Purification, reconstitution, and cloning of an NMDA receptor-ion channel complex from rat brain synaptic membranes: implications for neurobiological changes in alcoholism.
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Alcoholism↗
Biomedical subjects
M L Michaelis
Publications and source records attributed to M L Michaelis.
Regulation of calcium levels in brain tissue from adult and aged rats.
The possibility that regulation of Ca2+ levels in brain nerve terminals is altered as the brain ages was examined in synaptosomes from adult and aged Fischer 344 rats. Free intrasynaptosomal [Ca2+]i was monitored with fura-2 as synaptosomes were depolarized with KCl, veratridine and ibotenic acid. With all three depolarizing agents, synaptosomes from aged animals reached higher free Ca2+ levels, and the maximal Ca2+ increases (delta Ca2+) estimated from computer assisted-fitting of the curves, ranged from 35% to 80% greater in synaptosomes from aged animals. The total Ca2+ content of the brain and of synaptosomes was also found to be considerably higher in aged than in adult animals. These results suggest that the aging process in brain is accompanied by alterations in both dynamic aspects of Ca2+ handling in nerve endings and the overall content of Ca2+ in the brain and synaptic terminals.
Aging↗
Molecular aspects of glutamate receptors and sodium-calcium exchange carriers in mammalian brain: implications for neuronal development and degeneration.
N-Methyl-D-aspartate (NMDA) and L-glutamate activate membrane receptors that produce substantial permeation of Na+, K+ and Ca2+ through the neuronal membrane. These ionic fluxes are intimately linked to processes that regulate neuronal survival, growth and differentiation. Intracellular free Ca2+ concentrations are thought to be particularly important determinants of the vulnerability of neurons to excessive excitatory stimulation produced through activation of NMDA receptors. In order to understand the molecular events involved in both NMDA receptor activation and regulation of intracellular Ca2+ levels, we have purified and reconstituted the protein complexes that form the NMDA/glutamate receptors in rat brain synaptic membranes and those that constitute the Na(+)-Ca2+ antiporters in bovine brain synaptic membrane. The molecular properties of these protein complexes are described, and information from the most recent studies of exploration of the molecular structures of these receptors and transport carriers is summarized.
Animals↗
Purification of a synaptic membrane Na+/Ca2+ antiporter and immunoextraction with antibodies to a 36-kDa protein.
The conditions for optimal solubilization and reconstitution of bovine brain synaptic plasma membrane Na+/Ca2+ exchange activity were examined and a series of chromatographic procedures were used for the isolation of a protein involved in this transport activity. The zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate in the presence of 20% (vol/vol) glycerol led to optimal solubilization, and soybean phospholipids in low-pH medium were found to produce optimal reconstitution of activity after dialysis to remove the detergent. Sequential chromatography steps involving the use of gel filtration on Sephacryl S-400 HR, ion exchange on diethylaminoethyl-Sephacel, and metal chelate chromatography on tris-(carboxymethyl)ethylenediamine loaded with LaCl3 led to the isolation of a fraction highly enriched in both Na+/Ca2+ exchange activity and two protein bands identified by denaturing electrophoresis. The estimated molecular masses of the two proteins were 50 and 36 kDa. Development of polyclonal antibodies to the 36-kDa protein permitted immunoextraction of greater than 95% of the antiporter activity from solubilized synaptic plasma membranes. These antibodies cross-reacted with the electroeluted 50-kDa protein on enzyme-linked immunosorbent assays, suggesting a close relationship between the two proteins. These results indicate that the 36-kDa protein is at least a component of the brain membrane Na+/Ca2+ antiporter.
Animals↗
Some molecular properties of the synaptic plasma membrane Na(+)-Ca2+ exchanger.
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Brain Chemistry↗
Characterization of Na(+)-Ca2+ exchange activity in plasma membrane vesicles from postmortem human brain.
Procedures were developed for measurement of Na+/Ca2+ exchange in resealed plasma membrane vesicles from postmortem human brain. The vesicle preparation method permits use of stored frozen tissue with minimal processing required prior to freezing. Vesicles prepared in this manner transport Ca2+ in the presence of a Na+ gradient. The kinetic characteristics of the Na+/Ca2+ exchange process were determined in membrane vesicles isolated from hippocampus and cortex. The Kact for Ca2+ was estimated to be 32 microM for hippocampal and 17 microM for cortical tissue. The maximal rate of Ca2+ uptake (Vmax) was 3.5 nmol/mg protein/15 sec and 3.3 nmol/mg protein/15 sec for hippocampal and cortical tissue, respectively. Exchange activity was dependent on the Na+ gradient, and was optimal in the high pH range. Therefore, membranes in which Na(+)-dependent Ca2+ transport activity is preserved can be isolated from postmortem human brain and could be used to determine the influence of pathological conditions on this transport system.
Brain↗
Glutamate receptor changes in brain synaptic membranes from human alcoholics.
Brains from human alcoholics and non-alcoholics were obtained shortly after death. The hippocampus was dissected, homogenized, and processed for the isolation of a synaptic membrane-enriched fraction and the study of L-[3H]glutamic acid and 3-((+-)-2-carboxypiperazin-4-yl)-[1,2(3H)propyl-1-phosphonic acid ([3H]CPP) binding sites. The pharmacological characteristics of L-[3H]glutamic acid binding to synaptic membranes isolated from hippocampus corresponded to the labeling of a mixture of N-methyl-D-aspartate (NMDA), kainate and quisqualic acid receptor sites. Synaptic membranes prepared from the hippocampus of individuals classified as alcoholics had significantly higher density of glutamate binding sites than identically prepared membranes from non-alcoholic individuals. In addition, there was a clear definition of a population of L-glutamate binding sites (approx. 10% of total) in the membranes from alcoholics that had a higher affinity for the ligand than the major set of sites labeled in membranes from both alcoholics and non-alcoholics. Neither the age of the individuals at the time of death nor the time that elapsed between death and processing of brain tissue were significant factors in determining either recovery of purified synaptic membranes from brain homogenates or L-[3H]glutamate binding to synaptic membranes. In order to determine whether some of the changes in L-[3H]glutamic acid binding were due to alterations in binding at the NMDA receptor subtype, we also measured binding of [3H]CPP to extensively washed crude synaptosomal membranes. Membranes from brains of alcoholics had higher affinity (3-fold) for [3H]CPP but lower binding capacity (3-fold) when compared with those of non-alcoholics.(ABSTRACT TRUNCATED AT 250 WORDS)
Alcoholism↗
Ca2+ handling systems and neuronal aging.
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Aging↗
Studies on the ionic mechanism for the neuromodulatory actions of adenosine in the brain.
Although much evidence exists to support the modulation of neurotransmitter release by adenosine in both the central and peripheral nervous systems, the ionic mechanisms involved in this process are still not established. In our initial series of studies, the effects of adenosine analogs on depolarization-induced 45Ca influx into synaptosomes were examined, but no reduction in 45Ca uptake was observed in the presence of these agents, regardless of whether synaptosomes were depolarized by KCl or veratridine. The possibility that synaptosomal adenosine receptors are coupled directly to K+ channels and that an increase in K+ conductance is the primary ionic event initiated by adenosine was examined in resealed synaptic plasma membranes. We followed the movement of K+ through the use of the labeled lipophilic anion [35S]thiocyanate [( 35S]SCN-), which distributes across membranes according to the existing membrane potential. Both 2-chloroadenosine and adenosine-5'-cyclopropyl carboxamide produced dose-dependent increases in SCN- uptake, indicating enhanced K+ fluxes across the synaptic membranes. These effects of adenosine receptor agonists on membrane K+ conductance were blocked by receptor antagonists such as isobutylmethylxanthine. In addition, the placement of a K+-selective microelectrode in the caudate nuclei of intact, anesthetized rats revealed a substantial increase in the extracellular K+ concentration when adenosine was released onto the cells from a pipet in the assembly containing the ion-selective microelectrode. The results of these studies at both the membrane level and in the intact brain suggest that the initial event in the neuromodulatory actions of adenosine is an increase in the membrane conductance for K+ rather than inhibition of the voltage sensitive Ca2+ channels.
1-Methyl-3-isobutylxanthine↗
Effects of chronic alcohol administration on synaptic membrane Na+-Ca2+ exchange activity.
We have recently reported that ethanol and other n-alkanols added to in vitro assays inhibit the activity of the Na+-Ca2+ exchange system in brain synaptic plasma membrane vesicles. The present studies were undertaken to determine whether in vivo chronic ethanol administration leads to alterations in this Na+-Ca2+ antiporter that might be indicative of an adaptive response to alcohol. The Na+-dependent Ca2+ transport activity in the plasma-membrane fractions obtained was measured at various Ca2+ concentrations. Results of these experiments revealed that a 3-week ethanol regimen brought about a significant increase in the Na+-dependent Ca2+ transport activity only in the membrane fraction enriched in synaptic junctional complexes. These membranes showed a near doubling in the maximal transport activity of the antiporter in alcohol-treated compared with the control animals. Changes in the kinetic parameters were reversible as the Na+-Ca2+ exchange activity in these membranes from animals maintained on alcohol for 3 weeks and then withdrawn for 1 week was indistinguishable from that of membranes from control animals. Thus it appears that ethanol-treated animals make a reversible adaptation in their neuronal cell membranes to compensate for the acute effects of ethanol on the Na+-Ca2+ antiporter.
Animals↗
Characteristics of Mg2+-dependent, ATP-activated Ca2+ transport in synaptic and microsomal membranes and in permeabilized synaptosomes.
Synaptic plasma membranes isolated from rat brain exhibited a Ca2+ transport process that was strictly dependent on the presence of Mg2+ and activated by ATP hydrolysis. The characteristics of this ATP-activated transport process included a high affinity for Ca2+ and ATP with the Kact for these two substrates being 0.7 and 5 microM, respectively, and a lower affinity for Mg2+, Kact = 54 microM. The estimated constants for ATP-activated Ca2+ transport into synaptic membrane vesicles and the dependence of such transport on Mg2+ were indicative that such transport was related to the previously described high affinity (Ca2+ + Mg2+)-ATPase in synaptic membranes. An ATP- and Mg2+-dependent Ca2+ transport process with very similar kinetic characteristics was present also in a general microsomal membrane fraction obtained from brain tissue. The synaptic and microsomal membrane ATP-activated transport processes exhibited differences in their sensitivity to vanadate inhibition. Interaction with vanadate was fairly complex and best analyzed by a two-component model. Thus, the estimated Ki values for vanadate were 0.2 and 6.6 microM for the synaptic membranes and 0.7 and 13.8 microM for the microsomes. Since the microsomal membranes contain a substantial population of intraneuronal endoplasmic reticulum vesicles, the effects of vanadate on Ca2+ transport into intraneuronal membrane organelles, other than mitochondria, was determined in saponin-permeabilized synaptosomes. The estimated Ki values for vanadate inhibition of Ca2+ transport activity were 0.7 and 13 microM. The accumulation of Ca2+ into synaptic plasma membrane vesicles was readily reversed by activation of the Na+-Ca2+ exchange carrier, whereas the Ca2+ associated with intrasynaptosomal organelles was not affected by changes in [Na+]. Thus, there are at least two ATP-dependent Ca2+ transporting processes localized on two distinct neuronal membranes, one on the plasma membrane and the second on intraneuronal membranes.
Adenosine Triphosphatases↗
Characteristics of adenosine binding sites in atrial sarcolemmal membranes.
The studies reported here involve an exploration of the sites on atrial myocyte membranes with which adenosine interacts to produce its potent physiological effects in atrial muscle. Specific, high affinity binding of the stable adenosine analogs 2-chloro[3H]adenosine (2-ClAdo) and [3H]adenosine 5'-N-ethylcarboxamide (NECA) to atrial sarcolemmal membranes was measured in kinetic and equilibrium studies at 4 degrees C and 35 degrees C. Analysis of the [3H]2-ClAdo binding isotherm indicated the presence of two classes of binding site with equilibrium Kassoc values estimated to be 5.7 X 10(7) M-1 and 2.7 X 10(6) M-1. Displacement of bound [3H]2-ClAdo by adenosine 5'-N-cyclopropylcarboxamide (NCPCA) and by several N6-substituted adenosine analogs confirmed the presence of two classes of binding site. Analysis of the [3H]NECA binding also revealed the presence of two types of binding site for this ligand. The methylxanthines isobutylmethylxanthine and theophylline displaced bound [3H]2-ClAdo whereas adenosine uptake inhibitors and several other purines showed little activity. These atrial membrane binding sites exhibit many of the characteristics of the physiological adenosine receptors studied in intact atria. Furthermore, the [3H]2-ClAdo binding sites were sensitive to treatment with proteolytic enzymes, suggesting that these sites exist on sarcolemmal membrane proteins.
1-Methyl-3-isobutylxanthine↗
Effects of amphipathic drugs on L'[3H]glutamate binding to synaptic membranes and the purified binding protein.
Four amphipathic molecules with known local anesthetic activity, dibucaine, tetracaine, chlorpomazine, and quinacrine, inhibited the binding of L-[3H]glutamic acid to rat brain synaptic plasma membranes and to the purified glutamate binding protein. Neither haloperidol nor diphenylhydantoin had significant inhibitory effects on the glutamate binding activity of the membranes or of the purified protein. The amphipathic drugs apparently inhibited L-[3H]glutamate binding to synaptic membranes by a mixed type of inhibition. The inhibitory activity of quinacrine on glutamate binding to the synaptic membranes was greater in a low ionic strength, Ca2+-free buffer medium, than in a physiologic medium (Krebs-Henseleit buffer). Removal of Ca2+ from the Krebs solution enhanced quinacrine's inhibition of glutamate binding. Quinacrine up to 1 mM concentration did not inhibit the high affinity Na+-dependent L-glutamate transport in these membrane preparations. The importance of Ca2+ in the expression of quinacrine's effects on the glutamate binding activity of synaptic membranes and the observed tetracaine and chlorpromazine-induced increases in the transition temperature for the glutamate binding process of these membranes, were indicative of an interaction of the local anesthetics with the lipid environment of the glutamate binding sites.
Anesthetics, Local↗
High affinity Ca2+-stimulated Mg2+-dependent ATPase in rat brain synaptosomes, synaptic membranes, and microsomes.
High affinity Ca2+-stimulated Mg2+-dependent ATPase activity of nerve ending particles (synaptosomes) from rat brain tissue appears to be associated primarily with isolated synaptic plasma membranes. The synaptic membrane (Ca2+ + Mg2+)-ATPase activity was found to exhibit strict dependence on Mg2+ for the presence of the activity, a high affinity for Ca2+ (K0.5 = 0.23 microM), and relatively high affinities for both Mg2+ and ATP (K0.5 = 6.0 microM for Mg2+ and KM = 18.9 microM for ATP). These kinetic constants were determined in incubation media that were buffered with the divalent cation chelator trans-cyclohexane-1,2-diamine-N,N,N',N'-tetraacetic acid. The enzyme activity was not inhibited by ouabain or oligomycin but was sensitive to low concentrations of vanadate. The microsomal membrane subfraction was the other brain subcellular fraction with a high affinity (Ca2+ + Mg2+)-ATPase activity which approximated that of the synaptic plasma membranes. The two membrane-related high affinity (Ca2+ + Mg2+)-ATPase activities could be distinguished on the basis of their differential sensitivity to vanadate at concentrations below 10 microM. Only the synaptic plasma membrane (Ca2+ + Mg2+)-ATPase was inhibited by 0.25-10 microM vanadate. The studies described here indicate the possible involvement of both the microsomal and the neuronal plasma membrane (Ca2+ + Mg2+)-ATPase in high affinity Ca2+ transport across membranes of brain neurons. In addition, they suggest a means by which the relative contributions of each transport system might be evaluated based on their differential sensitivity to inhibition by vanadate.
Animals↗
Alcohol and local anesthetic effects on Na+-dependent Ca2+ fluxes in brain synaptic membrane vesicles.
Resealed synaptic plasma membrane vesicles exhibit Na+-dependent Ca2+ transport activity which may participate in regulation of free Ca2+ concentrations in nerve endings. Sodium chloride-loaded vesicles took up Ca2+ from the external medium (150 mM KCl-25 mM Tris/HCl) in the presence of an outward-directed Na+ gradient, a Ca2+ concentration of 38.6 microM producing half-maximal uptake at 23 degrees. Methanol (5-200 mM) and low concentrations of ethanol (5-25 mM) enhanced the Na+-dependent Ca2+ influx measured at 23 degrees. Higher ethanol concentrations (100-600 mM), as well as 1-propanol and 1-butanol (10-200 mM), produced only inhibition of Ca2+ fluxes. Dixon plot analysis of the inhibitory phase revealed that ethanol inhibited Ca2+ uptake in an apparently competitive manner with respect to Ca2+ concentration, and the Ki obtained from these experiments was 1.01 M ethanol. The inhibition of Ca2+ fluxes by butanol was non-competitive, and the Ki was 68.6 mM. The local anesthetics dibucaine and tetracaine also inhibited Ca2+ fluxes with IC50 values of 1.8 mM for tetracaine and 0.46 mM for dibucaine. The possible physiologic consequences of this inhibition of Na+-Ca2+ countertransport is synaptic membranes by the alcohols and local anesthetics are discussed with regard to neuronal transmission and membrane conductance.
Alcohols↗
DNA damage by PAH and repair in a marine sponge.
The sponge Tethya lyncurium from the Northern Adriatic has been used as an experimental species. A method is outlined for preparation of DNA which yields a highly purified DNA with a double-strand (ds) molecular weight of 25 M-dalton between single-strand (ss) breaks, which when properly damaged can be cut opposite to ss-breaks with nuclease S1. The molecular weights of the resulting ds-DNA pieces and their distribution has been evaluated by electron microscope photographs. Sponges exposed to benzo[a]pyrene (BaP) in the dark only incorporate BaP-derivatives (BaPD) in small amounts, if any. However, in the presence of light, derivatization to BaP derivatives enables effective coupling to occur, as shown previously (R.K. Zahn et al., 1981). Sponges were exposed to radiolabeled BaP in the presence of light. Coupling of BaPD to the DNA as well as the induction of ss-breaks were measured. Light-mediated coupling is concentration dependent from 0.01-20 ppb BaP with a correlation coefficient of r = 0.84. Under conditions of possible repair, ss-breaks completely disappear from sponge DNA in the course of three weeks while a substantial fraction of the BaP derivatives persists. Double label experiments show that substantial DNA synthesis occurs during this time. Pollution causes a decrease of the molecular weight of unnicked DNA, re-incubation in clean water an increase. A DNA species of 24 M-dalton seems to play a critical role. If its percentage in the DNA population drops below a critical level, recovery is not longer possible. DNA damage by PAH and repair in sponges seems to differ from that of most eucaryotes.
Animals↗
Alcohol effects on synaptic membrane calcium ion fluxes.
The effects of ethanol on Na+-dependent CA2+ fluxes have been examined in resealed synaptic membrane vesicles assayed at 3 different temperatures. Sodium chloride-loaded vesicles were preincubated with various concentrations of ethanol for 120 sec prior to being diluted into a 45CaCl2-containing medium in the presence or absence of an outward-directed Na+ gradient. The effect of ethanol on Na+-dependent Ca2+ transport measured at 23 degrees C was biphasic. However, when the assay was conducted either at 16 degrees C or at 35 degrees C, all ethanol concentrations tested (10-300 mM) produced only inhibition of Ca2+ influx. The role of membrane fluidization in the ethanol-induced inhibition was explored by determining the effects of incorporating various fatty acids into the membranes. Membrane fluidizing agents such as cis-vaccenic acid stimulated Ca2+ influx whereas trans-vaccenic and saturated fatty acids had little effect. The fluidizing effect of incorporating cis-vaccenic acid into the membranes was confirmed with electron paramagnetic resonance (EPR) spectroscopy. The data obtained from these studies suggest that the inhibition of Ca2+ fluxes produced by alcohol and local anesthetics is not the result of a general increase in bulk phase synaptic membrane fluidity.
Animals↗
Purification and molecular characterization of the brain synaptic membrane glutamate-binding protein.
A glutamate-binding protein from rat brain synaptic plasma membranes has been purified to apparent homogeneity. This protein has a Mr of 14,300 based on amino acid and carbohydrate analyses. The protein is enriched with tryptophan residues, which contribute substantially to its hydrophobic nature. It also has a relatively high content of acidic amino acids, which determine is low isoelectric point (4.82). The protein exhibits either a single, high-affinity class of sites for L-[3H]glutamate binding (KD = 0.13 microM) when binding is measured at low protein concentrations, or two classes of sites with high (KD = 0.17 microM) and low affinities (KD = 0.8 microM) when binding is measured at high protein concentrations. These observations suggest preferential binding of L-glutamate to a self-associating form of the protein. The displacement of protein-bound L-[3H]glutamic acid by other neuroactive amino acids has characteristics similar to those observed for displacement of L-glutamate from membrane binding sites. Chemical modification of the cysteine and arginine residues results in an inhibition of glutamate binding activity. The possible function of this protein in the physiologic glutamate receptor complex of neuronal membranes is discussed.
Amino Acids↗