TMP 600

January 8, 2014 15:49 WSPC/S1793-0480 204-BRL 1330006
Biophysical Reviews and Letters

Vol. 8, Nos. 3 & 4 (2013) 213–227

c World Scientific Publishing Company
DOI: 10.1142/S1793048013300065
NEURONAL MULTIFUNCTIONAL ATPase
SERGEY A. MENZIKOV
State Research Institute of General Pathology
and Pathological Physiology, 8, RAMS
Baltiyskaya str., Moscow, 125315 Russia
Biophys. Rev. Lett. 2013.08:213-227. Downloaded from www.worldscientific.com
sambrainic@gmail.com
Received 19 November 2013

Accepted 24 November 2013
by 89.222.227.217 on 02/20/14. For personal use only.
Published 14 January 2014
Here, we review the properties of a suggested mechanism for a neural ATPase complex
based on our recent experimental findings. The mechanism represents a multifunctional
ATPase: an enzyme that is a chloride pump and a GABA receptor. This enables new
views on the ways Cl− channel transports anions and its regulation by the intra- and
extracellular ions and molecules (in particular by glucose, ATP, HCO− 3 ). The hydrolytic
activity of this GABAA -coupled ATPase provides the Cl− /HCO− 3 transport process the
energy and determines a certain direction of ions flux across neuronal membrane. This
can help with the research regarding several diseases such as epilepsy.
Keywords: ATPase molecule; chlorine; bicarbonate; GABAA -receptor; animal

brain plasma membranes; GABAA -ergic ligands; phosphorylation/dephosphorylation;
GABAA -induced depolarization; receptor channel model.
Special Issue Comment: This project is about a multifunctional ATPase com-

plex. Experiments involving measuring & solving individual ATPases are
related with the Special Issue about FRET experiments,1 about enzymes,2 and about
treatments when solving single molecules.3,4 The model suggested here is simply tested
with these experimental and mathematical methods.
1. Introduction
In the neuronal cells, intracellular chloride concentration [Cl− ]i is important
in the regulation of a resting membrane potential and neuronal signaling sys-
tems.7–9 Intracellular [Cl− ]i concentrations are determined by operation of vari-
ous transporters expressed in the plasma membrane. These chloride transporter
molecules include: cation–Cl− cotransporter (CCCs),10 Cl− anion antiporters, Cl− –
H+ antiporters (ClC),11 a passive elerctrochemical coupling processes (members
of the SLC6 family)12 and primary active ATP-dependent system (Cl− -ATPase
and Cl− -pump).13,14 In addition, electrophysiological methods in the neuronal
213
214 S. A. Menzikov
membranes revealed a specific GABAA -regulated chloride pump.15 The GABAA -

receptor that binds the GABA on the inner side of the neuron induces the chloride
transport against the electrochemical gradient while utilizing ATP energy.16 This
process is inhibited by bicuculline. Therefore, the hypothesis of the existence of
ATPase coupled to GABAA -receptor and is involved in the stabilization of the func-
tion was expressed near three decades ago.17 In addition, in the plasma membranes
of neurons, a “basal” Mg2+ -ATPase has been detected. This complex shows its vital
role in neuronal processes and several diseases.18,19 We emphasize that the gene and
other such evidences about the multifunctional ATPases are missing. In the present
review, based on our recent experimental findings,20,21 we consider the possibility
that the plasma membrane of neurons containing the multifunctional ATPase: a
chloride pump that is a GABA receptor with an enzymatic activity (ATPase).

The results at least suggest the existence of a multi-entity activity working in a
synchronized way, yet the mechanism presented here is about a multifunctional
entity.
Localization - The ATPase activity was detected in membrane vesicles from ani-
mal brain as nonmitochondrial “basal” Mg2+ -ATPase with a maximal activity in
the presence of Cl− and HCO− 3.
21,22
Localization of ATPases in neuronal plasma
membranes have been established by marker studies after gradient centrifugation
of microsomes and enzyme cytochemistry. Applying biochemical and cytochemical
methods, it was established that ATPases having the elements of the GABAA -ergic
receptors and reside in dendro-dendrite synapses.22
The model - Our biological findings enabled us postulating a new model of activ-
ity of the ATPase complex: ATPase with multifunctionality — an enzyme that
is also a Cl− -pump and a receptor. This conclusion is based on the following
discoveries: the protein preferably hydrolyzes ATP and the covalent phosphory-
lation by ATP (directly or by kinase) during the transport cycle and dephospho-
rylation by anions.23 These findings enable suggesting the chloride transporting
ATPase: this ATPase is a chloride pump involved in the import of Cl− ions across
native24 and artificial liposome membranes.20 Moreover, the protein is a ligand-
driven Cl− -channel which is structurally and functionally associated with GABAA -
receptors.25
The organization of this review - In this article, we review our findings and
the model about the multifunctional ATPase complex. In part 2, we explain the
model involving the multifunctional ATPase in neurons: the enzyme has two stable
states — phosphorylated and dephosphorylated. These two important states vary
in the ATPase functional activity and modulate the kinetic of Cl− and HCO− 3-
transport. In part 3 of this article are various experimental results supporting the
existence of a multifunctional ATPase complex in neuronal membranes. Yet in
this part we try to prove that the suggested model is the proper one. Discussion
Neuronal Multifunctional ATPase 215
regarding several diseases such as epilepsy is made. Part 4 presents concluding

comments.
2. The Multifunctional ATPase

Based on our results,20,23 we proposed the following model for multifunctional
ATPase (see Fig. 1).
(a) (b)
Fig. 1. (a) The mechanism of the multifunctional ATPase complex in the neuronal membrane.
(b) The suggested scheme of Cl− transport through the neuronal membrane utilizing ATPase from
neuronal membranes at GABAA -induced depolarization in the low (a) or high (b) concentration
[Cl− ]i . We assume that the protein, which is detected at both low (10 mM Cl− /2 mM HCO− 3 ) and
high (40 mM Cl− /8 mM HCO− 3 ) concentrations, has different functional states. Hydrolytic activity
of this ATPase molecule provides energy for the transport process and determines a certain direc-
tion of Cl− flux: into or out of the neuron. This process depends not only on the intracellular con-
centrations of Mg2+ -ATP and Cl− and HCO− 3 ions, yet also on the concentration of glucose. (c and
d) the suggested model involves two functional state ATPase molecule (phosphorylated or dephos-
phorylated). (c) The ATPase complex is in the state of a phosphorylation and has the low “basal”
Mg2+ -ATPase activity. Here the enzyme is activated by Cl− and HCO− 3 ions and is involved in
ATP-dependent transport of anions. (d) The ATPase complex is in the state of a dephosphoryla-
tion and has the high “basal” Mg2+ -ATPase activity. Here the enzyme is not activated by Cl− and
HCO− 3 ions and is not involved in ATP-dependent transport of anions. (e) Skeleton mechanism
of the multifunctional ATPase complex a scheme parallel to the one in (a). However, in this case
the figure involves the representation of the channel which is activated by GABA molecules (black
triangles). In the first step, HCO− 3 ions (the black circle with a minus sign) is attracted to the
ionic channel cytoplasmic vestibule by an exposed positive charge. Also, in this phase the ATP
hydrolysis is joining ATP in the presence of Mg2+ . After the joining of and the GABA to receptor
change of the channel conformation, Cl− ions (the white circle with a minus sign) is attracted
to the channel vestibule by an exposed positive charge. A grossly over simplified Cl− pump is
composed of one (or two) nucleotide-binding domains attached to a membrane-spanning domain,
which binds chlorine and bicarbonate ions (as 5:1) between two “gates”. The protein molecule
closes the inner “gates”. Then ATP in the presence of Mg2+ phosphorylates the channel and
forms macroergic phosphorylated intermediate. Then, a dephosphorylation step by anions occurs
and cytoplasmic “gates” open. Finally, the system can go to the initial configuration and begins
a new cycle. ATPase can function in the absence (or bypass) of neurotransmitter: in which case
the enzyme may has three steps of operation. This scheme shows how the anions can be extruded
(in↔out) from a neuron via an Cl− channel if energy is provided to complex by ATP consumption.
216
S. A. Menzikov
(c)
Fig. 1.
(e)
(Continued )
(d)
1: E+ MgATP → E1 · MgATP.
(The first phase of ATP hydrolysis is joining ATP in the presence of Mg2+ )
kinase or directly ATP
2: E1 · MgATP −−−−−−−−−−−−−−→ E2 Pi + ADP.
(The second stage consists of ATP hydrolysis in the phosphorylation of the terminal
phosphate ATP in the presence of Mg2+ to form macroergic phosphorylated inter-
mediate of the protein molecule; here, inhibitors are oligomycin, Zn2+ , o-vanadate)
Cl− and HCO−
3: E2 Pi −−−−−−−−−−→ 3
E + Cl− + HCO− 3.
(The third stage of hydrolysis: its activated by Cl− and/or HCO−3 ions of dephos-
phorylation of the complex E2 Pi ; here, inhibitors are o-vanadate, GABAergic
ligands, hydroxylamine).
We propose that the multifunctional ATPase complex is closely related to

GABAA -receptor and therefore can exist either in a phosphorylated ATPase
complex-P (Fig. 1(c)) or dephosphorylated form (Fig. 1(d)). The former case has
a low “basal” Mg2+ -ATPase activity and its activated by Cl− and HCO− 3 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. In this case, it has a high “basal” Mg2+ -
ATPase activity and it is not activated by anions. This state of the enzyme is called
“collapsed”.
Such a form of the enzyme cannot participate in the ATP-dependent trans-
port of 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 GABAA -
receptor.28 Yet, our model demonstrates that after convulsant effect, the dephos-
phorylation of the enzyme also occurs. As a result the ATPase doesn’t participate in
the chloride transport (as the “collapsed” state), see Fig. 1(d). Our results suggest
that not only inhibitors (o-vanadate, okadaic acid, etc.) have effect on the phos-
phatases activity, yet also the GABAA -ergic ligands (particularly convulsant) have
an influence on the phosphatases activity.
This enzyme hydrolyzes ATP and participates in the GABAA -induced
Cl− /HCO− 3 -exchange process. It is known that glucose through the processes of
glycolysis provides the GABAA -receptors ATP, which is essential for their func-
tion. Yet, the data show the glucose and Mg2+ -ATP can directly regulate the mode
that will work at the multifunctional ATPase (see Fig. 1(b)).
The multifunctional ATPase has a subunit structure and exhibits cooperative
properties of the allosteric-dissociating enzyme system with kinetic properties dif-
ferent from Michaelis–Menten kinetics. Based on the suggested model, we can
conclude that under the influence of convulsants (particularly pentylenetetrazole)
appeared are oligomeric complex with the different ATPase activity and state of
phosphorylation.
218 S. A. Menzikov
3. Various Experimental Results Supporting the ATPase

Multifunctional Model
3.1. ATPase activity
The biochemical properties of the ATPase are20–22 : 1) pH optimum — 7.4. 2) ATP
being the most effective nucleotide hydrolyzed. 3) anions, stimulating the enzyme
activity are Cl− ≥ Br− > I− F− , and also HCO− 2− −
3 , SO3 , but inhibited by NO2
2− + +
and no effect elicited by NO3 . 4) Na , K and choline provide no effect. 5) it has
an absolute requirement for Mg2+ .
Additional properties - the detection of the Cl− -ATPase sensitivity to SH-reagents
(PCMB, DTNB), blockers phosphorylation (o-vanadate, oligomycin), as well as
substrate specificity, show that this ATPase transport is of the P-type.21,29,30 The
fact that the “basal” and Cl− -activated Mg2+ -ATPase are not sensitive to CoA,
NaF, ouabain, EGTA and their high sensitivity to oligomycin, enable us to conclude
that the investigated enzyme is not associated with transport ATPases P-type and
ecto-ATPases E-type.
3.2. Functional coupling of the ATPase molecule

to inhibitory receptors
The activity of the neuromediators and blockers on the activation of the “basal”
Mg2+ -ATPase by Cl− and/or HCO− 3 are either ligands affecting (GABA, glycine,
picrotoxin, strychnine) or those that do not affect this ATPase (acetylcholine, glu-
tamate, benzotropin, D-2-amino-5-phosphonovaleric acid) see Table 1.31
Thus, GABA and glycine completely eliminate the enzyme activation by anions.
The effect of mediators is eliminated by picrotoxin and strychnine. In the absence
of these mediators, the convulsants (bicuculline, picrotoxin, pentylenetetrazole and
others) inhibit the enzyme activation by anions. The modulators (anticonvulsants,
benzodiazepines, anesthetics) of GABAA /benzodiazepine receptor have a similar
GABA effect in a range of concentrations: 10−8 –10−3 M.31,32
3.3. Molecular weight and subunit composition of the ATPase

The ATPase from animal brain has molecular mass ∼260–300 kDa and has various
identical subunits in fish involving with molecular mass per subunit ∼56 kDa, and is
heterooligomer in rats involving subunits with molecular mass ∼56, 53 and 48 kDa
(see Table 2).21,25 The purified and reconstituted into liposomes enzyme retain the
sensitivity to GABAA -ergic ligands. Such method provides the evidence that the
ATPase is a receptor protein.33–35
3.4. Participation of the ATPase molecule in the Cl− /HCO−

3
transport processes
Although brief activation of dendritic GABAA -receptors hyperpolarizes the neu-
ronal membrane, prolonged activation or large amounts of GABA applied to the
Table 1. Effect of neuromediators and blockers of receptor proteins of ion channels on the “basal”
Mg2+ -ATPase activity in the absence and presence of 40 Cl− /8 mM HCO− 3 . Plasma membrane
samples (15–25 µg) preincubation for 15 min with a ligand were added to incubation medium
containing 10 mM Hepes-Tris (pH 7.4), 1 mM Tris-ATP, 1 mM MgSO4 , and ligand in the same
concentration as in the preincubation medium.
Ligand added “basal” Mg2+ -ATPase activity,

µmol Pi /h/mg protein
In the absence In the presence Effect
of Cl− /HCO− − − − −
3 of 40 Cl /8 mM HCO3 40 Cl /8 mM HCO3
Control 12.0 + 0.6 16.4 ± 0.9 5,4 ± 0.6

GABA (10−5 M) 19.6 ± 0.7∗ 19.0 ± 1.2 —
GABA (10−5 M) 11.2 ± 0.5 15.7 ± 0.3 4.5 ± 0.5
+ picrotoxin (2·10−5 M)
Picrotoxin(2·10−5 M) 16.0 ± 0.8∗ 15.4 ± 0.9 —

GABA (10−5 M) 18.1 ± 1.0∗ 18.0 ± 0.7 —
+ strychnine (2·10−5 M)
Glycine (10−5 M) 17.6 ± 1.1∗ 16.8 ± 0.8 —
Glycine (10−5 M)
11.4 ± 0.9 15.5 ± 0.6 4.1 ± 0.3

+ picrotoxin (2·10−5 M)
Strychnine (2·10−5 M) 15.0 ± 1.2∗ 15.0 ± 0.7 —
Glycine (10−5 M) 17.5 ± 0.7∗ 17.3 ± 0.5 —
+ strychnine (2·10−5 M)
Acetylcholine (10−5 M) 14.2 ± 1.0∗ 19.0 ± 1.1 4.8 ± 0.5
Acetylcholine (10−5 M) 9.5 ± 0.4∗ 14.5 ± 0.8 5.3 ± 0.6
+ benztropine (10−5 M)
Benztropine mesylate (10−5 M) 9.0 ± 0.8∗ 14.0 ± 0.9 5.0 ± 0.4
Glutamate (10−5 M) 14.7 ± 0.9∗ 20.0 ± 0.6 5.3 ± 0.5
Glutamate (10−5 M) + D-2-
amino-5-phosphonovaleric
acid(10−5 M) 10.2 ± 0.5 15.3 ± 0.7 5.2 ± 0.7
D-2-amino-5- 10.0 ± 0.8 15.0 ± 1.0 5.0 ± 0.6
phosphonovaleric acid
(10−5 M)
Note: Values are expressed as means ± S.D. (n = 3).

∗ Significantly different from values in the medium without ligands, p < 0.05.
resting membrane potential become depolarizing.26 The ionic mechanism and func-
tional significance of the depolarizing GABAA -receptor mediated responses are
uncertain. However, it was shown that the modulation of GABAA -receptor func-
tion was caused by the kinetics of Cl− and HCO−3 transport. Such GABAA -induced
− −
Cl /HCO3 -exchange process is inhibited by low concentrations of bicuculline and
acetazolamide.27 We investigated the combined effect of Cl− and HCO− 3 on the
“basal” Mg2+ -ATPase activity, see Fig. 2.21
In both the low (Fig. 2(a)) and high (Fig. 2(b)) anion concentrations, the
ATPase activities are higher than those in the presence of each anion alone. Bicu-
culline (7 µM) and acetazolamide (20 µM) abolished the activating effect of Cl−
and HCO− −
3 ions on the enzyme. ATP-induced Cl transport across the membranes
2+
of proteoliposomes was studied at Mg -ATP concentrations of 0.5–3.0 mM, see
220
Table 2. Comparison of biochemical properties of the neuronal multifunctional ATPase complex with GABAA -receptor and Cl− -ATPase/Cl− -pump from
neuronal membranes of various origins.
S. A. Menzikov
Biochemical Neuronal GABAA -receptor Cl− -ATPase/ Cl− pump

properties multifunctional ATPase Cl− -channel
Species Fish Rat Fish Rat Rat
Origin Brain Brain Brain
Molecular mass 260–300 kDa 240–300 kDa 520–580 kDa
Subunit structure 56 kDa 56, 53, 48 kDa 56 kDa 56, 53, 48 and 26 kDa 62, 60, 55, 51 kDa
Optimum pH 7.4 7.4 7.4
Antagonists Oligomycin o-vanadate, Oligomycin o-vanadate Ethacrynic acid, o-vanadate,
furosemide, picrotoxin bicuculline, furosemide, picrotoxin, SITS, SH-reagents
ethacrynic acid, o-vanadate, bicuculline, SITS
SITS, SH-reagents,
Agonists/modulators GABA, barbiturates, GABA, Barbiturates, Phosphatidylinositol-4-
benzodiazepines, Zn2+ ethanol benzodiazepines, Zn2+ ethanol, monophosphatc
Phosphorylation Protein kinase and ATP Protein kinase and ATP ATP
The high-energy Forms ——— Forms
phosphorylated intermediate
The substrate specificity ATP UTP>CTP>ADP ATP ADP ATP GTP>ITP>UTP>CTP
Specificity to divalent Cations Mg2+ > Mn2+ > Co2+ AI2+ > Cd2+ Mg2+ > Mn2+ > Ca2+
− − − −
Specificity to anions HCO−
3 > Cl = Br > I > F SCN− > l− > Br− > Cl− > F− > HCO−
3 Cl− > Br− > CH3 COO = I− >
2−
SO24 = HCO−
3 > SO3
The ions are transported Cl− and HCO−
3 Cl− and HCO−
3 Cl− and HCO−
3 Cl−
Fig. 2(c). Addition of 1.0–1.5 mM Mg2+ -ATP to the incubation medium was fol-
lowed by a decrease in fluorescence. The next experiments were performed to con-
firm the involvement of HCO− −
3 in ATP-dependent Cl -transport, see Fig. 2(d). The
effect was most pronounced at a substrate concentration of 2–3 mM. This ATPase
is involved in GABAA -receptor induced Cl− /HCO− 3 -exchange in neuronal mem-
branes.20
3.5. The GABAA -ergic ligands effects on the ATPase activity

Short neurotransmitter receptor interaction results in membrane hyperpolarization
and decreased neuronal excitability, see Fig. 3(d).7 The interaction of GABA with
the receptor in the postsynaptic membrane results in conformational changes in
14
13
Mg2+ -ATPase activity, µmol Pi/h /mg protein

13
12
12 11
11 10
10 9
9 8
←"basal " Mg2+-ATPase
←"basal " Mg2+-ATPase
8 7
7 6
6 5
HCO3-,mM -
0 2 4 6 8 0 20 40 60 80 100 120 140 HCO3 ,mM
0 8 6 4 2 Cl- ,mM 0 4 8 12 16 20 24 28 Cl-, mM
[ Anions ], mM [ Anions ], mM
(a) (b)
Fig. 2. The dependence of the “basal” Mg2+ -ATPase activity from animal brain plasma mem-
branes at low (a) and high (b) of Cl− and HCO− 3 concentration in the incubation medium (ratio
Cl− /HCO− 3 as 1:5). The inhibited action of the carbonic anhydrase inhibitor (acetazolamide), an
inhibitor of the GABAA -mediated depolarization, supports the idea that this ATPase is involved
in epileptic seizures. (c) The effect of ATP concentration on fluorescence of proteolyposomes with
the reconstituted enzyme from rat brain (proteoliposomes were resuspended in 0.7 ml 30 mM
HEPES-Tris buffer (pH 7.3) containing 0.125 mM EDTA and 0.1 mM phenylmethylsulfonyl fluo-
ride. Proteoliposomes were loaded with a fluorescent probe 6-methoxy-N-ethylquinolinium iodide
(MEQ; highly sensitive to Cl− ions) by the method of freezing/defrosting]; (d) The effect of HCO− 3
ions on the fluorescence of proteoliposomes with the reconstituted enzyme from rat brain in the
presence of 2 mM ATP over 5 min, in the absence and in the presence of 20 µM acetazolamide.
HCO− 3 ions had no effect on the ATP-dependent chloride transport into proteoliposomes. In the
experiments with chloride preloaded liposomes, addition of HCO− 3 ions to the incubation medium
caused the reversion of Cl− transport (ion efflux from liposomes). In the presence of acetazolamide,
the HCO− 3 effect on the ATPase is eliminated.
222 S. A. Menzikov
Not containing Cl -
Containing 30 mM Cl -
Containing 30 mM Cl - + 6 mM HCO 3-
Containing Cl - /HCO3- +15 µM acetazolamide
30 40
35
30
∆ F, % inhibition
20 25
20
15
10
10
0
0
1 2 3 4
0 1 2 3 4 Proteoliposomes with the reconstituted

[ ATP ], mM single ATPase molecule of neurons
(c) (d)
Fig. 2. (Continued )
the chloride channel: the channel is opened and the Cl− ions go through the neu-
ronal cell with the electrochemical gradient. Electrophysiological experiments have
shown that the opening of the channel requires connection of at least two molecules
of GABA.7 The energy profile of the ion channel is shown in Fig. 3(a), the potential
wells are consistent with the binding site of Cl− and/or HCO− 3 ions with the ion
7
channel. However, experiments with mature neurons showed that mechanism of
GABA on the membrane potential in the neuronal cells has the biphasic responses.
So, increase in GABA concentration is accompanied by the transition of neuronal
membrane inhibition into membrane excitation, see Fig. 3(d).27,36 The general opin-
ion of all scientists is that HCO−3 ions are involved in this process. At the same
−
time, the role of Cl ions in this mechanism is not entirely clear. Some authors
hypothesized that GABAA -induced Cl− /HCO− 3 -exchange is characterized by pas-
sive influx of Cl− ions into the neuron (in exchange for HCO− 3 ions). Other authors
reported that Cl− efflux from the cell occurs under conditions of GABAA -induced
depolarization. The question arises: does Cl− -ATPase have a role in ATP-dependent
Cl− transport into the cell differing from the Cl− channel and coupled to GABAA -
receptors? The existence of this ATPase is confirmed by published data on the
bicuculline-sensitive GABAA -receptor-regulated Cl− -channel in specific neurons of
the brain.15
In addition, the GABAA -receptors function of rat hippocampus shows the dual
nature of the GABA effect on the membrane potential, depending on its concentra-
tion. In the low (0.01 µM) and high (1–10 µM) concentrations the neurotransmitter
causes hyperpolarization of neurons, and in the average range concentration (0.1–

10 µM) it is not effective.37
The effect of different GABA concentrations (0.00001–100 µM) on the “basal”
and Cl− /HCO− 3 -activated Mg
2+
-ATPase activities follows sinusoidal curves, see
Cl-/HCO3- 16
Cl-/HCO3-
30
"Basal" Mg2+-ATPase
Activating by Cl-/HCO3-
25
20 12
Mg2+ -ATPase activity,µmol Pi/h /mg protein
Cl-/HCO3-
15 Cl-/HCO3-
10
8
5
∆G(kJ mol-1)
4
-5
+
-10 +
+
+
-15 0 //
0 0.5 1 0 -11 -10 -9 -8 -7 -6 -5 -4 -3
Electric distance - log [ GABA], µM
(a) (b)
Fig. 3. (a) A schematic representation of the charge properties of GABA channel.7 A: a section
through a channel with two fixed positive charges (+) to which Cl− can bind, free-energy profile
for a Cl− transport through an open channel in the absence of membrane voltage. Free energy
of chloride ions in intracellular and extracellular bulk solution was set arbitrarily to zero. This
profile was assumed to describe the single-file transport of chloride ions through the channel. The
ordinate is the change in free energy (kJ mol− ).7 The abscissa is the electric membrane distance.
(b) The dependence of the “basal” Mg2+ -ATPase activity of animal brain plasma membranes
from GABA concentration in the incubation medium in the absence and in the presence of 10
mM Cl− /2 mMHCO− 3 . (c) The effect of GABA on the “basal” Mg
2+ -ATPase activity of animal
brain plasma membranes in the absence and in the presence 10 mM Cl− /2 mM HCO− 3 at a time
when its not activated. (d) The mechanism of biphasic GABA responses: when GABAA -receptors
are activated at inhibitory synapses, or when a small amount of exogenous GABA is applied
from a pipette, many central neurons respond with a small hyperpolarization. However, when
large amounts of GABA are applied, responses become depolarizing. This figure is based on the
project from Ref. 36. (e) Convulsants (pentylenetetrazole and other) in the concentrations (15-20
µM) enhances the “basal” Mg2+ -ATPase activity and eliminated the activating effect of Cl− and
HCO− 3 on the enzyme both in vitro and in vivo.
224 S. A. Menzikov
20
"basal" Mg2+-ATPase
activating by Cl-/HCO3-
15 +
10
0
11 10 9 8 7 6 5
-log [ GABA], µM
(c) (d)
12
"basal" Mg2+-ATPase
activating by Cl-/HCO3-
10
Mg2+ -ATPase activity,µmol Pi/h /mg protein
0
Control Convulsants
(GABAA-ergic ligands)
(e)
Fig. 3. (Continued )
Fig. 3(b). With an increase in activity of the “basal” Mg2+ -ATPase, the
Cl− /HCO− 3 -activated Mg
2+
-ATPase activity decreased, and vice versa. The sinu-
soidal character of the “basal” Mg2+ -ATPase activity in the presence and in the
absence of Cl− /HCO− 3 and different GABA concentrations is similar to biphasic
effect mediator on the GABAA -regulated Cl− channel.36,37 The profile of the “basal”
Mg2+ -ATPase activity depending on the GABA concentration is associated with
the change of activation of the enzyme by anions, see Fig. 3(b).
The Cl− /HCO− 3 -ATPase that we detected in the animal brain has various
subunit structure and exhibits cooperative properties of the allosteric-dissociating
enzyme system. In the incubation medium containing at least 1.5 mM Mg2+ -ATP
and Hepes-tris buffer above 15 mM, the “basal” Mg2+ -ATPase activity of the
plasma membrane of animal brain could be 16.5 µmol Pi /h/mg protein and higher,
and the ATPase was not activated by chloride ions. After preincubation of the
enzyme to GABA in the range of concentrations 0.00001–1 µM, the “basal” Mg2+ -
ATPase activity fell from 16.5 to 3.7 µmol Pi /h /mg protein, see Fig. 3(c). The
action of neurotransmitter on the enzyme makes it sensitive to Cl− /HCO− 3 ions.
3.6. Role of Mg2+ -ATP and glucose in maintaining

the ATPase function
The effect of the glucose and Mg2+ -ATP on the ATPase was also investi-
gated. The glucose (5–10 mM) decreased the “basal” Mg2+ -ATPase activity and
completely abolished the enzyme activation by low concentrations of ions (i.e.
10 mM Cl− /2 mM HCO− 3 ).
38
The effect of glucose and Mg2+ -ATP on the acti-
vation of the “basal” Mg2+ -ATPase depending on the concentrations of various
anions was established. It was found that in the presence of Mg2+ -ATP in the
incubation medium > 1 mM, the enzyme was not activated by 10 mM Cl− /2 mM
HCO− 3 ions.
38
Yet, in the presence of glucose (10 mM) the inhibiting effect of the
2+
Mg -ATP on the enzyme disappears. In the presence of the high concentrations
in the incubation medium (40 mM Cl− /8 mM HCO− 3 ), the inhibiting effect of the
glucose and Mg2+ -ATP was insignificant.
4. Concluding Comments and Future Directions

Our results suggest the existence of a multifunctional ATPase complex in the neu-
ronal membranes. This is an enzyme that is also a chloride pump and a receptor.
Clear evidences were supplied about the involvement of ATP, phosphatases, protein
kinase, and glucose in the modulation and the maintaining of the GABAA -receptor
function in normal and pathological conditions.4,28,39 Yet, this review further clar-
ifies the involvement of the multifunctional ATPase complex in these molecular
activities. The participation of the GABAA coupled ATPase in the epileptic seizures
confirms the importance of the suggested ATPase in the CNS. It is vital to derive the
mechanism of the ATPase, and its effect regarding pathology. In particular, there
is an evidence that protein phosphatases blockers (o-vanadate and okadaic acid)
226 S. A. Menzikov
eliminate lowering function (run-down effect) of GABAA -receptors in the period

of seizure activity of the human brain.8 Further research about the functional role
of the corresponding GABAA -receptor population and structural dynamics of their
ATP-recognizing and hydrolyzing centers can help in the science involving epilepsy.
The results presented here can pave the way.
References
1. M. J. Ruedas-Rama, J. M. Alvarez-Pez and A. Orte, Biophys. Rev. Lett. 8, 161–190
(2013).
2. S. K. Jørgensen and N. S. Hatzakis, Biophys. Rev. Lett. 8, 137–160 (2013).
3. O. Flomenbom, Biophys. Rev. Lett. 8, 109–136 (2013).
4. C. Nicolai and F. Sachs, Biophys. Rev. Lett. 8, 191–211 (2013).
5. E. Palma, C. Roseti, F. Maiolino et al., Proc. Natl. Acad. Sci. USA 104, 52 (2007).
6. R. Pumain and J. Laschet, J. Crit. Rev. Neurobiol. 18, 1–2 (2006).
7. J. Bormann, O. P. Hamill and B. Sakmann, J. Physiol. Lond. 385, (1987).
8. E. Palma, M. Amici, F. Sobrero et al., Proc. Natl. Acad. Sci. USA 103, 22 (2006).
9. K. L. Perkins, J. Neurophysiol. 82 (1999).

10. J. A. Payne, Curr. Top. Membr. 70 (2012).
11. E. McMains, V. Krishnan et al., PLOS ONE (2011), doi: 10.1371/jour-
nal.pone.0017647.
12. N. Svichar et al., J. Neurosci. 29, 10 (2009).
13. F. J. Alvarez-Leefmans and E. Delpire, Physiology and Parthology of Chloride Trans-
porters and Channels in the Nervous System. From Molecules to Diseases (Elsevier-
Academic Press, San Diego, CA, 2009).
14. C. Inagaki, M. Hara and X. T. Zeng, J. Exp. Zool. 275, 262–268 (1996); C. Inagaki
et al., J. Exp. Zool. 289, 224–231 (2001).
15. A. Cupello, Amino Acids 24, 4 (2003).
16. H. Hyden, A. Cupello and M. V. Rapallino, Neuroscience 89 (1999).
17. A. Stelzer, A. R. Kay and R. K. Wong, Science, 241 (1988).
18. T. Tsakiris et al., Int. J. Sports Med. 27, 19 (2006).
19. U. V. Gopalaswamy, Chem. Biol. Interac. 103, 1 (1997).
20. S. A. Menzikov, M. N. Karpova and M. V. Kalinina, Bull. Exp. Biol. Med. 152,
1 (2011).
21. S. A. Menzikov and O. V. Menzikova, Zh. Evol. Biokhim. Fiziol. 43, 3 (2007); S. A.
Menzikov and O. V. Menzikova, Ukr. Biokhim. Zh. 76, 2 (2004).
22. S. A. Menzikov et al., Zh. Evol. Biokhim. Fiziol. 36, 3 (2000); S. A. Menzikov and O.
V. Menzikova, Izv. Akad. Nauk. Ser. Biol. 5 (1999).
23. S. A. Menzikov, O. V. Menzikova, Ukr. Biokhim. Zh. 78, 1 (2006); S. A. Menzikov
and O. V. Menzikova, Dokl. Biochem. Biophys. 407 (2006).
24. S. A. Menzikov et al., Biochemistry (Mosc) 56, 6 (1991).
25. S. A. Menzikov and O. V. Menzikova, Dokl. Biochem. Biophys. 401, (2005); S. A.
Menzikov and O. V. Menzikova, Biochemistry (Mosc) 70, 12 (2005).
26. K. J. Staley and I. Mody, J. Neurophysiol. 68 (1992).
27. K. J. Staley, B. L. Soldo and W. R. Proctor, Science 269 (1995); K. J. Staley and
W. R. Proctor, J. Physiol. Lond. 519, 3 (1999).
28. Q. X. Chen, A. Stelzer, A. R. Kay and R. K. Wong, J. Physiol. 420 (1990).
29. S. A. Menzikov and O. V. Menzikova, Zh. Evol. Biokhim. Fiziol. 40, 4 (2004).
30. S. A. Menzikov and O. V. Menzikova, Dokl. Biol. Sci. 382, 6 (2002); S. A. Menzikov
and O. V. Menzikova, Dokl. Biol. Sci. 382, 1 (2002).
31. S. A. Menzikov and O. V. Menzikova, Biochemistry (Mosc) 67, 2 (2002); S. A. Men-

zikov and O. V. Menzikova, Dokl. Biochem. Biophys. 396 (2004).
32. S. A. Menzikov and O. V. Menzikova, Neuroscince Letters 334 (2002).
33. S. A. Menzikov and O. V. Menzikova, Dokl. Biol. Sci. 396, 2 (2004).
34. L. Deng et al., J. Neurochem. 56 (1991).
35. C. Mamalaki et al., J. Neurochem. 52, 1 (1989).
36. N. Lambert and L. Grover, Science 269 (1995).
37. P. K. Plinkert, A. H. Gitter et al., Eur. Archiv. Oto-Rhino-Laryn. 250, 351–357 (1993).
38. S. A. Menzikov, M. N. Karpova and M. V. Kalinina, Patol. Fiziol. Eksp. Ter. 1,
(2013).
39. T. Shirasaki, K. Aibara and N. Akaike, J. Physiol. 449, (1992).
40. M. Chebib and G. A. Johnston, J. Med. Chem. 43, 8 (2000).
41. L. A. Baker and J. L. Rubinstein, Biophys. Rev. Lett. 05, 59 (2010).
42. E. Gerritsma and P. Gaspard, Biophys. Rev. Lett. 05, 04 (2010).

43. C. L. Cole and H. Qian, Biophys. Rev. Lett. 06, 01n02 (2011).

TMP 600

Uploaded by

Document Information

Original Description:

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

TMP 600

Uploaded by

Copyright:

Available Formats

January 8, 2014 15:49 WSPC/S1793-0480 204-BRL 1330006

Biophysical Reviews and Letters

NEURONAL MULTIFUNCTIONAL ATPase

Received 19 November 2013

Published 14 January 2014

Keywords: ATPase molecule; chlorine; bicarbonate; GABAA -receptor; animal

Special Issue Comment: This project is about a multifunctional ATPase com-

membranes revealed a speciﬁc GABAA -regulated chloride pump.15 The GABAA -

chloride pump that is a GABA receptor with an enzymatic activity (ATPase).

Neuronal Multifunctional ATPase 215

regarding several diseases such as epilepsy is made. Part 4 presents concluding

2. The Multifunctional ATPase

Neuronal Multifunctional ATPase 217

We propose that the multifunctional ATPase complex is closely related to

3. Various Experimental Results Supporting the ATPase

3.2. Functional coupling of the ATPase molecule

3.3. Molecular weight and subunit composition of the ATPase

3.4. Participation of the ATPase molecule in the Cl− /HCO−

Neuronal Multifunctional ATPase 219

Ligand added “basal” Mg2+ -ATPase activity,

Control 12.0 + 0.6 16.4 ± 0.9 5,4 ± 0.6

Picrotoxin(2·10−5 M) 16.0 ± 0.8∗ 15.4 ± 0.9 —

11.4 ± 0.9 15.5 ± 0.6 4.1 ± 0.3

Note: Values are expressed as means ± S.D. (n = 3).

Biochemical Neuronal GABAA -receptor Cl− -ATPase/ Cl− pump

Neuronal Multifunctional ATPase 221

3.5. The GABAA -ergic ligands eﬀects on the ATPase activity

Mg2+ -ATPase activity, µmol Pi/h /mg protein

0 1 2 3 4 Proteoliposomes with the reconstituted

Neuronal Multifunctional ATPase 223

causes hyperpolarization of neurons, and in the average range concentration (0.1–

Neuronal Multifunctional ATPase 225

action of neurotransmitter on the enzyme makes it sensitive to Cl− /HCO− 3 ions.

3.6. Role of Mg2+ -ATP and glucose in maintaining

4. Concluding Comments and Future Directions

eliminate lowering function (run-down eﬀect) of GABAA -receptors in the period

9. K. L. Perkins, J. Neurophysiol. 82 (1999).

Neuronal Multifunctional ATPase 227

31. S. A. Menzikov and O. V. Menzikova, Biochemistry (Mosc) 67, 2 (2002); S. A. Men-

42. E. Gerritsma and P. Gaspard, Biophys. Rev. Lett. 05, 04 (2010).

You might also like