The work is a study of the influence of Ca2+ (0.01 - 1 mM) on neuronal CI-, HCO3-, -ATPase complex: an enzyme that is a CI--pump which is functionally and structurally coupled to GABAA-receptors. It is found that influence of Ca2+ on the multifunctional complex starts at concentration of 50·M and at concentration of 0.1 mM, it reduces the “basal” one and increases the CI-, HCO3-, -stimulated Mg2+-ATPase activities. GABA (0.1 - 100μM) activates the “basal” Mg2+-ATPase activity in the ab-sence of calcium. The effect of GABA on the enzyme in the presence of 0.01 ·M Ca2+ does not change. At the same time, 1 mM Ca2+eliminates the GABA effect on the “basal” Mg2+-ATPase activity. Competitive blocker of GABAA-receptors bicuculline (5 - 20 μM) in the absence of Ca2+ ions elimi-nates the stimulation of the “basal” Mg2+-ATPase by anions. When 0.25 mM Ca2+ is added to the in-cubation medium the inhibitory bicuculline effect on the enzyme does not appear. We found that 0.1 mM o-vanadate (protein tyrosine phosphatase blocker) reduces the GABA-activated ATPase activity. At the same time, 0.1 mM genistein (a protein tyrosine kinase blocker) has no effect on enzyme activity. In the presence of Ca2+ (0.25 mM), the effect of o-vanadate on the “basal” and CI-, HCO3-, -ATPase activities does not appear. It is shown for the first time that high concentrations of Ca2+prevent the action of GABAA-ergic ligands on the study ATPase. It is assumed that there is the involvement of protein kinases and protein phosphatases in the modulation of the enzyme activity by calcium. The observed effect of calcium on the ATPase may play an important role in the study of the mechanisms of epileptogenesis and seizure activity.
Cl−-ATPase/Cl−-pump in plasma membrane from various cells (including neurons) is a “molecular machine” participating in the transportation of
Furthermore, we have found that such ATPase is involved in rat convulsant-induced seizure activity [
Experiments were performed on male Wistar rats weighing 180 - 200 g. Animals were maintained under standard vivarium conditions with free access to water and food. The experiment was conducted under the “Rules of work with experimental animals” FGBU “NIIOPP” RAS, which comply with the World Society for the Protection of Animals (WSPA) and the European Convention for the protection of experimental animals.
All procedures were performed at 0˚C - 4˚C. After decapitation of animals, the brain was isolated, homogenized in 8 vol. of ice-cold buffer solution containing 0.25 M sucrose, 1 mM ethylenediaminetetraacetic acid-Tris (hydroxymethyl) aminomethane (EDTA-Tris, pH 7.4), 12.5 mM N-(2-Hydroxyethyl)piperazine-N’-(2-ethanesul- fonic acid) (HEPES-Tris, pH 7.4), 1 mM phenylmethylsulfonyl fluoride (PMSF) and 50 units/ml aprotinin and centrifuged in a Beckman ultracentrifuge (SW-28 bucket rotor) at 10,000 × g and 4˚C for 25 min. The supernatant was centrifuged at 100,000 × g and 4˚C for 1 h. The supernatant was discarded and microsomal fraction enriched plasma membranes (pellet) was resuspended in 1 mM EDTA-Tris (pH 7.4), 12.5 mM HEPES-Tris (pH 7.4), stirred for 15 min and centrifuged (100,000 × g, 45 min). The resulting pellets were resuspended in 12.5 mM HEPES-Tris (pH 7.4) and frozen at −80˚C. This plasma membrane rich fraction was used for further measurements of the ATPase activity.
The enzyme preparation (20 - 25 μg) was added to 0.5 ml incubation medium containing 12.5 mM HEPES-Tris buffer (pH 7.4), 1.0 mM MgSO4, 1.0 mM ATP-Tris, 10 mM NaCl/2 mM NaHCO3 and 60 mM NaNO3 (neutral salt) to measure enzyme activity. The specific activity of ATPase was estimated from the increase in the content of inorganic phosphorus (Pi) in 0.5 ml incubation medium at 30˚C for 30 min. Phosphorus concentration in samples was measured by the method of Chen and expressed in μmol Pi/h/mg protein [
The enzyme activity in the presence of chemicals (Ca2+, EGTA, GABA, bicuculline, picrotoxine, o-vanadate, genistein) was determined as described before [
All drugs were prepared as stock solutions in water unless otherwise stated. GABA, picrotoxin, bicuculline methochloride, CaCl2, EGTA, Tris, Hepes, Na2ATP, o-vanadate, genistein were by Sigma-Aldrich.
The data are expressed with mean ± standard error where appropriate. The experimental data are statistically processed using one-way ANOVA test program “Statistica 7.0”. Evaluation of the significance of differences was carried out at p < 0.05 (n = 4).
We showed earlier that the multifunctional ATPase complex is the enzyme system, including “basal” Mg2+- ATPase, which is stimulated by anions and regulated (activated/inhibited) by GABAА-ergic ligands. In the samples of plasma membrane from rat brain studied by us, the activity of the “basal” Mg2+-ATPase is 8.8 μmol Pi/h/mg protein. This ATPase activity is stimulated by ions 10 mM Cl−/2 mM
To verify that the enzymatic activity under study is a GABAA-coupled ATPase, we added GABAA-ergic ligands (GABA, bicuculline, picrotoxin) to the incubation medium. GABA (10 mM) activated the “basal” Mg2+- ATPase, while no
The literature shows that Ca2+ modulates the activity of the transport ATPase P-type of different cells. In particular, it was shown that EGTA (EDTA) and Ca2+ can modify the neuronal membrane Na+, K+-ATPase [
(a) The “basal” (I) and, -stimulated (II) ATPase activities of rat brain plasma membranes in the absence (1) or in the presence of 10 mM GABA (2), 10 mM GABA + 20 mM bicuculline (3), 10 mM GABA + 50 mM pirotoxin (4) and (b) The “basal” (I) and, -stimulated (II) Mg2+-ATPase activities in the presence of the different Ca2+ concentrations. Plasma membrane samples (20 - 25 mg) were added to incubation medium containing 12.5 mM HEPES-Tris (pH 7.4) and GABAAergic drugs and preincubated at 30˚C for 20 min. The reaction was started by addition of substrate (Mg2+-ATP) in the incubation medium
“basal” Mg2+-ATPase decreased to 55% and amounted to 4.4 mmol Pi/h/mg protein. Along with this, the
The observed Ca2+ concentrations (0.5 - 1 mM) that cause the greatest change in the activity of the ATPase under study are similar to concentrations that inhibit the GABAA-induced
Furthermore, it was found that [Ca2+]i accelerates reduction of function (run-down effect) of GABAA-induced
Bicuculline is known to competitively interact with the allosteric binding site close to the
Effect of GABA on the “basal” Mg2+-ATPase activity of rat brain plasma membranes in the absence (1) and in the presence of 0.01 mM (2) or 0.25 mM (3) Ca2+ in the incubation medium. Plasma membrane samples (20 - 25 mg) were added to incubation medium containing 12.5 mM HEPES-Tris (pH 7.4) and GABAA-ergic drugs and preincubated at 30˚C for 20 min. The reaction was started by addition of substrate (Mg2+-ATP) in the incubation medium
We have found that bicuculline inhibits the investigated activity starting with the concentration of 2.5 μM, and shows the greatest effect at the concentration of 15 μM. Ca2+ (0.25 mM) eliminates the inhibitory effect of bicuculline on the
These data suggest that Са2+ has protective properties against the action of the GABAA-receptor blocker on the enzyme. In this regard, it seemed appropriate to investigate the effect of different concentrations of Ca2+ (0.01 - 1 mM) on the
It is known from the literature that the function of the GABAA-receptors is supported by processes of
Effect of bicuculline (a) or calcium (b) on the, -stimulated ATPase activity of rat brain plasma membranes in the absence and in the presence of Ca2+ or bicuculline, respectively. Plasma membrane samples (20 - 25 mg) were added to incubation medium containing 12.5 mM HEPES-Tris (pH 7.4) and GABAAergic drugs and preincubated at 30˚C for 20 min. The reaction was started by addition of substrate (Mg2+-ATP) in the incubation medium
phosphorylation of receptor molecule or tightly bound regulatory molecules [
To investigate the possible involvement of these phosphatases in the action of Ca2+ on the investigated ATPase, we added o-vanadate and Ca2+ to the incubation medium (
Previously, Hyden and colleagues showed the existence on rabbit Deiters’ neuron membrane of molecular (protein) machineries which recognize intracellular GABA and extrude chloride [
(a) Effect of GABA on the “basal» Mg2+-ATPase activity of rat brain plasma membranes in the absence (1) and in the presence of 0.1 mM genistein (2) or 0.1 mM o-vanadate (3); (b) The “basal” (I) and, -stimulated (II) ATPase activities of the neuronal membrane in the absence (1) and in the presence 0.25 mM Ca2+ (2), 0.1 mM o-vanadate (3) and 0.25 mM Ca2+ + 0.1 mM o-vanadate (4). Plasma membrane samples (20 - 25 mg) were added to incubation medium containing 12.5 mM HEPES-Tris (pH 7.4) and GABAAergic drugs and preincubated at 30˚C for 20 min. The reaction was started by addition of substrate (Mg2+-ATP) in the incubation medium
properties have suggested to these authors that these receptors are GABAA-activated
Analogously, the results of this work and preliminary our studies have shown that the investigated ATPase was inhibited by picrotoxin (or bicuculline) and regulated by modulators (anticonvulsants, benzodiazepines, anesthetics) [
Cations of calcium play a vital role in the function of cells of various origin (including neurons). The concentration gradient of calcium across the plasma membrane of neuronal cells is a very high, from ~10−3 M Ca2+ outside, to ~10−7 M Ca2+ inside [
Calcium has been shown to exert a powerful inhibitory effect on the Na+, K+-ATPase of cell membranes [
Our data demonstrate, for the first time, the sensitivity of the investigated multifunctional ATPase complex to calcium cations. This conclusion is well demonstrated by our results on reduction of the activity of “basal” Mg2+-ATPase and stimulation of the
In this paper, we have found that in the presence of Са2+ in the incubation medium, no effect of bicuculline on the
This study provides an additional biochemical characterization of multifunctional ATPase. Furthermore, cations of calcium regulate the ATPase activity in the absence of drugs. Consequently, the modulation of the multifunctional ATPase activity by GABAA-ergic drugs is a Ca2+-dependent process. It is important to understand the basic properties of this new multifunctional ATPase system and how it responds to changes in its environment. The obtained results seem to have an important functional significance in the study of the mechanisms of epileptogenesis and convulsant-induced seizure activity.