GABAA-Coupled Cl − / 3 HCO −-ATPase from Plasma Membrane of the Rat Brain : Role of 3 HCO − in the Enzyme Activation

This work examines the influence of Cl− (2.5 125 mM) and 3 HCO − (2 30 mM) on the Cl−/ 3 HCO − ATPase complex of the neuronal membrane and this enzyme is a Cl−-pump that is coupled to GABAA receptors. The greatest (44%) activating effect on the enzyme is found with 3 HCO − (20 30 mM), while the maximum activity occurs in the presence of a ratio of ~25 mM 3 HCO − /~5mM Cl−. Blockers of the GABAA receptor, namely bicuculline (10 50 μM) and picrotoxin (50 100 μM), inhibit this anion activation, whereas the 3 HCO − -ATPase activity is not sensitive to these ligands. Autoradiographic analysis of the spectrum of the partially purified enzyme phosphorylated with [γ-32P]ATP allowed us to distinguish three major 32P-labeled protein whose molecular weight are about 57, 53, and 48 kDa. In the presence of 5 mM Cl−/25mM 3 HCO − and 100 μM picrotoxin, the intensity of the phosphorylation of bands significantly decreased, thereby confirming the assumption about coupled of binding sites for anions and GABAA-ergic ligands. It was suggested scheme of Cl−-transport through the plasma membrane by utilizing neuronal Cl−/ 3 HCO − -ATPase in the low (5 mM) Cl− and high (25 mM) 3 HCO − concentrations. The data demonstrated for the first time that the GABAA-coupled Cl−/ 3 HCO − -ATPase from rat brain neuronal membranes is maximally activated at a Cl−/ 3 HCO − ratio of 1:5 and it remains stable at high concentrations of substrate and buffer. Corresponding author. S. A. Menzikov et al.


Introduction
The Cl − -ATPase/Cl − -pump (EC 3.6.3.11-Cl− -transporting ATPase) from the plasma membrane of different cells (including neurons) is a "molecular machine" that participates in Cl − -transport against an electrochemical gradient [1]- [3].We have determined that neuronal membranes from animal brain contain a Cl − -ATPase that is functionally coupled with the GABA A /benzodiazepine receptor/Cl − -channel complex [4]- [6].Cl − -transport through this ATPase in liposomal membranes depends on the presence of 3 HCO − ions in the incubation me- dium [7].In our preliminary studies, we relied on data from electrophysiological studies of GABA A receptor function to identify enzymatic activity.In particular, the literature showed that Cl − and 3 HCO − ions are trans- ported through the GABA A receptor/Cl − -channel in a 5:1 ratio, respectively [8].Indeed, the presence of Cl − and 3 HCO − ions (in a 5:1 ratio) in the incubation medium resulted in greater Cl − / 3 HCO − -ATPase activity than that was seen in the presence of either of these anions alone [5] [6].Synergistic activation of the ATPase by anions was also observed in both low (~10 mM/~2mM) and high (~50 mM/~10mM) concentrations of Cl − and 3 HCO − ions, respectively [5] [6].These data suggest the participation of the enzyme in GABA A -induced Cl − / 3 HCO − ex- change processes and, as a consequence, in the hyperpolarization/depolarization of the neuronal membrane [9]- [12].At the same time, there are other reasons that require the search for new concentration ratios anions activating ATPase-low enzyme activity and the lack of stability of the enzyme activity in the presence of high concentrations of the substrate and buffer molarity.The literature also indicated that the concentrations of Cl − and 3 HCO − ions both inside and outside fully developed neurons were 6 mM and 120 mM, and 16 mM and 27 mM, respectively, which suggested an important role for 3 HCO − ions in the modulation of Cl − -transport [13] [14].In view of this, the goal of this study was to examine effect of Cl − and/or 3 HCO − on the ATPase and its response to anion activation by asking two questions: whether there are other ratios of anions concentrations optimally activating the enzyme and whether such ATPase activity can be sensitive to GABA A -ergic ligands.To address these questions, we performed the phosphorylation of the partially purified enzyme by [γ-32 P]ATP in the absence and in the presence of anions and GABA A -ergic blocker.Here, our biological findings enabled us to postulate a hypothetical scheme of operation of the GABA A -coupled Cl − / 3 HCO − -ATPase complex in the neu- ronal membrane.

Animals
Male Wistar rats (180 -200 g) were obtained from vivarium of the Federal State Scientific Institution "Research Institute of General Pathology and Pathophysiology".Animals were housed in a climate-controlled room on a 12 -22 hour light/dark cycle and where free access to water and food.The experiment was conducted under the "Rules of work with experimental animals" FSSI "RIGPP", which complied with the World Society for the Protection of Animals (WSPA) and the European Convention for the protection of experimental animals.

ATP Hydrolysis Assay
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/2mM 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 [15] [16].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, 2 -125 mM NaCl, 2 -30 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 activity of the GABA A -coupled ATPase of neuronal membrane was calculated as the difference between the ATPase activities in the presence and absence of GABA A -ergic blockers (picrotoxin or bicuculline) in the incubation medium containing 12.5 mM HEPES-Tris buffer (pH 7.4), 1.0 mM MgSO 4 , 1.0 mM Tris-ATP, 5 mM NaCl, 25 mM NaHCO 3 and 60 mM NaNO 3 .The figures show values of the enzyme activity averaged from the results of at least four determinations.

Protein Purification and Phosphorylation
To obtain soluble form of enzyme, plasma membranes were incubated with 1% sodium deoxycholate at room temperature for 20 min and centrifuged at 100,000 g and 4˚C for 30 min.Cl − , 3 HCO − -ATPase was isolated by the method of preparative gel filtration and concentrated using ultrafiltration through Centricon YM-100 filters (Amicon, USA) [6].
Enzyme preparations were frozen at -20˚C and used within 30 days.The membrane preparation was phosphorylated in 30 μl of incubation medium containing 25 mM HEPES-Tris (pH 7.2), 3 mM MgSO 4 , and protein (~25 μg).The reaction of phosphorylation was started by the addition to the incubation medium of 70 μM [γ-32 P]ATP (specific radioactivity, 5 × 10 -6 dpm/nmol) (Amersham, Biosciences).The mixture was incubated at 0˚C -1˚C for 2 min.To study the effect of 5 mM Cl/25 mM HCO 3 and 100 μM picrotoxin on the phosphoprotein formation, the membrane preparation was preincubated with the ligands at 0˚C -1˚C for 15 min [17].

The Enzyme Identification by SDS-PAGE and Autoradiography
Briefly, phosphorylation of protein preparations was stopped by the addition of an equal volume of a buffer containing 100 mM Tris-HCl (pH 6.8), 2% SDS, 2% 2-mercaptoethanol, and 10% sucrose.Electrophoresis was performed in 12% SDS-containing polyacrylamide gel by the method of Laemmli at a current strength of 35 mA [6].Electrophoretograms were stained with 0.1% Coomassie Brilliant Blue R-250.Stained and dried gels were placed in a chamber for autoradiography (Sigma, United States) on a Hyperfilm ™ MP film (Amersham, United States) and exposed at room temperature for 72 -96 h.The film was developed using the standard developer to obtain the maximum contrast image.The molecular weight of proteins was determined by the conventional procedure by comparing their electrophoretic mobility with that of the standard protein markers (BSA, ovalbumin, chymotrypsinogen A, myoglobin, and cytochrome c).

Assay of Action of Chemicals on the Enzyme
The enzyme activity in the presence of GABA A -ergic chemicals (bicuculline, picrotoxin) was determined as described before [6].Membrane samples were preincubated at 30˚C for 20 min with the relevant chemical in incubation medium containing 25 mM HEPES-Tris buffer (pH 7.4), 2.5 -125 mM NaCl/2 -30 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).

Results and Discussion
We have previously found that the activity of the GABA A -coupled ATPase is a "basal" Mg 2+ -ATPase activity that is stimulated by anions [6] [7].The activity of this "basal" Mg 2+ -ATPase from rat brain plasma membranes is 6.7 μmol P i /h/mg protein.Addition of Cl − (0 -125 mM) stimulates the activity of this enzyme (Figure 1(A)), with a maximal effect (9%) observed at 15 mM Cl − .At higher Cl − ion concentrations, no enzyme activation is observed.Addition of 3 HCO − (20 mM) in the absence of Cl − ions stimulated the "basal" Mg 2+ -ATPase activity by 44%.A study of the combined action of these anions on the "basal" Mg 2+ -ATPase revealed a synergistic effect observed under conditions of low Cl − (2.5 -7 mM) in the incubation medium.In this case, the stimulatory effect on the ATPase activity under the simultaneous action of Cl − + 3 HCO − exceeds the sum of the activating effects of each anion.Higher concentrations of Cl − (>10 mM) inhibit the activating effect of 3 HCO − ions on the enzyme activity.Therefore, we investigated the effect of a range of 3 HCO − concentrations (2 -30 mM) on the "basal" Mg 2+ -ATPase activity at low constant concentrations of Cl − (2.5 mM and 5) in the incubation medium (Figure 2(A)).Under these conditions, the maximal stimulation of enzymatic activity occurred upon introduction of Our preliminary studies showed that the stimulatory effect of Cl − / 3 HCO − (at a concentration ratio of 5:1) on the "basal" Mg 2+ -ATPase activity is observed at low concentrations (<1 mM) of substrate (Mg 2+ -ATP) and does not occur at higher concentrations [4]- [6].In this work, we investigated the effect of Mg 2+ -ATP (0.5 -4 mM) on the anion stimulation at the established optimal concentration ratio of 5 mM Cl − /25mM 3 HCO − (1:5) (Figure 3).Both  of 7.4 in imidazole-HCl buffer (100 mM) [2].At lower or higher concentrations of [H + ], the enzyme activity is reduced.In our study, the maximum Cl − / 3 HCO − -ATPase activity is also observed in the physiological pH range 7.3 -7.4 and no activity is seen at lower (<6.0) and higher (>9.0) pH values (data not shown).Increasing concentrations of Hepes-Tris buffer, along with an increase of the "basal" Mg 2+ -ATPase activity, causes a persistent stimulating effect of anions on the enzyme (data not shown).At 50 mM buffer, the Cl − / 3 HCO − -ATPase activity is 4.5 μmol P i /h/mg protein.
We confirmed that the enzyme activity under study is the GABA A -coupled ATPase by investigating the effect of antagonists of GABA A receptors (picrotoxin and bicuculline) on its activity (Figure 4).The GABA A receptor blockers, when supplied in a range of study concentrations (20 -100 µM) stimulate the "basal" Mg 2+ -ATPase activity by ~30% (data not shown), whereas addition of picrotoxin (100 µM) and bicuculline (25 -50 µM) completely eliminate the stimulatory effect of 3 HCO − + Cl − on the enzyme.The 3 HCO − -ATPase activity, by con- trast, is not sensitive to these ligands (Figure 3).Thus, inhibition of Cl − / 3 HCO − -ATPase activity by specific in- hibitors of GABA A receptor blockers confirms its identity as a GABA A -coupled ATPase [18].
Earlier studies of the Cl − / 3 HCO − -ATPase from fish brain plasma membranes showed that it was phosphory- lated by [γ-32 P]ATP in the presence of Mg 2+ [17].In our study, the ATPase from rat brain plasma membranes are also phosphorylated with [γ-32 P]ATP in the presence Mg 2+ .We have previously shown that the Cl − / 3 HCO − -ATPase from rat brain has molecular mass ~260 -300 kDa and it is heterooligomer involving subunits with molecular mass ~56, 53 and 48 kDa [6].In this work the AT-Pase studied was identified by SDS-PAGE polyacrylamide gel and autoradiography.On the electrophoretogram, the original preparation of partially purified ATPase treated with SDS was represented three major proteins and the small number of minor subunits (data not shown).Autoradiographic analysis of the spectrum of the enzyme phosphorylated with [γ-32 P]ATP allowed us to distinguish three major 32 P-labeled proteins whose molecular weight was ~57, 53 and 48 kDa (Figure 4).In the presence of Cl − / enzyme in the course of its phosphorylation (Figure 4).Thus, the results of this study indicate that similar subunits of Cl − / 3 HCO − -ATPase from rat brain neuronal membranes can be directly phosphorylated by ATP and de- phosphorylated in the presence of anions and GABA A -ergic ligands.
Earlier studies that incorporated the purified of the enzyme into proteoliposomes showed a differentiation of the detected properties of the ATPase system and omnidirectional Cl − -transport that depended on the concentration of anions in the intracellular and extracellular medium [7].In particular, the presence of high intracellular concentrations of Cl − and 3 HCO − in the incubation medium reverses ATP-dependent Cl − -transport and its exit from the cell.These data are consistent with electrophysiological studies on the function of GABA A receptors.In fully developed neurons, GABA interacts with the GABA A receptors to cause an increase in Cl − conductance inside the cells, which results in hyperpolarization of membrane potential [8].Furthermore, experiments with mature neurons showed that an increase in GABA concentration or incidence of receptor exposure to GABA was accompanied by the transition of neuronal membrane inhibition into membrane excitation [9] [19].All previous studies have noted the important role of 3 HCO − ions in this process, but no consensus has been reached regarding the role of Cl − ions.Some researchers have suggested that the GABA A -induced Cl − / 3 HCO − exchange process involves the passive entry of Cl − ions into the neuron in exchange for HCO 3 ions [10].However, conclusive evidence in support of this assumption remains to be produced.Other authors have considered that the Cl − ions exit from the cell upon the GABA-induced depolarization, which raises the question regarding the existence of ATP-dependent transport of Cl − into the cell by a Cl − -ATPase other than the Cl − -pump and coupled with the GABA A receptors [11] [12].The possible existence of this type of an ATPase is evident from electrophysiological data from rat brain neurons showing the presence of a bicuculline-sensitive GABA A -regulated Cl −pump that, upon binding to GABA, induces the ATP-dependent Cl − -transport against the electrochemical gradient [20].This Cl − / 3 HCO − -ATPase, which is revealed by treatment with a high concentration of Clearly, the hydrolytic activity of the ATPase under study plays an important role not only because this system provides metabolic energy but also because it provides a definite direction of flow of chloride ions, which depends not only on the intracellular ATP and Cl − concentrations, but also on the presence of a specific concentration of 3 HCO − ions.Further investigation of the properties of GABA A -coupled Cl − /HCO 3 -ATPase and its role in the transport of anions through the neuronal membrane may be important in clarifying the pathogenesis of several diseases, such as epilepsy [21] [22].

Conclusion
In the present work, we described a new approach for the detection of anion-stimulated ATPase which is functionally coupled with GABA A /benzodiazepine receptor/Cl − -channel complex.The presence of physiological anion concentrations in the incubation medium not only increases the enzyme activity but also contributes to the stability of the activity in the presence of high concentrations of substrate and buffer molarity.This is an important aspect in the further study of molecular properties of the enzyme.

Figure 1 . 3 HCO − -and 3 HCO
Figure 1.(A) Effect of Cl − concentration on the the "basal" Mg 2+ -ATPase activity of rat brain plasma membranes in the absence or in the presence of 20 mM 3 HCO − and (B) The Cl − -, Cl − +

3 HCO − ( 20 - 3 HCO − -and 3 HCO
30 mM) to the incubation medium.The absolute values of the Cl − -, Cl − / − -sti- mulated ATPase activities obtained in the presence of corresponding anions in the incubation medium are on Figure2(B).These results indicate that maximization of the stimulatory effect on enzyme activity requires the simultaneous presence of both Cl − and 3 HCO − .In addition, the optimal ratio for these anions is 5 mM Cl − /25 mM 3 HCO − for stimulation of the "basal" Mg 2+ -ATPase, leading to the conclusion that the main contributor to enzyme activation is 3 HCO − , not Cl − as suggested earlier[5] [7].

3 HCO
− and Cl − + 3 HCO − ions stimulated the "basal" Mg 2+ -ATPase activity across the entire range of substrate concentrations (i.e., high Mg 2+ -ATP concentrations did not impair the enzyme activation by anions).Previous studies showed that the Cl − -ATPase/Cl − -pump from rat brain plasma membrane has a pH optimum

3 HCO 3 HCO
− (~25 mM) and a low concentration of Cl − (~5 mM), is an enzyme that hydrolyzes ATP and participates in Cl − / 3 HCO − - exchange process.The suggested hypothetical scheme of Cl − -transport through the neuronal membrane by utilizing ATPase from neuronal membranes at GABA A -induced depolarization in the low (5 mM) Cl − and high (25 mM) − (Figure5).Hydrolytic activity of this Cl − / 3 HCO − -ATPase molecule provides energy for the transport process and determines a certain direction of Cl − flux: into of the neuron.

Figure 5 .
Figure 5. Operational model of the GABA A -coupled Cl − /