COMPARATIVE STUDIES ON THE CHARACTERISTIC PROPERTIES OF TWO FORMS OF BRAIN ACTIN SEPARABLE BY ISOELECTRIC FOCUSSING

Abstract— Actin isolated from 10-day embryonic chick brain produced a single band indistinguishable from that of muscle actin when electrophoresed in sodium dodecyl sulfate-polyacrylamide gel. Isoelectric focussing in polyacrylamide gel showed that the isolated protein was composed of two components–the β and γ forms of actin previously detected in other mammalian nonmuscle cells and tissues–in a molar ratio of 1.1/1.0. The same ratio was observed in a sonicate of 10-day embryonic chick brain and in various actin-containing fractions at each step of an actin purification procedure which involved gel filtration chromatography and polymerization-depolymerization of the protein. Additionally, the two forms of actin were found to co-precipitate with muscle myosin and to bind to a DNase I-agarose affinity column in this ratio. In contrast, a third isoelectrically distinct protein with the same electrophoretic mobility as actin was found in the whole brain sonicate, but not in any of the actin-containing fractions examined. When cellular protein in neuronal and non-neuronal cell populations isolated from 10-day embryonic chick brain was analyzed, it was found that β and β actins were also present in each class of cells in the molar ratio of 1.1/1.0. However, this ratio decreased slightly during development of the chick brain. We conclude from this study that the β and γ forms of brain actin are very similar in several characteristic properties of actin, and that it is unlikely that brain cells utilize different forms of the protein for different types of cell motility.     

Molecular Determinants of General Anesthetic Action: Role of GABAA Receptor Structure

Abstract: Using receptors expressed from mouse brain mRNA in Xenopus oocytes, we found that enhancement of type A γ-aminobutyric acid (GABA_A) receptor-gated Cl⁻ channel response is a common action of structurally diverse anesthetics, suggesting that the GABA_A receptor plays an important role in anesthesia. To determine if GABA_A receptor subunit composition influences actions of anesthetics, we expressed subunit cRNAs in Xenopus oocytes and measured effects of enflurane on GABA-activated Cl⁻ currents. Potentiation of GABA-activated currents by enflurane was dependent on the composition of GABA_A receptor protein subunits; the order of sensitivity was α₁β₁ > α₁β₁γ_2s=α₁β₁γ_2L > total mRNA. The results suggest that anesthetics with simple structures may act on the GABA_A receptor protein complex to modulate the Cl⁻ channel activity and provide a molecular explanation for the synergistic clinical interactions between benzodiazepines and general anesthetics.     

RAT BRAIN TUBULIN: SUBUNIT HETEROGENEITY AND PHOSPHORYLATION

Abstract— Evidence for multiple forms of the α and β subunits of tubulin isolated from rat brain has been obtained by means of SDS polyacrylamide gel electrophoresis, isoelectric focusing, and SDS hydroxylapatite column chromatography. Fourteen distinct bands, localized near pH 5.4, were formed when tubulin was subjected to isoelectric focusing in a gradient established with a very narrow range ampholyte mixture. Three tubulin subunits, a₁., α₂, and β, were separated by sodium dodecyl sulfate polyacrylamide slab gel electrophoresis in a second dimension. The β subunit was more acidic than the α subunits. Brain sections were incubated in tissue culture medium containing ³²P₁ and radiolabeled tubulin was subsequently isolated and subjected to electrophoresis. Only the β subunit was labeled. All radioactivity was associated with two or three adjacent bands on isoelectric focusing gels.     

The Subunit Composition of a GABAA/Benzodiazepine Receptor from Rat Cerebellum

Abstract: The pentameric subunit composition of a large population (36%) of the cerebellar granule cell GABA_A receptors that show diazepam (or clonazepam)-insensitive [³H]Ro 15-4513 binding has been determined by immunoprecipitation with subunit-specific antibodies. These receptors have α₆, α₁, γ_2S, γ_2L, and β₂ or β₃ subunits colocalizing in the same receptor complex.     

UPTAKE OF [14C]ASPARTIC ACID AND [14C]GLUTAMIC ACID BY RETINAL SYNAPTOSOMAL FRACTIONS

Abstract— Uptake systems for [¹⁴C]aspartate and [¹⁴C]glutamate were characterized in two distinct synaptosomal fractions solated from rabbit retina. The P, synaptosomal fraction was highly enriched in large photoreceptor cell synaptosomes but contained very few conventional sized synaptosomes from amacrine, horizontal or bipolar cells. In contrast, the P₂ synaptosomal fraction contained numerous conventional sized synaptosomes and was virtually free of photoreceptor cell synaptosomes. Both synaptosomal fractions took up [¹⁴C]aspartate and [¹⁴C]glutamate with high affinity [ K _m= 1–2μM). Uptake characteristics were similar to those described for high affinity uptake systems in brain synaptosomes, i.e. saturation kinetics; temperature and Na⁺ dependence. Although the presence of a high affinity uptake system is not a definitive criterion for demonstration of functional neurotransmitter systems, it is an important and necessary prerequisite and can thus be considered as supportive evidence for the involvement of asparate and glutamate in neurotransmission in rabbit retina.     

CaMKII associates with CaV1.2 L-type calcium channels via selected β subunits to enhance regulatory phosphorylation

Calcium/calmodulin-dependent kinase II (CaMKII) facilitates L-type calcium channel (LTCC) activity physiologically, but may exacerbate LTCC-dependent pathophysiology. We previously showed that CaMKII forms stable complexes with voltage-gated calcium channel (VGCC) β_1b or β_2a subunits, but not with the β₃ or β₄ subunits ( Grueter et al. 2008 ). CaMKII-dependent facilitation of Ca_V1.2 LTCCs requires Thr498 phosphorylation in the β_2a subunit ( Grueter et al. 2006 ), but the relationship of this modulation to CaMKII interactions with LTCC subunits is unknown. Here we show that CaMKII co-immunoprecipitates with forebrain LTCCs that contain Ca_V1.2α₁ and β₁ or β₂ subunits, but is not detected in LTCC complexes containing β₄ subunits. CaMKIIα can be specifically tethered to the I/II linker of Ca_V1.2 α₁ subunits in vitro by the β_1b or β_2a subunits. Efficient targeting of CaMKIIα to the full-length Ca_V1.2α₁ subunit in transfected HEK293 cells requires CaMKII binding to the β_2a subunit. Moreover, disruption of CaMKII binding substantially reduced phosphorylation of β_2a at Thr498 within the LTCC complex, without altering overall phosphorylation of Ca_V1.2α₁ and β subunits. These findings demonstrate a biochemical mechanism underlying LTCC facilitation by CaMKII.     

Glutamine, Glutamate, and Other Possible Regulators of α-Ketoglutarate and Malate Uptake by Synaptic Terminals

Abstract: The uptake of a-ketoglutarate and malate by rat brain synaptosomal preparations was found to be affected by a variety of substances at physiologically relevant concentrations. Glutamine altered the uptake of γ-ketoglutarate by causing an apparent reduction in the substrate-carrier affinity and an increase in V_max. In contrast, glutamine did not appear to affect the V_max of malate uptake, but it did increase markedly the uptake velocity at low concentrations of malate. L-Glutamate and L-as-partate were comparatively strong inhibitors of γ-keto-glutarate and malate uptake. N-Acetylaspartate was a weak inhibitor of γ-ketoglutarate uptake, a finding that contrasts with our previous observation that this compound potently inhibited γ-ketoglutarate uptake by synaptosomes obtained from the cerebellum of 8- to 14-day-old mice. Ca²⁺ exhibited a variable effect but usually enhanced the uptake of γ-ketoglutarate. The addition of small amounts of postmicrosomal supernatant to the incubation medium enhanced the uptake of γ-ketoglutarate by low-density synaptosomes. By comparison, the uptake of glutamate, glutamine, γ-aminobutyric acid, and several other amino acids was not affected. The enhancement of γ-ketoglutarate uptake by the supernatant was due to a heat labile substance that was retained by dialysis tubing (MW cutoff = 8,000) and Amicon filter cones (CF 25), and was precipitated by ammonium sulfate at 60% saturation. In experiments in which the metabolic conversion of [U-^-14C] γ-ketoglutarate to glutamate, as-partate, glutamine, and aminobutyric acid was determined, the presence of glutamine and glutamate in the incubation medium did not affect the pattern of labelling appreciably.     

Identification and characterization of a novel amphioxus dopamine D1-like receptor

Dopamine receptors function to control many aspects of motor control and other forms of behaviour in both vertebrates and invertebrates. They can be divided into two main groups (D₁ and D₂) based on sequence similarity, ligand affinity and effector coupling. However, little is known about the pharmacology and functionality of dopamine receptors in the deuterostomian invertebrates, such as the cephalochordate amphioxus ( Branchiostoma floridae) which has recently been placed as the most basal of all the chordates. A bioinformatic study shows that amphioxus has at least three dopamine D₁-like receptor sequences. One of these receptors, AmphiD₁/β, was found to have high levels of sequence similarity to both vertebrate D₁ receptors and to β-adrenergic receptors. Here, we report on the cloning of AmphiD₁/β from an adult amphioxus cDNA library, and its pharmacological characterization subsequent to its expression in both mammalian cell lines and Xenopus oocytes. It was found that AmphiD₁/β has a similar pharmacology to vertebrate D₁ receptors, including responding to benzodiazepine ligands. The pharmacology of the receptor exhibits 'agonist-specific coupling' depending upon the second messenger pathway to which it is linked. Moreover, no pharmacological characteristics were observed to suggest that AmphiD₁/β may be an amphioxus orthologue of vertebrate β-adrenergic receptors.     

Binding of ATP to Brain Glutamate Decarboxylase as Studied by Affinity Chromatography

Abstract: The interactions of two forms of porcine brain glutamate decarboxylase (β-GAD and γ-GAD) with the effector ATP were studied by affinity chromatography. A third form, γk-GAD, was only slightly retarded by the affinity matrix and was eluted in the buffer wash. The interaction of GAD with the ATP affinity matrix was qualitatively similar to its interaction with free ATP as reported in previous kinetic studies. The rank order of adenine nucleotides as eluting agents and affinity ligands was ATP > ADP > AMP. GAD was also eluted by its cofactor, pyridoxal 5'-phosphate, and this was enhanced by 1 mM P_i In contrast, a high concentration (140 mM) of P_i by itself was required to elute the enzyme. GAD remained active while bound to the affinity column and was eluted in the holoenzyme form by ATP, indicating that the affinity ligand did not bind in the active site and did not displace catalytically active cofactor from the enzyme.     

(+)-Anatoxin-a Is a Potent Agonist at Neuronal Nicotinic Acetylcholine Receptors

Abstract: The effects of the nicotinic agonist (+)-anatoxin-a have been examined in four different preparations, representing at least two classes of neuronal nicotinic receptors. (+)-Anatoxin-a was most potent (EC₅₀= 48 n M ) in stimulating ⁸⁶Rb⁺ influx into M10 cells, which express the nicotinic receptor subtype comprising α4 and β2 subunits. A presynaptic nicotinic receptor mediating acetylcholine release from hippocampal synaptosomes was similarly sensitive to (+)-anatoxin-a (EC₅₀= 140 n M ). α-Bungarotoxin-sensitive neuronal nicotinic receptors, studied using patch-clamp recording techniques, required slightly higher concentrations of this alkaloid for activation: Nicotinic currents in hippocampal neurons were activated by (+)-anatoxin-a with an EC₅₀ of 3.9 γ M , whereas α7 homooligomers reconstituted in Xenopus oocytes yielded an EC₅₀ value of 0.58 γ M for (+)-anatoxin-a. In these diverse preparations, (+)-anatoxin-a was between three and 50 times more potent than (–)-nicotine and ˜20 times more potent than acetylcholine, making it the most efficacious nicotinic agonist thus far described.     

Posttranslational Processing of Brain Actin

Abstract: Two short-lived isoforms of actin, named δ- and ε-actin, have been detected in brain extracts from rats labeled in vivo with [³⁵S]methionine. These two molecular species have PI values slightly more basic than β- and γ-actin, the stable isoforms of the protein found in brain tissue. Under the appropriate incubation conditions δ- and ε-actin, synthesized in vivo , can be converted in vitro into β- and γ-actin. This posttranslational processing of δ- and ε-actin requires acetyl coenzyme A, suggesting that an acetylation step, presumably of the NH₂-terminal end, is involved in the transformation of these proteins into β- and γ-actin.     

Sodium and Chloride Ion-Dependent Transport of β-Alanine Across the Blood-Brain Barrier

Abstract: The characteristics of β-alanine transport at the blood-brain barrier were studied by using primary cultured bovine brain capillary endothelial cells. Kinetic analysis of the β-[³H]alanine transport indicated that the transporter for β-alanine functions with K_t of 25.3 ± 2.5 µ M and J _max of 6.90 ± 0.48 nmol/30 min/mg of protein in the brain capillary endothelial cells. β-[³H]Alanine uptake is mediated by an active transporter, because metabolic inhibitors (2,4-dinitrophenol and NaN₃) and low temperature reduced the uptake significantly. Furthermore, the uptake of β-[³H]alanine required Na⁺ and Cl⁻ in the external medium. Stoichiometric analysis of the transport demonstrated that two sodium ions and one chloride ion are associated with one β-alanine molecule. The Na⁺ and Cl⁻-dependent uptake of β-[³H]alanine was stimulated by a valinomycin-induced inside-negative K⁺-diffusion potential. β-Amino acids (β-alanine, taurine, and hypotaurine) inhibited strongly the uptake of β-[³H]alanine, whereas α- and γ-amino acids had little or no inhibitory effect. In ATP-depleted cells, the uptake of β-[³H]alanine was stimulated by preloading of β-alanine or taurine but not l -leucine. These results show that β-alanine is taken up by brain capillary endothelial cells, via the secondary active transport mechanism that is common to β-amino acids.     

Role of G Protein βγ Subunits in Muscarinic Receptor-Induced Stimulation and Inhibition of Adenylyl Cyclase Activity in Rat Olfactory Bulb

Abstract: In the olfactory bulb, muscarinic receptors exert a bimodal control on cyclic AMP, enhancing basal and G_s-stimulated adenylyl cyclase activities and inhibiting the Ca²⁺/calmodulin- and forskolin-stimulated enzyme activities. In the present study, we investigated the involvement of G protein βγ subunits by examining whether the muscarinic responses were reproduced by the addition of βγ subunits of transducin (βγ_t) and blocked by putative βγ scavengers. Membrane incubation with βγ_t caused a stimulation of basal adenylyl cyclase activity that was not additive with that produced by carbachol. Like carbachol, βγ_t potentiated the enzyme stimulations elicited by vasoactive intestinal peptide and corticotropin-releasing hormone. RT-PCR analysis revealed the expression of mRNAs encoding both type II and type IV adenylyl cyclase, two isoforms stimulated by βγ synergistically with activated G_s. In addition, βγ_t inhibited the Ca²⁺/calmodulin- and forskolin-stimulated enzyme activities, and this effect was not additive with that elicited by carbachol. Membrane incubation with either one of two βγ scavengers, the GDP-bound form of the α subunit of transducin and the QEHA fragment of type II adenylyl cyclase, reduced both the stimulatory and inhibitory effects of carbachol. These data provide evidence that in rat olfactory bulb the dual regulation of cyclic AMP by muscarinic receptors is mediated by βγ subunits likely acting on distinct isoforms of adenylyl cyclase.     

Concanavalin A Receptors Associated with Rat Brain Synaptic Junctions Are High Mannose-Type Oligosaccharides

Abstract: Glycoproteins were isolated from a rat brain synaptic junction fraction by affinity chromatography on Concanavalin A-agarose. The isolated glycoproteins were digested with pronase and radiolabeled with ¹²⁵I-Bolton Hunter reagent, and ¹²⁵I-Concanavalin A-binding glycopeptides were isolated by chromatography on Concanavalin A-agarose. Treatment of the ¹²⁵I-Concanavalin A-binding glycopeptides with either α-mannosidase or endo-β- N -acetylglucosaminidase-C₁₁ abolished their interaction with Concanavalin A. The pronase digest was reacted with endo-β-N-acetylglucosaminidase-C₁₁ and released oligosaccharides were reduced with NaB³H₄. Following affinity chromatography on Concanavalin A-agarose, Concanavalin A-binding [³H]oligosaccharides were chromatographed on Biogel P₄. Two major oligosaccharides corresponding to standard carbohydrates containing eight and five mannose residues were identified. Treatment of these oligosaccharides with α-mannosidase converted them to smaller saccharides having a mobility on Biogel P₄ columns equal to the standard disaccharide mannose-β-1-4- N '-acetylglucosamine. These results demonstrate that the Concanavalin A receptor activity associated with CNS synaptic junctions resides in asparaginelinked oligosaccharides of the high-mannose type.     

Differential Effects of 4'-Chlorodiazepam on Expressed Human GABAA Receptors

Abstract: The interactions of the atypical benzodiazepine 4'-chlorodiazepam (Ro 5-4864) with functionally expressed human GABA_A receptor cDNAs were determined. Cotransfection of human α₂, β₁, and γ₂ subunits was capable of reconstituting a 4'-chlorodiazepam recognition site as revealed by a dose-dependent potentiation of t -[³⁵S]butylbicyclophosphorothionate ([³⁵S]TBPS) binding to the GABA-activated chloride channel. This site is found on GABA_A receptor complexes containing sites for GABA agonist-like benzodiazepines and neuroactive steroids. The importance of the α subunit was further demonstrated as substitution of either α₁ or α₃ for the α₂ subunit did not reconstitute a 4'-chlorodiazepam recognition site that was capable of modulating [³⁵S]TBPS binding under the same experimental conditions. The 4'-chlorodiazepam modulatory site was shown to be distinct from the benzodiazepine site, but the phenylquinolines PK 8165 and PK 9084 produced effects similar to 4'-chlorodiazepam, consistent with the previous analysis of the 4'-chlorodiazepam site in brain homogenates. Further analysis of the subunit requirements revealed that coexpression of α₂ and β₁ alone reconstituted a 4'-chlorodiazepam recognition site. It is interesting, however, that the 4'-chlorodiazepam site was found to inhibit [³⁵S]TBPS binding to the GABA-activated chloride channel. Thus, the 4'-chlorodiazepam site may be reconstituted with only the α and β polypeptides.     

Distinct Loci Mediate the Direct and Indirect Actions of the Anesthetic Etomidate at GABAA Receptors

Abstract: Most general anesthetics produce two distinct actions at GABA_A receptors. Thus, these drugs augment GABA-gated chloride currents (referred to as an indirect action) and, at higher concentrations, elicit chloride currents in the absence of GABA (referred to as a direct action). Because a β subunit appears to be required for the direct action of intravenous anesthetics in recombinant GABA_A receptors, site-directed mutagenesis of the β3 subunit was performed to identify amino acid residues that are critical for this action. In HEK293 cells expressing a prototypical GABA_A receptor composed of α1β3γ2 subunits, mutation of amino acid 290 from Asn to Ser dramatically reduced both etomidate-induced chloride currents and its ability to stimulate [³H]flunitrazepam binding. By contrast, the ability of etomidate to augment GABA-gated chloride currents and GABA-enhanced [³H]flunitrazepam binding was retained. The demonstration that the direct, but not the indirect, actions of etomidate are dependent on β3(Asn²⁹⁰) indicates that the dual actions of this intravenous anesthetic at GABA_A receptors are mediated via distinct loci.     

SIMILARITIES OF β-BUNGAROTOXIN AND PHOSPHOLIPASE A2 AND THEIR MECHANISM OF ACTION

Abstract— β-Bungarotoxin, a presynaptic neurotoxin isolated from the venom of Bungarus multicinctus , has been shown to initially cause an increase in the frequency of miniature endplate potentials and subsequently block neuromuscular transmission by inhibiting nerve impulse induced release of acetylcholine. In rat brain synaptosomes it causes a Ca²⁺-dependent release of acetylcholine together, with a strong inhibition of the high affinity choline uptake system. In this report we demonstrate that β-bungarotoxin acts as a phospholipase A₂ (phosphatide 2-acyl hydrolase, EC 3.1.1.4), liberating fatty acids from synaptic membrane phospholipids. It also exhibits a striking similarity in a number of neurochemical properties with that of a purified phospholipase A₂ from Naja naja siamensis. In addition, both agents produce a marked depolarization of synaptosomal preparations as measured by a fluorescent dye. We propose that disruption of the membrane phospholipids by phospholipase activity can lead to depolarization of the synaptosomal preparation which promotes both transmitter release and inhibition of the energy-dependent high affinity choline uptake system. With this decreased supply of choline, the acetylcholine content of the cell would be gradually depleted leading to a decrease in transmission.     

Brain β-Spectrin Phosphorylation: Phosphate Analysis and Identification of Threonine-347 as a Heparin-Sensitive Protein Kinase Phosphorylation Site

Abstract: Phosphorylation of brain spectrin was studied by a combination of in vivo and in vitro approaches. Chemical analysis of phosphate groups on electrophoretically purified mouse brain β-spectrin yielded a stoichiometry of 3.2 ± 0.18 mol of PO₄/mol of β-spectrin. The spectrin isolated by chromatographic methods from mouse brain, pig brain, and human erythrocytes yielded 4.1, 5.6, and 3.2 mol of PO₄/mol of spectrin heterodimer, respectively. The ³²P labeling of spectrin in retinal ganglion cell neurons or NB 2a/d1 neuroblastoma cells with [³²P]orthophosphate showed phosphorylation of only β-spectrin in vivo. Two-dimensional phosphopeptide map analyses showed that most of the in vivo sites on β-spectrin were phosphorylated by either a heparin-sensitive endogenous cytoskeleton-associated protein kinase or protein kinase A. Phosphoamino acid analysis of in vivo and in vitro phosphorylated β-spectrin showed that [³²P]phosphate groups were incorporated into both serine (>90%) and threonine residues. In vitro, phosphate groups were incorporated into threonine residues by the heparin-sensitive endogenous protein kinase. The amino acid sequence VQQQLQAFNTY of an α-chymotryptic ³²P-labeled peptide phosphorylated by the heparin-sensitive cytoskeleton-associated endogenous protein kinase corresponded to amino acid residues 338–348 on the β1 repeat of β-spectrin_G (βSPIIa) gene. These data suggest that phosphorylation of Thr³⁴⁷, which is localized on the presumptive synapsin I binding domain of β-spectrin_G, may play a role in synaptic function by regulating the binding of spectrin to synaptic vesicles.