Effects of Calcium on the GABAA-Coupled 3 Cl HCO − −-ATPase from Plasma Membrane of Rat Brain

The work is a study of the influence of Ca2+ (0.01 1 mM) on neuronal Cl−, 3 HCO − -ATPase complex: an enzyme that is a Cl−-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 μM and at concentration of 0.1 mM, it reduces the “basal” one and increases the Cl−, 3 HCO − -stimulated Mg2+-ATPase activities. GABA (0.1 100 μM) activates the “basal” Mg2+-ATPase activity in the absence of calcium. The effect of GABA on the enzyme in the presence of 0.01 μM 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 eliminates the stimulation of the “basal” Mg2+-ATPase by anions. When 0.25 mM Ca2+ is added to the incubation 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 Cl−, 3 HCO − -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.


Introduction
Cl − -ATPase/Cl − -pump in plasma membrane from various cells (including neurons) is a "molecular machine" participating in the transportation of Cl − ions against the electrochemical gradient [1] [2].Earlier, we showed the existence of the anion-sensitive Mg 2+ -ATPase in developed neurons of animal brain with maximum activity in the presence of Cl − / 3 HCO − ions (in the ratio of as 5:1) [3].These data are in line with the electrophysiological studies demonstrating that Cl − , 3 HCO − anions are transported through the GABA А -receptor Cl − -channel in the ratio as 5:1, respectively [4].Besides, this enzyme is functionally and structurally coupled with the GABA А / benzodiazepine receptor complex [3].With the use of biochemical and cytochemical methods, it has been established that Cl − , 3 HCO − -ATPase is localized in elements of GABA А -ergic dendro-dendritic synapses [5].The hydrolytic activity of this GABA А -coupled ATPase provides the Cl − / 3 HCO − -transport processes with energy and determines the direction of ion transport through neuronal membrane [6].This conclusion is based on the following findings: the protein preferably hydrolyzes ATP and is covalently phosphorylated by ATP (directly or with the participation of protein kinase) during the transport cycle and is dephosphorylated by anions [7].These data allow us to suppose that this is a chloride transporting ATPase: this multifunctional ATPase is a Cl − -pump participating in transporting chloride ions through the native and artificial membranes of liposomes.
Furthermore, we have found that such ATPase is involved in rat convulsant-induced seizure activity [8].It is known that the pathogeneses of epilepsy and convulsive states are associated not only with impairment of the GABA A -receptor function [9] [10] but also with calcium homeostasis [11].It is known that the level of intracellular free calcium [Ca 2+ ] i in the nerve cells is vital for the process of neuronal transmission [12].Moreover, cations of calcium and magnesium are required to maintain the functional activity of many receptors and enzyme systems (including the GABA A -receptor) [13].The influence of [Ca 2+ ] i on the GABA A -receptors in the brain is exerted either directly through binding sites on the receptor molecule [14] or via Ca 2+ -dependent enzymatic processes (including protein kinases or protein phosphatases) [15].In this regard, in the present work we have studied the influence of Ca 2+ on the multifunctional ATPase activity of the neuronal membranes in the absence and in the presence of GABA A -ergic ligands and blockers of protein kinases and protein phosphatases.

Animals
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.

HCO − -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 MgSO 4 , 1.0 mM ATP-Tris, 10 mM NaCl/2 mM NaHCO 3 and 60 mM NaNO 3 (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" Mg 2+ -ATPase was calculated as the difference between the ATPase activities in the presence and absence of MgSO 4 in the incubation medium containing 12.5 mM HEPES-Tris buffer (pH 7.4), 1.0 mM MgSO 4 , 1.0 mM Tris-ATP and 60 mM NaNO 3 .The Cl − , 3 HCO − -activated Mg 2+ -ATPase was determined in the presence of Cl − / 3 HCO − ions in the incubation medium containing 12.5 mM HEPES-Tris buffer (pH 7.4), 1.0 mM MgSO 4 , 1.0 mM Tris-ATP, 10 mM NaCl/2 mM NaHCO 3 and 60 mM NaNO 3 .The enzyme activation by anions was calculated as the difference between the "basal" Mg 2+ -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.

Assay of the Action of Chemicals on the ATPase Activity
The enzyme activity in the presence of chemicals (Ca 2+ , 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 NaHCO 3 and 60 mM NaNO 3 .The reaction was started by addition of the substrate (Mg 2+ -ATP) to the incubation medium.

Statistics
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).

HCO − -ATPase Activity
We showed earlier that the multifunctional ATPase complex is the enzyme system, including "basal" Mg 2+ -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" Mg 2+ -ATPase is 8.8 μmol P i /h/mg protein.This ATPase activity is stimulated by ions 10 mM Cl − /2 mM 3 HCO − , the stimulation effect ( Cl − , 3 HCO − -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 µM) activated the "basal" Mg 2+ -ATPase, while no Cl − , 3 HCO − -ATPase activity could be detected.This effect of the mediator on the enzyme was eliminated by bicuculline (20 µM) and picrotoxin (50 µM) (Figure 1(a)).These data confirm that the AT-Pase 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" Mg 2+ -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 Ca 2+ modulates the activity of the transport ATPase P-type of different cells.In particular, it was shown that EGTA (EDTA) and Ca 2+ 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" Mg 2+ -ATPase or "basal" Mg 2+ -ATPase that are insensitive to ouabain [21] [22].Therefore, we investigated the effect of Ca 2+ on the "basal" and Cl − , "basal" Mg 2+ -ATPase decreased to 55% and amounted to 4.4 µmol P i /h/mg protein.Along with this, the Cl − , 3 HCO − -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 Ca 2+ chelator causes an increase activity of "basal" Mg 2+ -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 µM) in the incubation medium, the activity of "basal" Mg 2+ -ATPase was increased by 65% [20].

Effect of Ca 2+ on the ATPase Activity in the Presence of GABAA-Ergic Drugs
The observed Ca 2+ 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 Cl − -current.It was shown that intracel- lular [Ca 2+ ] i , depending on the concentration, has a multidirectional effect on the GABA A -receptors.Low (0.01 mM) concentrations of Ca 2+ cause potentiation of the GABA A -induced Cl − -current, while high (500 µM) con- centrations reduce their functional activity by decreasing the opening time of the GABA A -receptor Cl − -channel [13].Furthermore, it was found that [Ca 2+ ] i accelerates reduction of function (run-down effect) of GABA A -induced Cl − -current in hippocampal neurons in rats [23] [24].Therefore, the next step in our work was to study the ef- fect of Ca 2+ on the GABA A -activated Mg 2+ -ATPase activity (Figure 2).We found that low concentrations of Са 2+ (0.01 µM) do not affect this enzymatic activity.At the same time, high concentrations of Са 2+ (0.25 mM) eliminate activation of the "basal" Mg 2+ -ATPase by GABA.
Bicuculline is known to competitively interact with the allosteric binding site close to the Cl − -channel of the GABA A /benzodiazepine receptor complex.This interaction results in a change in the conformation of Cl − - channel and reduction of Cl − -conductance in the neuron [24].The results of our earlier studies showed the func- tional and structural conjugation of the investigated neuronal membrane ATPase with GABA A /benzodiazepine  Cl − -channel receptor complex [18].Therefore, we studied the effect of different concentrations (2.5 -20 µM) of bicuculline on the Cl − , 3 HCO − -ATPase activity in the absence and in the presence of Ca 2+ (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.Ca 2+ (0.25 mM) eliminates the inhibitory effect of bicuculline on the Cl − , 3 HCO − -ATPase activity.These data suggest that Са 2+ 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 Ca 2+ (0.01 -1 mM) on the Cl − , 3 HCO − -ATPase activity in the presence of bicuculline (20 µM).It was found that Ca 2+ 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.

Role of Phosphatases on Modulation of the ATPase Activity by Ca 2+
It is known from the literature that the function of the GABA A -receptors is supported by processes of HCO − -stimulated ATPase activity of rat brain plasma membranes in the absence and in the presence of Ca 2+ or bicuculline, respectively.Plasma membrane samples (20 -25 µg) were added to incubation medium containing 12.5 mM HEPES-Tris (pH 7.4) and GABA A ergic drugs and preincubated at 30˚C for 20 min.The reaction was started by addition of substrate (Mg 2+ -ATP) in the incubation medium.
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 36 Cl 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 -induced Mg 2+ -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 Ca 2+ on the investigated AT-Pase, we added o-vanadate and Ca 2+ to the incubation medium (Figure 4).It was found that independent action of each of the two substances reduces the "basal" Mg 2+ -ATPase and increases the Cl − , 3 HCO − -ATPase activity.The combined action of these substances does not result in the increase of their inhibitory effect on the "basal" Mg 2+ -ATPase activity.These results are in a good agreement with the data obtained in the study of vanadatesensitive alkaline phosphatase conjugated with GABA A -receptors [13].Its role in the regulation of the GABA Areceptor 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 Cl − after postsy- naptic 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 Cl − -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 Cl − -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 Cl − -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 AT-Pase complex-P (first functional state) or dephosphorylated form (second functional state).The former case has a low "basal" Mg 2+ -ATPase activity and it is activated by Cl − / 3 HCO − 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" Mg 2+ -ATPase activity and it is not activated by anions.The dephosphorylation process is catalyzed by a vanadate-sensitive phosphatase.Thus, with the provision of Mg 2+ -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 Cl − -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 Ca 2+ 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 concen-tration gradient of calcium across the plasma membrane of neuronal cells is a very high, from ~10 −3 M Ca 2+ outside, to ~10 −7 M Ca 2+ 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 Ca 2+ (1 -5 mM) decreased the number of GABA binding sites in rat cortical synaptic membranes [14].Increases in [Ca 2+ ] i (>1 µM) were reported in some works to reduce the open time [31] or to cause depression [23] of GABA A -activated Cl − -channels in pituitary cells and dentate granule cells, respectively.In contrast, maintenance of a low level of [Ca 2+ ] i (<0.1 µM) was required for full activation of GABA A -induced Cl − -cur- rent in guinea pig hippocampal neurons [32].An increased intracellular Ca 2+ concentration (10 nM -34 µM) caused a transient augmentation of the GABA A -induced Cl − -current [15].Moreover, it was established, that Ca 2+ (depending on the concentration) has a biphasic effect on synaptic GABA A receptor Cl − -channel [23].So, the amplitude of GABA A -induced Cl − -current recorded with 1 mM internal CaCl 2 and 10 mM EGTA (10 nM free Ca 2+ ) decayed by less than 30% of control.At the same time, increasing the CaCl 2 concentration to 10 mM (34 µM free Ca 2+ ) 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, Ca 2+ (10 −6 -3 × 10 −3 M) always exerts an inhibitory effect on the Na + , K + -ATPase [34].Addition of Ca 2+ to the incubation medium in the absence of EDTA caused no change in the "basal" Mg 2+ -ATPase activity at >10 −3 M Ca 2+ .However at low (1 -3 µM) Ca 2+ in the media there was a significant stimulation of the Na + , K + -ATPase activity and decreasing as Ca 2+ increased.So, at 10 −3 M Ca 2+ 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, Ca 2+ (>50 μM) inhibits the activity of basal Mg 2+ -ATPase and greatly increases the Cl − , 3 HCO − -ATPase activity.However the effects seen at millimolar Ca 2+ 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" Mg 2+ -ATPase and stimulation of the Cl − , 3 HCO − -ATPase activity with the increase of Ca 2+ concentration in the incubation medium.Interdependent multidirectional response of these two ATPase activities to change in concentration of Ca 2+ confirms the conjugation between investigated enzymes, and their association to the same complex.Furthermore, it indicates their involvement in Ca 2+ -dependent processes.This is confirmed by the results of the study of the effect of Ca 2+ on the "basal" and Cl − , 3 HCO − -activated Mg 2+ -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 Ca 2+ 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, Ca 2+ can interact directly with the receptors either through binding sites on the molecule, or through receptor-conjugated enzyme systems (in particular Ca 2+ /calmodulin-dependent protein phosphatase, or via Ca 2+ -dependent protein kinase).In our study, the conjugation of the investigated ATPase activities and their involvement in Ca 2+ -dependent processes is also demonstrated by the effect of protein tyrosine phosphatase and protein tyrosine kinase blockers.Specifically, we have shown that the GABA-induced Cl − -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 Ca 2+ inhibited the GABA A -induced "basal" Mg 2+ -ATPase activity, and genistein did not affect the effect of the GABA on the enzyme.Therefore, although the "basal" and Cl − , gation of their anticonvulsive therapeutic effect in experiments in vivo.

Conclusion
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 GABA A -ergic drugs is a Ca 2+ -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.

Figure 2 .
Figure 2. Effect of GABA on the "basal" Mg 2+ -ATPase activity of rat brain plasma membranes in the absence (1) and in the presence of 0.01 µM (2) or 0.25 mM (3) Ca 2+ in the incubation medium.Plasma membrane samples (20 -25 µg) were added to incubation medium containing 12.5 mM HEPES-Tris (pH 7.4) and GABA A -ergic drugs and preincubated at 30˚C for 20 min.The reaction was started by addition of substrate (Mg 2+ -ATP) in the incubation medium.

Figure 3 .
Figure 3.Effect of bicuculline (a) or calcium (b) on the Cl − ,