GABAA-Coupled Cl-/ HCO3--ATPase from Plasma Membrane of the Rat Brain: Role of HCO3- in the Enzyme Activation
Affiliation(s)
Federal State Scientific Institution “Research Institute of General Pathology and Pathophysiology”, Moscow, Russia.
Federal State Scientific Institution “Research Institute of General Pathology and Pathophysiology”, Moscow, Russia.
ABSTRACT
This work examines the influence of Cl- (2.5 - 125 mM) and HCO3- (2 - 30 mM) on the Cl-/HCO3- - 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 HCO3- (20 - 30 mM), while the maximum activity occurs in the presence of a ratio of ~25 mM HCO3- /~5mM Cl-. Blockers of the GABAA receptor, namely bicuculline (10 - 50 μM) and picrotoxin (50 - 100 μM), inhibit this anion activation, whereas the HCO3- -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 HCO3- 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-/ -HCO3- ATPase in the low (5 mM) Cl- and high (25 mM) HCO3- concentrations. The data demonstrated for the first time that the GABAA-coupled Cl-/ HCO3- -ATPase from rat brain neuronal membranes is maximally activated at a Cl-/HCO3- ratio of 1:5 and it remains stable at high concentrations of substrate and buffer.
This work examines the influence of Cl- (2.5 - 125 mM) and HCO3- (2 - 30 mM) on the Cl-/HCO3- - 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 HCO3- (20 - 30 mM), while the maximum activity occurs in the presence of a ratio of ~25 mM HCO3- /~5mM Cl-. Blockers of the GABAA receptor, namely bicuculline (10 - 50 μM) and picrotoxin (50 - 100 μM), inhibit this anion activation, whereas the HCO3- -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 HCO3- 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-/ -HCO3- ATPase in the low (5 mM) Cl- and high (25 mM) HCO3- concentrations. The data demonstrated for the first time that the GABAA-coupled Cl-/ HCO3- -ATPase from rat brain neuronal membranes is maximally activated at a Cl-/HCO3- ratio of 1:5 and it remains stable at high concentrations of substrate and buffer.
KEYWORDS
Rat Brain, Plasma Membrane, Mg2+-ATPase, Chloride, Bicarbonate, Mg2+-ATP, Picrotoxin, Bicuculline, Autoradiography, Molecular Weight, Subunits
Rat Brain, Plasma Membrane, Mg2+-ATPase, Chloride, Bicarbonate, Mg2+-ATP, Picrotoxin, Bicuculline, Autoradiography, Molecular Weight, Subunits
Cite this paper
Menzikov, S. , Karpova, M. , Kuznetsova, L. and Klishina, N. (2015) GABAA-Coupled Cl-/ HCO3--ATPase from Plasma Membrane of the Rat Brain: Role of HCO3- in the Enzyme Activation. Advances in Enzyme Research, 3, 9-18. doi: 10.4236/aer.2015.31002.
Menzikov, S. , Karpova, M. , Kuznetsova, L. and Klishina, N. (2015) GABAA-Coupled Cl-/ HCO3--ATPase from Plasma Membrane of the Rat Brain: Role of HCO3- in the Enzyme Activation. Advances in Enzyme Research, 3, 9-18. doi: 10.4236/aer.2015.31002.
References
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http://dx.doi.org/10.1007/s10863-005-9470-3 [4] Menzikov, S.A. (2013) Neuronal Multifunctional ATPase. Biophysical Reviews and Letters, 8, 213-227. http://dx.doi.org/10.1142/S1793048013300065 [5] Menzikov, S.A. and Menzikova, O.V. (2004) Effect of Chloride and Bicarbonate Ions on Mg2+-ATPase of Plasma Membranes of Bream (Abramis brama L.) Brain Sensitive to GABAA-Ergic Compounds. Ukrainskii Biokhimicheskii Zhurnal, 76, 53-58. [6] Menzikov, S.A. and Menzikova, O.V. (2007) Comparative Properties of Sensitive to GABAA-ergic Ligands, Cl-, HCO3--Activated Mg2+-ATPase from Brain Plasma Membranes of Fish and Rats. Zhurnal Evoliutsionnoi Biokhimii i Fiziologii, 43, 246-253. [7] Menzikov, S.A., Karpova, M.N. and Kalinina, M.V. (2011) Effect of HCO3- Ions on the ATP-Dependent GABAA Receptor-Coupled Cl-- Channel in Rat Brain Plasma Membranes. Bulletin of Experimental Biology and Medicine, 152, 38-42. http://dx.doi.org/10.1007/s10517-011-1448-z [8] Bormann, J., Hamill, O.P. and Sakmann, B. (1987) Mechanism Of Anion Permeation through Channels Gated by Glycine and Gamma-Aminobutyric Acid in Mouse Cultured Spinal Neurons. The Journal of Physiology, 385, 243-286. http://dx.doi.org/10.1113/jphysiol.1987.sp016493 [9] Staley, K.J., Soldo, B.L. and Proctor, W.R. (1995) Ionic Mechanisms of Neuronal Excitation by Inhibitory GABAA Receptors. Science, 269, 977-981. http://dx.doi.org/10.1126/science.7638623 [10] Staley, K.J. and Proctor, W.R. (1999) Modulation of Mammalian Dendritic GABAA Receptor Function by the Kinetics of Cl- and HCO3- Transpоrt. The Journal of Physiology, 519, 693-712. [11] Perkins, K.L. and Wong, R.K.S. (1996) Ionic Basis of the Postsynaptic Depolarizing GABA Response in Hippocampal Pyramidal Cells. Journal of Neurophysiology, 76, 3886-3894. [12] Perkins, K.L. (1999) Сl-Accumulation Does Not Account for the Depolarizing Phase of the Synaptic GABA Response in Hippocampal Pyramidal Cells. Journal of Neurophysiology, 82, 768-777. [13] Ashmarin, I.P., et al. (1999) Biochemistry of Brain. St. Petersburg State University, St. Petersburg. [14] Glebov, R.N. and Kryzhanovsky, G.N. (1978) The Functional Biochemistry of Synapses. Medicine, Moscow. [15] Bradford, M.M. (1976) A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principal of Protein-Dye Binding. Analytical Biochemistry, 72, 248-254.
http://dx.doi.org/10.1016/0003-2697(76)90527-3 [16] Chen, P.S., Toribara, T.Y. and Warner, H. (1956) Microdetermination of Phosphorus. Analytical Chemistry, 28, 1756- 1758. http://dx.doi.org/10.1021/ac60119a033 [17] Menzikov, S.A. and Menzikova, O.V. (2006) Phosphorylation of Cl-, HCO3- -Activated Mg2+-ATPase from Carp (Cyprinus carpio L.) Brain Plasma Membranes. Doklady Biochemistry and Biophysics, 407, 64-67.
http://dx.doi.org/10.1134/S1607672906020050 [18] Menzikov, S.A. and Karpova, M.N. (2011b) Cl--Transporting Mechanisms in Neuronal Membranes and Role of ATP in the Functioning. Pathogenesis, 1, 4-10. [19] Lambert, N. and Grover, L. (1995) The Mechanism of Biphasic GABA Responses. Science, 269, 928-929. http://dx.doi.org/10.1126/science.7638614 [20] Hyden, H., Cupello, A. and Rapallino, M.V. (1999) Chloride Permeation across the Deiters’ Neuron Plasma Membrane: Activation by GABA on the Membrane Cytoplasmic Side. Neuroscience, 89, 1391-1400.
http://dx.doi.org/10.1016/S0306-4522(98)00357-1 [21] Alvarez-Leefmans, F.J. and Delpire, E. (2009) Physiology and Pathology of Chloride Transporters and Channels in the Nervous System: From Molecules to Diseases. Elsevier-Academic Press, San Diego. [22] Menzikov, S.A., Karpova, M.N. and Kalinina, M.V. (2012) Effect of Pentylenetetrazole on the GABAA-Coupled Cl-, HCO3- -Activated Mg2+-ATPase Activity of the Plasma Membrane from Rat Brain both in Vitro and in Vivo Experiences. Pathological Physiology and Experimental Therapy, 98-102.
[1] Gerencser, G.A. and Zhang, J.L. (2003) Chloride ATPase Pumps in Nature: DO They Exist? Biological Reviews, 78, 197-218. http://dx.doi.org/10.1017/S146479310200605X [2] Inagaki, C., Hara, M. and Zeng, X.T. (1996) A Cl--Pump in Rat-Brain Neurons. The Journal of Еxperimental Zoology, 275, 262-268. [3] Pedersen, P.L. (2005) Transport ATPases: Structure, Motors, Mechanism and Medicine: A Brief Overview. Journal of Bioenergetics and Biomembranes, 37, 349-357.
http://dx.doi.org/10.1007/s10863-005-9470-3 [4] Menzikov, S.A. (2013) Neuronal Multifunctional ATPase. Biophysical Reviews and Letters, 8, 213-227. http://dx.doi.org/10.1142/S1793048013300065 [5] Menzikov, S.A. and Menzikova, O.V. (2004) Effect of Chloride and Bicarbonate Ions on Mg2+-ATPase of Plasma Membranes of Bream (Abramis brama L.) Brain Sensitive to GABAA-Ergic Compounds. Ukrainskii Biokhimicheskii Zhurnal, 76, 53-58. [6] Menzikov, S.A. and Menzikova, O.V. (2007) Comparative Properties of Sensitive to GABAA-ergic Ligands, Cl-, HCO3--Activated Mg2+-ATPase from Brain Plasma Membranes of Fish and Rats. Zhurnal Evoliutsionnoi Biokhimii i Fiziologii, 43, 246-253. [7] Menzikov, S.A., Karpova, M.N. and Kalinina, M.V. (2011) Effect of HCO3- Ions on the ATP-Dependent GABAA Receptor-Coupled Cl-- Channel in Rat Brain Plasma Membranes. Bulletin of Experimental Biology and Medicine, 152, 38-42. http://dx.doi.org/10.1007/s10517-011-1448-z [8] Bormann, J., Hamill, O.P. and Sakmann, B. (1987) Mechanism Of Anion Permeation through Channels Gated by Glycine and Gamma-Aminobutyric Acid in Mouse Cultured Spinal Neurons. The Journal of Physiology, 385, 243-286. http://dx.doi.org/10.1113/jphysiol.1987.sp016493 [9] Staley, K.J., Soldo, B.L. and Proctor, W.R. (1995) Ionic Mechanisms of Neuronal Excitation by Inhibitory GABAA Receptors. Science, 269, 977-981. http://dx.doi.org/10.1126/science.7638623 [10] Staley, K.J. and Proctor, W.R. (1999) Modulation of Mammalian Dendritic GABAA Receptor Function by the Kinetics of Cl- and HCO3- Transpоrt. The Journal of Physiology, 519, 693-712. [11] Perkins, K.L. and Wong, R.K.S. (1996) Ionic Basis of the Postsynaptic Depolarizing GABA Response in Hippocampal Pyramidal Cells. Journal of Neurophysiology, 76, 3886-3894. [12] Perkins, K.L. (1999) Сl-Accumulation Does Not Account for the Depolarizing Phase of the Synaptic GABA Response in Hippocampal Pyramidal Cells. Journal of Neurophysiology, 82, 768-777. [13] Ashmarin, I.P., et al. (1999) Biochemistry of Brain. St. Petersburg State University, St. Petersburg. [14] Glebov, R.N. and Kryzhanovsky, G.N. (1978) The Functional Biochemistry of Synapses. Medicine, Moscow. [15] Bradford, M.M. (1976) A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principal of Protein-Dye Binding. Analytical Biochemistry, 72, 248-254.
http://dx.doi.org/10.1016/0003-2697(76)90527-3 [16] Chen, P.S., Toribara, T.Y. and Warner, H. (1956) Microdetermination of Phosphorus. Analytical Chemistry, 28, 1756- 1758. http://dx.doi.org/10.1021/ac60119a033 [17] Menzikov, S.A. and Menzikova, O.V. (2006) Phosphorylation of Cl-, HCO3- -Activated Mg2+-ATPase from Carp (Cyprinus carpio L.) Brain Plasma Membranes. Doklady Biochemistry and Biophysics, 407, 64-67.
http://dx.doi.org/10.1134/S1607672906020050 [18] Menzikov, S.A. and Karpova, M.N. (2011b) Cl--Transporting Mechanisms in Neuronal Membranes and Role of ATP in the Functioning. Pathogenesis, 1, 4-10. [19] Lambert, N. and Grover, L. (1995) The Mechanism of Biphasic GABA Responses. Science, 269, 928-929. http://dx.doi.org/10.1126/science.7638614 [20] Hyden, H., Cupello, A. and Rapallino, M.V. (1999) Chloride Permeation across the Deiters’ Neuron Plasma Membrane: Activation by GABA on the Membrane Cytoplasmic Side. Neuroscience, 89, 1391-1400.
http://dx.doi.org/10.1016/S0306-4522(98)00357-1 [21] Alvarez-Leefmans, F.J. and Delpire, E. (2009) Physiology and Pathology of Chloride Transporters and Channels in the Nervous System: From Molecules to Diseases. Elsevier-Academic Press, San Diego. [22] Menzikov, S.A., Karpova, M.N. and Kalinina, M.V. (2012) Effect of Pentylenetetrazole on the GABAA-Coupled Cl-, HCO3- -Activated Mg2+-ATPase Activity of the Plasma Membrane from Rat Brain both in Vitro and in Vivo Experiences. Pathological Physiology and Experimental Therapy, 98-102.