investigated by equilibrium radioligand binding assay. 2. Kinetic analysis of [3H]-kainic acid binding demonstrated the existence of two kainate binding sites in this tissue preparation and yielded equilibrium dissociation constants for [3H]-kainic acid of KD = 0.4 nM and KD = 20.8 nM. 3. Kainic acid and domoic acid both appeared to displace [3H]-kainic acid from a single binding site with equilibrium binding constants of KD = 19.4 nM and Ki = 14.5 nM respectively. Acromelic acid A exhibited a biphasic inhibition of [3H]-kainic acid binding to synaptic membranes with binding affinities of Ki = 15.1 nM and Ki = 1.49 microM. 4. In the absence of chaotropic ions, the order of potency of inhibition of [3H]-AMPA binding was acromelic acid A (Ki = 26 nM) greater than AMPA (KD = 184 nM) greater than domoic acid (Ki = 499 nM). 5. The inclusion of 100 mM thiocynanate ion in the [3H]-AMPA binding assay resulted in a change in the order of potency to: AMPA (KD = 160 nM) greater than acromelic acid A (Ki = 289 nM) greater than domoic acid (Ki = 9.02 microM). 6. These results show that acromelic acid A distinguishes two kainate binding sites in rat brain synaptic plasma membranes and in addition, that in the absence of chaotropic ions, acromelic acid A is the most potent displacer of [3H]-AMPA binding yet described.

The inhibitory effect of Mn 2+ on the ATP-dependent Ca 2+ pump in rat brain synaptic plasma membrane vesicles

Synaptic plasma membrane (SPM) vesicles take up Ca 2+ when both ATP and Mg 2+ are added to the reaction medium. Maximal ATP-dependent Ca 2+ uptake is obtained with between 3 and 5mM Mg 2+. Higher [Mg 2+] results in a slight decrease in the ATP-dependent Ca 2+ influx, which at 10 mM Mg 2+ is equal to 70% of the maximal uptake. [Mn 2+] up to 0.3 mM, Ca 2+n support ATP-dependent Ca 2+ uptake as efficiently as equimolar [Mg 2+]. Maximal ATP-dependent Ca 2+ uptake in the presence of Mn 2+ is obtained between 1 and 2 mM but its extent is only 70% of the maximal uptake obtained in the presence of Mg 2+. As the [Mn 2+] increases the ATP-dependent Ca 2+ uptake decreases and at 10 mM Mn 2+ it is only 40% of that obtained in the presence of Mg 2+. The reduction in ATP-dependent Ca 2+ uptake in the presence of Mn 2+ is obtained also when Mg 2+ is present in the reaction medium. The effect is independent of the [Mg 2+] used and depends on the [Mn 2+ ] alone. Studying the relationship between [Mn 2+] and [Ca 2+] revealed three patterns: at 0.3 mM Mn 2+ this ion could replace Mg 2+ over the entire [Ca 2+] range tested, 5–200 μM, supporting ATP-dependent Ca 2+ uptake. When the [Mn 2+] was increased to 1.8 mM, it exhibited a competitive behaviour with Ca 2+ which resulted in an increase in apparent K m to Ca 2+ of the SPM Ca 2+ pump from 10.69 μM (SD = 3.49) to 28.88 μM (SD = 21.08). Four millimolar Mn 2+ inhibited ATP-dependent Ca 2+ uptake to 50% of that obtained in equimolar Mg 2+ over the entire [Ca 2+], 5-200 μM, tested. No transport of 54Mn 2+ by the SPM Ca 2+ pump could be detected. Inclusion of Mn 2+ in the reaction medium led to excessive phosphorylation of SPM proteins. The excessive levels of phosphorylation persisted also when Ca 2+ and/or Mg 2+ were present in the reaction media.

Na +-Ca 2+ exchange activity in synaptic plasma membranes derived from the electric organ of Torpedo ocellata

Synaptic plasma membranes obtained by hypo-osmotic treatment of purified Torpedo ocellata synaptosomes, contain an electrogenic Na +-Ca 2+ exchange system. The dependence ofof the initial reaction rate on [Ca 2+] reveals a single binding site for Ca 2+ with an average apparent K m of 13.66 (S.D.= 12.07) μM [Ca 2+] and maximal reaction velocity of V max = 11.33 (S.D.= 5.93) nmol/mg protein per s. The dependence of the initial rate of the Na + gradient dependent Ca 2+ influx on the internal [Na +] exhibits a sigmoidal curve which reaches half-maximal reaction rate at 170.8 (S.D.=19.9) mM [Na +]. Addition of ATPγS does not change the K 0.5 to Na +. The average Hill coefficient is 3.09 (S.D.=0.86) indicating that 3–4 Na + ions are exchanged for each Ca 2+. Na + gradient dependent Ca 2+ uptake in Torpedo SPMs takes place also in the absence of K + suggesting that K + co-transport is not obligatory. The temperature dependence of the initial and steady-state rates of Na + gradient dependent Ca 2+ influx reveal that maximal reaction velocities of the Torpedo exchanger are attained between 15 and 20°C. The energy of activation between 0 and 20°C is 20 826 cal/mol. In comparison, rat brain synaptic plasma membrane Na + Na 2+ exchanger reaches maximal reaction rates between 30 and 40°C. Reconstitution of Torpedo or rat brain Na +-Ca 2+ exchangers into a membrane composed of either Torpedo or brain phospholipids, does not alter the temperature dependence of the native Torpedo or rat brain Na +-Ca 2+ exchangers; inspite of considerable differences in the composition of the fatty acyl chains that are esterified to brain and Torpedo phospholipid head groups and differences in membrane fluidity that were detected. An ATP-dependent Ca 2+ pump, which is insensitive to FCCP, is also present in the same synaptic membrane.

The modulation of rat brain Na(+)-Ca2+ exchange by K+

The involvement of potassium ions in the Na(+)-Ca2+ exchange process was studied in rat brain synaptic plasma membrane (SPM) vesicles. Addition of equimolar [K+] to the intravesicular and the extravesicular medium led to a stimulation of the Na+ gradient-dependent Ca2+ influx; this stimulation was noticeable already at 0.5 mM and reached its maximum at 2 mM K+. The magnitude of the K+ stimulation was between 1.3-2.5-fold in different SPM preparations. K+ ions also stimulated the Na(+)-dependent Ca2+ efflux. K+ stimulation of Na(+)-Ca2+ exchange is of considerable specificity, since it is not mimicked by either Li+ or H+. The following lines of evidence suggest that K+ modulation of Na(+)-Ca2+ exchange involves the catalytic moiety of the transporter itself and not an unrelated K+ channel which modulates the membrane potential. 1) K+ stimulation of the transport process was conserved following reconstitution of the transporter into phospholipid-rich liposomes, an experimental condition which presumably separates the native membrane proteins among different vesicular structures. 2) K+ stimulation of Na+ gradient-dependent Ca2+ influx persists also when the build up of negative inside membrane potential is prevented by addition of carbonyl cyanide p-trifluoromethoxy phenylhydrazone which renders the membrane highly permeable to protons both in the native and the reconstituted preparation. 3) K+ stimulation of Na+ gradient-dependent Ca2+ influx is obtained also when tetraethylammonium chloride, 2,3-diaminopyridine and Cs+ are added to the Ca2+ uptake medium. Reconstituted SPM vesicles take up 86Rb+ in response to activation of Na+ gradient-dependent Ca2+ influx. The ratio of Ca2+ taken up by SPM vesicles in a Na+ gradient-dependent manner to the corresponding amounts of Rb+ taken up varies between 8-5 in different SPM preparations. If the stoichiometry of the process is 1 Rb+/1 Ca2+, then Rb+ cotransport is mediated by 10-20% of the transporters present in the preparation.

Inhibition of CA2+Efflux by Pyridine Nucleotides

The effects of pyridine nucleotides on the Mg-dependent ATP-stimulated Ca2+ pump and on the ATP-independent Na(+)-Ca2+ exchanger were investigated in rat brain synaptic plasma membranes. Both Ca2+ efflux mechanisms are inhibited by pyridine nucleotides, in the order NADPH greater than NADP greater than NADH greater than NAD with IC50 = ca. 3-4 mM for NADP or NADPH and ca. 5 mM for the other pyridine nucleotides in the case of the ATP-driven Ca(2+)-pump, and with IC50 = 8 to 10 mM for the Na(+)-Ca2+ exchanger. Oxidizing agents such as DCIP or FeCN also affect the Ca(2+)-efflux mechanisms. DCIP and FeCN inhibit the ATP-driven Ca2+ pump but not the Na(+)-Ca2+ exchanger. Inhibition of the ATP-dependent Ca2+ pump is optimal when both a reduced pyridine nucleotide and an oxidizing agent (e.g. DCIP or FeCN) were added together. Under similar experimental conditions the pyridine nucleotide-mediated inhibition of the Na(+)-Ca2+ exchanger is partially removed. Therefore Ca(2+)-efflux mechanisms appear to be controlled in part through the redox environment, probably by means of transplasma membrane dehydrogenases.

The expression of rat brain synaptic plasma membrane Na +-Ca 2+ exchange activity in Xenopus oocytes

Injection of Xenopus oocytes with mRNA isolated from 1-day-old rat brains leads to expression of Na + gradient-dependent Ca 2+ uptake activity. Size fractionation of the mRNA by sucrose density gradient centrifugation reveals that an mRNA fraction enriched in 14–18 S mRNA is responsible for the transport activity. Plasma membrane proteins isolated from oocytes injected with total or 14–18 S enriched fraction of rat brain mRNA contain proteins of about 70-kDa molecular mass recognized by a polyclonal antibody prepared against the purified 70-kDa rat brain Na +-Ca 2+ exchanger. In control H 2O-injected oocytes, no proteins are recognized by the anti-70-kDa antibody.

A neutral phospholipase D activity from rat brain synaptic plasma membranes. Identification and partial characterization.

Rapid activation of phospholipase D (PLD) in response to cell stimulation was recently demonstrated in many systems, raising the hypothesis that PLD participates in transduction of extracellular signals across the plasma membrane. In the present study, we describe the identification of a neutral PLD activity in purified rat brain synaptic plasma membranes, and the in vitro conditions required to assay its catalytic activity with exogenous [3H]phosphatidylcholine as substrate. Production of [3H]phosphatidic acid, the natural lipid product of PLD and of [3H]phosphatidylethanol, catalyzed by PLD in the presence of ethanol via transphosphatidylation, were measured. The synaptic membrane PLD exhibited its highest activity at pH 7.2 and was thus defined as a neutral PLD. Enzyme activity was absolutely dependent on the presence of sodium oleate and was strongly activated by Mg2+ ions (at 1 mM). Ca2+ at concentrations up to 0.25 mM was as stimulatory as Mg2+, but at 2 mM it completely inhibited enzyme activity. Mg2+ extended the linear phase of PLD activity from 2 to 15 min, suggesting that it may stabilize the enzyme under our assay conditions. The production of [3H]phosphatidylethanol was a saturable function of ethanol concentration. Production of [3H] phosphatidic acid was inversely related to the concentration of ethanol and to the accumulation of phosphatidylethanol, indicating that the two phospholipids are indeed produced by the competing hydrolase and transferase activities of the same enzyme. beta,beta-Dimethylglutaric acid, utilized previously as a buffer in studies of rat brain PLD, inhibited enzyme activity at neutral pH but not at acidic pH. The properties of the neutral synaptic membrane PLD and its relationships with other in vitro, acid, and neutral PLD activities, as well as with the signal-dependent PLD detected in intact cells, are discussed.

Role of the phospholipid environment in modulating the activity of the rat brain synaptic plasma membrane calcium ATPase

The role of the phospholipid environment in modulating the activity of the rat brain synaptic plasma membrane (SPM) Ca2(+)-ATPase was investigated by its reconstitution into different phospholipids. Retention of activity of the solubilized Ca2(+)-ATPase depended on addition of exogenous phospholipids. As the cholate concentration used for solubilization of native SPM increased, a larger excess of exogeneous phospholipids, relative to membrane protein, had to be added to maintain optimal activity. Highest ATP-dependent Ca2+ transport activity was obtained when reconstitution was carried out in calf brain phospholipids (BPLs) followed by soybean phospholipids (SPLs) and the lowest in egg PC; reconstitution at a 40:1 weight ratio of exogenous phospholipids to native SPM protein resulted in ATP-dependent Ca2+ transport of 40.0 +/- 4.16, 23.4 +/- 8.48, and 11.54 +/- 2.31 nmol of Ca2+ (mg of protein)-1 (5 min)-1, respectively. Partial substitution of egg PC with BPLs led to an increase in the activity of the reconstituted Ca2+ pump. The highest ATP-dependent Ca2+ uptake was obtained when ratios of 15:25 or 10:30 egg PC to BPLs were used. Testing the individual phospholipids participating in the BPL mixture showed that addition of PS to egg PC led to a consistent increase in Ca2+ pump activity. Substitution of 50% of the PC with PS resulted in a 3.8-fold higher ATP-dependent Ca2+ uptake than that obtained in egg PC alone. No other phospholipid tested--PE, SM, or PI--had a similar effect. Increasing the proportion of PS within the BPL mixture above its original content led to a gradual decrease in the reconstituted SPM Ca2+ pump activity. Enrichment of asolectin with PS led first to increased Ca2+ pump activity; then, as the proportion of PS increased, Ca2+ transport of the reconstituted pump decreased. An increased proportion of PE, SM, or PI within the BPLs or asolectin, above their original contents, resulted in decreased Ca2+ transport. These results indicate that optimal SPM Ca2+ pump activity requires the combined presence of a critical amount of PC and PS within the reconstituted membrane.

Evidence for a functional coupling of the NMDA and glycine recognition sites in synaptic plasma membranes

Activation of the N-methyl-D-aspartate (NMDA) receptor complex is subject to modulation via interactions at a coupled [ 3H]glycine recognition site in rat brain synaptic plasma membranes (SPM). We examined the effect of the potent and specific glycine site antagonists, 1-hydroxy-3-amino-2-pyrrolidone (HA-966) and 1-aminocyclobutane-1-carboxylate (ACBC), on the NMDA recognition site. These glycine analogs were found to significantly stimulate the binding of the competitive NMDA antagonist, [ 3H]3-(2-carboxypiperazin-4-yl)propyl-1-phosphonate ([ 3H]CPP) in a dose-dependent fashion, whereas both compounds inhibited NMDA-specific L-[ 3H]glutamate (agonist) binding. Additionally, both glycine antagonists reduced the binding of [ 3H]1-[1-(2-thienyl)cyclohexyl]piperidine ([ 3H]TCP) to SPM, a functional assessment of activation of the NMDA receptor-channel complex. The glycine site agonists, glycine and serine reversed these effects in a dose-dependent manner, with the serine reversal being stereospecific for D-serine. The relative potencies of these compounds in reversing the glycine antagonist effects on the NMDA recognition site corresponded with their ability to competitively displace strychnine-insensitive [ 3H]glycine binding. These results provide evidence for a functional coupling between the glycine and NMDA recognition sites and further, may provide a mechanism by which compounds interacting at the glycine recognition site may modulate NMDA receptor activity.

Docichol alters GABA uptake and high affinity binding of agonist to rat brain synaptic plasma membranes

The effects of dolichol on high affinity [3H]muscimol binding to synaptic plasma membrane (SPM) and [3H]GABA uptake into synaptosomes from rat brain were analyzed. Membranes were enriched with dolichol, by preincubation, in the presence of bovine serum albumin (BSA) as a vehicle (40-100 micrograms of dolichol/mg protein + 5% BSA). The rate of dolichol incorporation into the membrane was determined using [1-3H]dolichol C95, and it was in the range of 5-7 nmol/mg protein/h for synaptosomes and SPM, respectively. The uptake of [3H]GABA into synaptosomes enriched with dolichol decreased significantly by about 30%. Dolichol alone added into the incubation medium produced only a negligible effect. Specific binding of [3H]muscimol, which was higher than 90% of total binding, was significantly reduced to SPM enriched with dolichol as compared to controls. The Kd of the high affinity sites was significantly elevated by about 30% in SPM enriched with dolichol (10.8 +/- 0.3 nM vs 7.3 +/- 0.2 nM in control). This difference was more pronounced for SPM isolated from cerebellum (Kd increased by about 50%). The Bmax value was not changed. Dolichol alone did not alter the agonist binding. These results indicate for the first time that the higher level of dolichol in SPM might influence the GABAergic transmission system. An increase in dolichols in membranes may be an important factor in the decline of brain function during aging.

Glutamate Transport and Not Glutamate Receptor Binding Is Stimulated by Gangliosides in a Ca2+‐Dependent Manner in Rat Brain Synaptic Plasma Membranes

Crude as well as purified synaptic plasma membrane (SPM) preparations were analyzed for the influence of the ganglioside galactosyl-N-acetylgalactosaminyl-(N-acetylneuraminyl)-galactosylgluc osyl ceramide (GM1) on high-affinity binding of L-[3H]glutamate. Assayed in two different buffer systems, SPM consistently exhibited increased (40-50%) binding upon incubation with GM1 plus Ca2+, as compared to controls without GM1. Incorporation experiments with 3H-labeled GM1 proved trypsin-stable insertion of GM1 into SPM, with a maximum incorporation of four times the endogenous amount (35 nmol/mg of protein). The observed increase in glutamate binding was not due to a change in the affinity of the binding sites, but to a change in the number of binding sites, and it was absolutely dependent on the presence of Ca2+. A pharmacological profile of the GM1/Ca2+-stimulated glutamate binding is presented. The original classification of the stimulatory effect as an effect on glutamate receptor binding had to be revised to take into account the observed temperature sensitivity of the ganglioside effect, its sensitivity to high osmolarity and to ultrasonication, and the lack of binding stimulation after detergent treatment of membranes or after receptor solubilization. Vesicular space measured in both SPM preparations was found to be around 7 microliters/mg of protein, in ganglioside-treated as well as in control membranes. From the data, it is concluded that a special, Na+- and Cl- -independent form of glutamate transport into resealed membrane vesicles is stimulated by gangliosides in the presence of Ca2+.

Characterization df a [3H]Glycine Recognition Site as a Modulatory Site of the N‐Methyl‐D‐Aspartate Receptor Complex

A [3H]glycine recognition site in rat brain synaptic plasma membranes (SPM) has been identified, having characteristics expected of a modulatory component of the N-methyl-D-aspartate receptor complex. Incubation of SPM with [3H]glycine for 10 min at 2 degrees C results in saturable, reversible binding with a KD of 0.234 microM and a Bmax of 9.18 pmol/mg. A pharmacological analysis of this binding site indicates that D-serine (Ki = 0.27 microM), D-alanine (Ki = 1.02 microM), and D-cycloserine (Ki = 2.33 microM) are potent inhibitors of binding, whereas the corresponding L isomers have significantly less activity (Ki = 25.4 microM, 15.9 microM, and greater than 100 microM, respectively). Inactive at concentrations of up to 100 microM were strychnine, L-valine, N,N-dimethylglycine, aminomethylphosphonate, and aminomethylsulfonate. The active compounds were analyzed further for their ability to stimulate [3H]1-[1-(2-thienyl)cyclohexyl]piperidine [( 3H]TCP) binding to Triton X-100-washed SPM. Results indicate that the affinity of the compounds for the [3H]glycine recognition site correlates with the ability of these analogues to stimulate [3H]TCP binding.

Characterization and Partial Purification of a Chloride- and Calcium-Dependent Glutamate-binding Protein from Rat Brain

A glutamate-binding protein was solubilized from rat brain synaptic plasma membranes using sodium cholate. Its properties were characterized after addition of exogenous phospholipids and formation of proteoliposomes. Glutamate binding was dependent on calcium and chloride ions with maximal binding at concentrations of 10(-5) M calcium and 10 mM chloride ions. The effects of the two ions were synergistic rather than additive. In addition, glutamate binding was not affected by inhibitors specific for N-methyl-D-aspartate and kainate receptor subtypes, but was inhibited by quisqualate (Ki = 50 microM) and DL-2-amino-4-phosphonobutyrate (Ki = 1.3 mM). Furthermore, binding was abolished by 100 microM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid and 1 mM dithiothreitol. These properties resemble those of the chloride- and calcium-dependent binding site. Starting from the detergent extract, the glutamate-binding protein was purified 123-fold using fractionated ammonium sulfate precipitation, chromatography on hydroxyapatite and on DEAE-Sephacel as sequential purification steps. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified protein fraction showed two major bands migrating with Mr values of 51,000 and 105,000. The properties of the partially purified binding protein were similar to those of the detergent extract. Glutamate binding to the partially purified protein is not due to a sequestration process or product binding to N-acetylated alpha-linked dipeptidase. Thus, the functional role of the binding protein remains to be established.