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 (P_i) 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 P_i/h/mg protein [16] [17] . The activity of the “basal” Mg²⁺-ATPase was calculated as the difference between the ATPase activities in the presence and absence of MgSO₄ in the incubation medium containing 12.5 mM HEPES-Tris buffer (pH 7.4), 1.0 mM MgSO₄, 1.0 mM Tris-ATP and 60 mM NaNO₃. The, -activated Mg²⁺-ATPase was determined in the presence of / ions in the incubation medium containing 12.5 mM HEPES-Tris buffer (pH 7.4), 1.0 mM MgSO₄, 1.0 mM Tris-ATP, 10 mM NaCl/2 mM NaHCO₃ and 60 mM NaNO₃. The enzyme activation by anions was calculated as the difference between the “basal” Mg²⁺-ATPase activities in the presence and absence of anions (chloride/bicarbonate) in the incubation medium. The figures show values of the enzyme activity averaged from the results of at least four determinations.

The enzyme activity in the presence of chemicals (Ca²⁺, EGTA, GABA, bicuculline, picrotoxine, o-vanadate, genistein) was determined as described before [3] . Membrane samples were preincubated at 30˚C for 20 min with the relevant chemical in incubation medium containing 12.5 mM HEPES-Tris buffer (pH 7.4), 10 mM NaCl/2 mM NaHCO₃ and 60 mM NaNO₃. The reaction was started by addition of the substrate (Mg²⁺-ATP) to the incubation medium.

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 GABA_А-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 P_i/h/mg protein. This ATPase activity is stimulated by ions 10 mM Cl⁻/2 mM, the stimulation effect (,-ATPase activity) is 2.4 μmol P_i/h/mg protein.

To verify that the enzymatic activity under study is a GABA_A-coupled ATPase, we added GABA_A-ergic ligands (GABA, bicuculline, picrotoxin) to the incubation medium. GABA (10 mM) activated the “basal” Mg²⁺- ATPase, while no, -ATPase activity could be detected. This effect of the mediator on the enzyme was eliminated by bicuculline (20 mM) and picrotoxin (50 mM) (Figure 1(a)). These data confirm that the ATPase activity under study belongs to the same enzyme—GABA_A-coupled ATPase complex. Therefore, if in the presence of an activator the activity of the “basal” Mg²⁺-ATPase achieves high levels when the molecular turnover is maximal, then an additional activation of the enzyme by anions cannot take place.

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 [19] [20] . At the same time, there was observed a change in the activity of both transport P-type ATPases and “total” Mg²⁺-ATPase or “basal” Mg²⁺-ATPase that are insensitive to ouabain [21] [22] . Therefore, we investigated the effect of Ca²⁺ on the “basal” and, -activated Mg²⁺-ATPase activities of the neuronal membranes. In our experiments, Ca²⁺ (0.01 - 1 mM) was shown to reduce the activity of “basal” Mg²⁺-ATPase starting from the concentration of 0.05 mM (Figure 1(b)). In the highest studied concentration of Ca²⁺ (1 mM) the activity of the

“basal” Mg²⁺-ATPase decreased to 55% and amounted to 4.4 mmol P_i/h/mg protein. Along with this, the, -ATPase activity doubled. In the presence of EGTA (0.1 mM), the activating effect of calcium ions on the enzyme activity does not occur (data not shown). Furthermore, this Ca²⁺ chelator causes an increase activity of “basal” Mg²⁺-ATPase by about 17%. These data indicate the presence of free calcium in the incubation medium similar to the literature data. So, in the presence of EDTA (40 mM) in the incubation medium, the activity of “basal” Mg²⁺-ATPase was increased by 65% [20] .

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 GABA_A-induced -current. It was shown that intracellular [Ca²⁺]_i, depending on the concentration, has a multidirectional effect on the GABA_A-receptors. Low (0.01 mM) concentrations of Ca²⁺ cause potentiation of the GABA_A-induced -current, while high (500 mM) concentrations reduce their functional activity by decreasing the opening time of the GABA_A-receptor -channel [13] .

Furthermore, it was found that [Ca²⁺]_i accelerates reduction of function (run-down effect) of GABA_A-induced -current in hippocampal neurons in rats [23] [24] . Therefore, the next step in our work was to study the effect of Ca²⁺ on the GABA_A-activated Mg²⁺-ATPase activity (Figure 2). We found that low concentrations of Са²⁺ (0.01 mM) do not affect this enzymatic activity. At the same time, high concentrations of Са²⁺ (0.25 mM) eliminate activation of the “basal” Mg²⁺-ATPase by GABA.

Bicuculline is known to competitively interact with the allosteric binding site close to the -channel of the GABA_A/benzodiazepine receptor complex. This interaction results in a change in the conformation of - channel and reduction of -conductance in the neuron [24] . The results of our earlier studies showed the functional and structural conjugation of the investigated neuronal membrane ATPase with GABA_A/benzodiazepine

-channel receptor complex [18] . Therefore, we studied the effect of different concentrations (2.5 - 20 mM) of bicuculline on the, -ATPase activity in the absence and in the presence of Ca²⁺ (Figure 3(a)).

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, -ATPase activity.

These data suggest that Са²⁺ has protective properties against the action of the GABA_A-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, -ATPase activity in the presence of bicuculline (20 mM). It was found that Ca²⁺ eliminates the inhibitory effect of bicuculline on the ATPase activity starting from 50 μM (Figure 3(b)). The greatest increase of the effect of calcium cations is observed in the concentration range of 0.1 - 0.5 mM.

It is known from the literature that the function of the GABA_A-receptors is supported by processes of

phosphorylation of receptor molecule or tightly bound regulatory molecules [13] [25] [26] . In the developed nervous system a high activity of protein tyrosine kinases and protein tyrosine phosphatases was shown, suggesting that protein tyrosine phosphorylation is an important factor for neuronal function. It has been shown that inhibitors of these enzymes regulate the functional activity of receptors involved in excitatory and inhibitory processes [10] . Thus, o-vanadate, blocker of protein phosphatases and transport ATPases P-type, increased the effect of GABA on the GABA_A-receptors. Genistein and tyrphostin, blockers of protein tyrosine kinases, inhibited the GABA_A-induced accumulation of ³⁶Cl by brain membrane vesicles in mice, and GABA_A-induced Cl^--current in brain neuronal membranes in rats [27] [28] . These data suggest an important role of these enzymes in the maintenance of GABA_A-receptor function. In our study, o-vanadate (0.1 mM) reduces the GABA_A-in- duced Mg²⁺-ATPase activity. At the same time, genistein (0.1 mM) has no effect on this ATPase activity.

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 (Figure 4). It was found that independent action of each of the two substances reduces the “basal” Mg²⁺-ATPase and increases the, -ATPase activity. The combined action of these substances does not result in the increase of their inhibitory effect on the “basal” Mg²⁺-ATPase activity. These results are in a good agreement with the data obtained in the study of vanadate- sensitive alkaline phosphatase conjugated with GABA_A-receptors [13] . Its role in the regulation of the GABA_A- receptor function was confirmed by addition of the enzyme to intracellular perfusate, which caused complete decline (run-down effect) of their function. Inhibition of such phosphatase by o-vanadate induced recovery of the GABA_A-receptor function [10] [27] [28] .

Previously, Hyden and colleagues showed the existence on rabbit Deiters’ neuron membrane of molecular (protein) machineries which recognize intracellular GABA and extrude chloride [29] . It was suggested that these structures are devices that at the expense of ATP consumed in their phosphorylation, extrude after postsynaptic GABA uptake into the Deiters’ neurons. The GABA effect was blocked by classical GABA_A antagonists picrotoxin (100 µM) and bicuculline (10 µM) and also activated in a biphasic manner by pentobarbitone. Such

properties have suggested to these authors that these receptors are GABA_A-activated -pumps, where the energy for chloride extrusion is provided by ATP in a phosphorylation step within the extrusion cycle. The core mechanism is the inversion of two energy peaks along the permeation pathway. However, the role of ATP in the phosphorylation step of GABA_A-regulated -pump is not conclusively established.

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) [18] . Our biochemical and cytochemical findings enabled us postulating a new model of activity of the multifunctional ATPase complex—an enzyme that is also a -pump and a receptor. We propose that the ATPase complex is closely related to GABA_A-receptor and therefore can exist either in a phosphorylated ATPase complex-P (first functional state) or dephosphorylated form (second functional state). The former case has a low “basal” Mg²⁺-ATPase activity and it is activated by / ions. This state enables the protein to participate in the ATP-dependent transport of anions. Phosphorylation is opposed by a dephosphorylation process which renders the ATPase complex as nonfunctional (the enzyme cannot participate in the ATP-dependent transport of anions). In this case, it has a high “basal” Mg²⁺-ATPase activity and it is not activated by anions. The dephosphorylation process is catalyzed by a vanadate-sensitive phosphatase. Thus, with the provision of Mg²⁺-ATP, a protein kinase (or directly ATP) phosphorylates the molecular complex and maintains the ATPase functional form. A similar cycle has been suggested to play a role in the regulation of GABA_A-receptor [28] and GABA_A-activated -pump. Yet, our results demonstrates that after convulsant effect, the dephosphorylation of the enzyme also occurs. As a result the ATPase doesn’t participate in the chloride transport (as the “collapsed” state). Our results suggest that not only inhibitors (o-vanadate, genistein, convulsants) have effect on the phosphatase activity, but also Ca²⁺ ions have an influence on the enzyme activity, as a result of their effect on the state of the protein phosphorylation.

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 [30] . The free calcium concentration in neurons is supported by various mechanisms (buffering systems, compartmentation and extrusion from the neuronal cells). In earlier studies it was shown that Ca²⁺ (1 - 5 mM) decreased the number of GABA binding sites in rat cortical synaptic membranes [14] . Increases in [Ca²⁺]_i (>1 µM) were reported in some works to reduce the open time [31] or to cause depression [23] of GABA_A-activated -channels in pituitary cells and dentate granule cells, respectively. In contrast, maintenance of a low level of [Ca²⁺]_i (<0.1 µM) was required for full activation of GABA_A-induced -cur- rent in guinea pig hippocampal neurons [32] . An increased intracellular Ca²⁺ concentration (10 nM - 34 µM) caused a transient augmentation of the GABA_A-induced -current [15] . Moreover, it was established, that Ca²⁺ (depending on the concentration) has a biphasic effect on synaptic GABA_A receptor -channel [23] . So, the amplitude of GABA_A-induced -current recorded with 1 mM internal CaCl₂ and 10 mM EGTA (10 nM free Ca²⁺) decayed by less than 30% of control. At the same time, increasing the CaCl₂ concentration to 10 mM (34 µM free Ca²⁺) induced a transient potentiation of the GABA_A-current [33] .

Calcium has been shown to exert a powerful inhibitory effect on the Na⁺, K⁺-ATPase of cell membranes [19] [34] . In particular, it was shown that in the presence of EDTA, Ca²⁺ (10⁻⁶ - 3 × 10⁻³ M) always exerts an inhibitory effect on the Na⁺, K⁺-ATPase [34] . Addition of Ca²⁺ to the incubation medium in the absence of EDTA caused no change in the “basal” Mg²⁺-ATPase activity at >10⁻³ M Ca²⁺. However at low (1 - 3 µM) Ca²⁺ in the media there was a significant stimulation of the Na⁺, K⁺-ATPase activity and decreasing as Ca²⁺ increased. So, at 10⁻³ M Ca²⁺ in the incubation medium an inhibition by 61% of the Na⁺, K⁺-ATPase activity occurred. Calcium concentrations that affect Na⁺, K⁺-ATPase are similar to concentrations that are effective on the ATPase we studied here. In our study, Ca²⁺ (>50 μM) inhibits the activity of basal Mg²⁺-ATPase and greatly increases the, -ATPase activity. However the effects seen at millimolar Ca²⁺ levels may not be seen in the cell except, perhaps, transiently.

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, -ATPase activity with the increase of Ca²⁺ concentration in the incubation medium. Interdependent multidirectional response of these two ATPase activities to change in concentration of Ca²⁺ confirms the conjugation between investigated enzymes, and their association to the same complex. Furthermore, it indicates their involvement in Ca²⁺-dependent processes. This is confirmed by the results of the study of the effect of Ca²⁺ on the “basal” and, -activated Mg²⁺-ATPase activities in the presence of GABA_A-ergic ligands, which are presented in this paper and in earlier studies. They also show a multidirectional GABA_A-regulation of the ATPase activity. These data are in good agreement with the published data on the effect of Ca²⁺ on the functional activity of the GABA_A-receptors [14] [15] . The authors demonstrated that GABA_A-receptor activity varies depending on intracellular cation concentration. Moreover, Ca²⁺ can interact directly with the receptors either through binding sites on the molecule, or through receptor-conjugated enzyme systems (in particular Ca²⁺/calmodulin-dependent protein phosphatase, or via Ca²⁺-dependent protein kinase). In our study, the conjugation of the investigated ATPase activities and their involvement in Ca²⁺-depen- dent processes is also demonstrated by the effect of protein tyrosine phosphatase and protein tyrosine kinase blockers. Specifically, we have shown that the GABA-induced -ATPase activity is not sensitive to o-vanadate blocker but is inhibited by genistein blocker [35] . In contrast, in this work, o-vanadate in the absence of Ca²⁺ inhibited the GABA_A-induced “basal” Mg²⁺-ATPase activity, and genistein did not affect the effect of the GABA on the enzyme. Therefore, although the “basal” and, -activated Mg²⁺-ATPase activities are structurally conjugated, their functional activities can be dissociated by means of blockers of Ca²⁺-dependent protein kinases and protein phosphatases. This process can involve GABA_Aergic ligands.

In this paper, we have found that in the presence of Са²⁺ in the incubation medium, no effect of bicuculline on the, -ATPase activity could be observed. Based on these data, it can be concluded that Са²⁺ plays an ATPase-protective role. In therapeutic practice bicuculline is known as a convulsant. Therefore, our results can be important in the study of mechanisms of epileptogenesis, which are caused by various neurological or systemic disorders. This largely relates to electrolyte imbalance, especially calcium and magnesium imbalance. In particular, abnormalities of Са²⁺ homeostasis can affect the neuromuscular excitability, forming convulsive readiness in the brain, and hypocalcemia is often a side effect of anticonvulsant treatment [11] . In most cases, patients with epilepsy have hypocalcemia and hypomagnesemia [36] [37] . Our results on the protective role of Ca²⁺ for multifunctional ATPase during the action of convulsants suggest the use of these cations in the investigation of their anticonvulsive therapeutic effect in experiments in vivo.