Interaction of calmodulin with synaptic plasma membranes isolated from sheep brain

Calmodulin binding to a membrane fraction enriched in synaptic plasma membranes of sheep brain cells was investigated with [¹²⁵I]calmodulin. Calmodulin binding to these membranes is Ca²⁺-dependent with a half maximal saturation at the pCa value of about 5.5. The binding is reduced by replacing Ca²⁺ with Mg²⁺, but it is significantly enhanced when both cations are present in the medium. Cation-dependent binding is specific and saturable with an apparent K_D of about 47–50 nM and a maximal capacity of about 4 pmol mg⁻¹ protein. The results indicate that synaptic plasma membranes isolated from sheep brain cells interact with calmodulin in a Ca²⁺-dependent, Mg²⁺-facilitated manner.     

High-affinity kainate and domoate receptors in rat brain.

Mammalian brain expresses receptors which bind the potent neurotoxins, kainate and domoate, with high affinity, and which form a subclass of ionotropic glutamate receptors. A new member of these receptors, expressed in both adult and embryonic CNS is compared in its ligand binding properties to its closely sequence-related homologs.     

Interaction of isolated brain synaptic vesicles with flat bilayer membranes in the rat

The conductivity of planar bilayer membrane comprising asolectin and phosphatidylserine (concentration ratio 9:1) in a buffer solution increased sharply in the presence of synaptic vesicles (SV) isolated from the rat brain and added to one side of the membrane only. The bilayer remained stable upon modification, and the conductivity increment was dependent on SV concentration in the range from 4 to 16 mu of the total protein per ml. If I mM CaCl2 was present in the buffer solution, the conductivity increased by 2 to 3 orders of magnitude upon the addition of SV at a final concentration of 3-4 mu protein per ml. The membrane was unstable and its rupture occurred often at an early stage of conductivity changes. In the absence of SV addition the membrane was stable, with its conductivity remaining unchanged for 2 h and more. With I mM CaCl2 addition to the solution already containing SV, no conductivity changes were observed, the cause perhaps, being Ca2+-induced SV aggregation.     

The role of RNA editing of kainate receptors in synaptic plasticity and seizures

The ionotropic glutamate receptor subunit GluR6 undergoes developmentally and regionally regulated Q/R site RNA editing that reduces the calcium permeability of GluR6-containing kainate receptors. To investigate the functional significance of this editing in vivo, we engineered mice deficient in GluR6 Q/R site editing. In these mutant mice but not in wild types, NMDA receptor-independent long-term potentiation (LTP) could be induced at the medial perforant path-dentate gyrus synapse. This indicates that kainate receptors with unedited GluR6 subunits can mediate LTP. Behavioral analyses revealed no differences from wild types, but mutant mice were more vulnerable to kainate-induced seizures. Together, these results suggest that GluR6 Q/R site RNA editing may modulate synaptic plasticity and seizure vulnerability.     

Solubilization and characterization of kainate receptors from goldfish brain

The binding of [3H]kainate to goldfish brain membrane fragments was investigated. Scatchard analysis revealed a single class of binding sites in Tris-HCl buffer with a Kd of 352 nM and a Bmax of 3.1 pmol/mg wet weight. In Ringer's saline, [3H]kainate bound with a Bmax of 1.8 pmol/mg wet weight and a Kd of 214 nM. Binding in Ringer's saline, but not Tris-HCl buffer, displayed positive cooperativity with a Hill coefficient of 1.15. The [3H]kainate binding sites were solubilized in Ringer's saline using the nonionic detergent n-octyl-beta-D-glucopyranoside. Approximately 30-50% of the total number of membrane-bound binding sites were recovered on solubilization. The Kd of [3H]kainate for solubilized binding sites was approximately 200 nM. The rank order of potency for glutamatergic ligands at inhibiting [3H]kainate binding was identical and the competitive ligands had similar Ki values in both membranes and solubilized extracts. In membrane preparations, [3H]kainate displayed a two component off-rate with koff values of 0.97 min-1 and 0.07 min-1; in solubilized extracts, however, only a single off-rate (koff = 0.52 min-1) was observed. The hydrodynamic properties of n-octyl-beta-D-glucopyranoside solubilized [3H]kainate binding sites was investigated by sucrose density centrifugation. A single well defined peak was detected which yielded a sedimentation coefficient of 8.3 S. The results presented in this report suggest that goldfish brain may provide an ideal system in which to study kainate receptor biochemistry.     

Effects of paradoxical sleep deprivation on the activity of opiate receptors isolated from synaptic membranes of the rat brain]

The specific binding of 3H-naloxone with opiate receptors isolated from brain synaptic membranes of control and paradoxical sleep deprived rats were studied. This extreme state was shown to reduce the naloxone-binding activity of synaptic membranes by 35% and isolated receptors by 25-28%. The values of Kd and Bmax were calculated for isolated opiate receptors at different stages of purification. Considerable decrease of 3H-naloxone binding sites density (2 times) in the isolated opiate receptors with simultaneous increase of their affinity (3-4,5 times) was found following 24 hours paradoxical sleep deprivation. These findings suggest development of fixed alterations in the structure and functions of integral receptor proteins under extreme influences.     

Trimethyltin-induced loss of NMDA and kainate receptors in the rat brain

Summary Adult rats exposed acutely to trimethyltin (TMT) manifest a number of behavioral alterations, in conjunction with neuronal degeneration in the limbic system. In the present study, changes in³H-TCP binding to N-methyl-D-aspartate (NMDA) receptors and³H-kainic acid (KA) binding to kainate receptors were studied by autoradiographic methods following TMT exposure (8 mg/kg, i.p.) in adult Sprague Dawley rats. No significant alterations were found at 4 hours after exposure. An extensive loss of³H-TCP and³H-KA binding was seen in the hilar region of the CA3 field at 2 and 12 weeks after TMT exposure. Also, the³H-TCP binding was decreased in piriform cortex and in striatum. Thus, TMT exposure leads to a major and regional selective loss of NMDA and kainate receptors in the limbic system, alterations that may be involved in the neuropathology and behavioral sequelae of TMT toxicity.Abbreviations TMT trimethyltin - NMDA N-methyl-D-aspartate - KA Kainic acid - TCP N-(1-2-thienylcyclohexyl)-3,4-piperidine     

Sodium-independent taurine binding to brain synaptic membranes

Saturable sodium-independent taurine binding to mouse and rat brain synaptic membranes was exposed after two freezing-thawing cycles combined with Triton X-100 treatments. The amount of saturable taurine binding was fairly low but was enhanced after depletion of brain taurine. Saturable taurine binding was displaceable by some convulsants and anticonvulsants but is specificity still remains to be established.     

Structural reorganization of the brain synaptic membranes and aging

Results of the authors' studies and data from literature underlie the development of a notion on structural rearrangement of the brain synaptic membranes in aging. Reorganization results in conformational changes of the key membrane-bound enzymes and receptors underlying the age alterations of neuronal functions.     

Lysophosphoinositide-specific phospholipase C in rat brain synaptic plasma membranes

A membrane preparation from rat brain catalyzed the hydrolysis of [2-³H]glycerol-labeled lysophosphatidylinositol (lysoPI) to yield monoacylglycerol (MG) and inositolphosphates. This phospholipase C activity had an optimal pH of 8.2. The membrane preparation did not require the addition of Ca²⁺ for its maximum activity, but the activity was inhibited by addition of 0.1 mM EDTA to the assay mixture and was restored by simultaneous addition of 0.2 mM Ca²⁺. The activity was found to be localized in synaptic plasma membranes prepared by Ficoll and Percoll density gradients. The phospholipase C was highly specific for lysoPI; diacylglycerol formation from phosphatidylinositol, and MG formation from lysophosphatidylcholine, lysophosphatidylethanolamine, and lysophosphatidylserine were below 5% of that observed with lysoPI under the conditions used. We concluded that there is a pathway for phosphatidylinositol metabolism in brain synaptic membranes which is different from the well-characterized phosphoinositide-specific phospholipase C pathway.Abbreviations PI phosphatidylinositol - lysoPI lysophosphatidylinositol - lysoPI-PLC lysophosphoinositide-specific phospholipase C - PI-PLC phosphoinositide-specific phospholipase C - MG monoacylglycerol - PLC phospholipase C To whom to address reprint requests.     

Interaction of isolated synaptic vesicles with synaptic contacts in the rat brain

The effects of Mg-ATP, EGTA, EDTA and dicyclohexylcarbodiimide on the changes in the intensity of light scattering were studied in rat brain synaptic vesicles (SV) suspended in saccharose-buffer medium. Specific interactions between SV and isolated synaptic junctional complex were observed in the presence of Mg-ATP and calmodulin. An in vitro model of exocytosis is discussed.     

High-affinity glutamic acid binding to brain synaptic membranes

Rat brain homogenate preparations exhibited two types of glutamine binding, one a high-affinity (K₁ = 0.2 μM) and the other a low-affinity type (K₂ = 4.4 μM). The high-affinity binding was primarily associated with the plasma membrane subcellular fractions and in particular with the synaptic membrane subfraction. This l-glutamate binding was found to be strongly stereospecific for the l-form and was almost totally reversible. The synaptic membrane glutamate binding was partialy inhibited by neuro-excitatory and neuro-inhibitory amino acids but was not affected by amino acids lacking in neuropharmacologic activity. The membrane-associated l-glutamate binding system could be solubilized by Triton X-100 without loss of its high-affinity binding activity. The chemical nature of this glutamate binding component was found to be that of a glycolipoprotein. It is proposed that this glutamate binding system represents the physiologic receptor on neuronal membranes of this amino acid.