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 ions in the incubation medium [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 ions are transported through the GABA_A receptor/Cl⁻-channel in a 5:1 ratio, respectively [8] . Indeed, the presence of Cl⁻ and ions (in a 5:1 ratio) in the incubation medium resulted in greater Cl⁻/-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 ions, respectively [5] [6] . These data suggest the participation of the enzyme in GABA_A-induced Cl⁻/ 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 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 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 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-³²P]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⁻/-ATPase complex in the neuronal membrane.

We have previously found that the activity of the GABA_A-coupled ATPase is a “basal” Mg²⁺-ATPase activity that is stimulated by anions [6] [7] . The activity of this “basal” Mg²⁺-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 (20 mM) in the absence of Cl⁻ ions stimulated the “basal” Mg²⁺-ATPase activity by 44%. Figure 1(B) clearly shows the absolute values of the Cl⁻-, Cl⁻/- and -stimulated ATPase activities obtained in the presence of Cl⁻, Cl⁻ + and ions in the incubation medium, respectively.

A study of the combined action of these anions on the “basal” Mg²⁺-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⁻ + exceeds the sum of the activating effects of each anion. Higher concentrations of Cl⁻ (>10 mM) inhibit the activating effect of ions on the enzyme activity. Therefore, we investigated the effect of a range of concentrations (2 - 30 mM) on the “basal” Mg²⁺-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 (20 - 30 mM) to the incubation medium. The absolute values of the Cl⁻-, Cl⁻/- and -sti- mulated ATPase activities obtained in the presence of corresponding anions in the incubation medium are on Figure 2(B). These results indicate that maximization of the stimulatory effect on enzyme activity requires the simultaneous presence of both Cl⁻ and. In addition, the optimal ratio for these anions is 5 mM Cl⁻/25 mM for stimulation of the “basal” Mg²⁺-ATPase, leading to the conclusion that the main contributor to enzyme activation is, not Cl⁻ as suggested earlier [5] [7] .

Our preliminary studies showed that the stimulatory effect of Cl⁻/ (at a concentration ratio of 5:1) on the “basal” Mg²⁺-ATPase activity is observed at low concentrations (<1 mM) of substrate (Mg²⁺-ATP) and does not occur at higher concentrations [4] -[6] . In this work, we investigated the effect of Mg²⁺-ATP (0.5 - 4 mM) on the anion stimulation at the established optimal concentration ratio of 5 mM Cl⁻/25mM (1:5) (Figure 3). Both and Cl⁻ + ions stimulated the “basal” Mg²⁺-ATPase activity across the entire range of substrate concentrations (i.e., high Mg²⁺-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

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⁻/-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” Mg²⁺-ATPase activity, causes a persistent stimulating effect of anions on the enzyme (data not shown). At 50 mM buffer, the Cl⁻/-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” Mg²⁺-ATPase activity by ~30% (data not shown), whereas addition of picrotoxin (100 µM) and bicuculline (25 - 50 µM) completely eliminate the stimulatory effect of + Cl⁻ on the enzyme. The -ATPase activity, by contrast, is not sensitive to these ligands (Figure 3). Thus, inhibition of Cl⁻/-ATPase activity by specific inhibitors of GABA_A receptor blockers confirms its identity as a GABA_A-coupled ATPase [18] .

Earlier studies of the Cl⁻/-ATPase from fish brain plasma membranes showed that it was phosphorylated by [γ-³²P]ATP in the presence of Mg²⁺ [17] . In our study, the ATPase from rat brain plasma membranes are also phosphorylated with [γ-³²P]ATP in the presence Mg²⁺.

We have previously shown that the Cl⁻/-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 ATPase 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-³²P]ATP allowed us to distinguish three major ³²P-labeled proteins whose molecular weight was ~57, 53 and 48 kDa (Figure 4). In the presence of Cl⁻/ ions or picrotoxin, the intensity of the major bands significantly decreased, thereby confirming the dephosphorylating effect of these ligands on the enzyme in the course of its phosphorylation (Figure 4).Thus, the results of this study indicate that similar subunits of Cl⁻/-ATPase from rat brain neuronal membranes can be directly phosphorylated by ATP and dephosphorylated 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 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 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⁻/ exchange process involves the passive entry of Cl⁻ ions into the neuron in exchange for HCO₃ 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⁻/-ATPase, which is revealed by treatment with a high concentration of (~25 mM) and a low concentration of Cl⁻ (~5 mM), is an enzyme that hydrolyzes ATP and participates in Cl⁻/- 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) (Figure 5). Hydrolytic activity of this Cl⁻/-ATPase molecule provides energy for the transport process and determines a certain direction of Cl⁻ flux: into of the neuron.

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 ions. Further investigation of the properties of GABA_A-coupled Cl⁻/HCO₃-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] .

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.

Sergey A.Menzikov,Margarita N.Karpova,Lada V.Kuznetsova,Natalya Y.Klishina， (2015) GABA_A-Coupled Cl-/ HCO₃^--ATPase from Plasma Membrane of the Rat Brain: Role of HCO^3- in the Enzyme Activation。 Advances in Enzyme Research，03，9-18. doi: 10.4236/aer.2015.31002