From the Department of Physiology, Faculty of
Medicine, Kyushu University 3-1-1 Maidashi Higashi-ku Fukuoka, 812-82, Japan and the ¶ Research Institute of Biological Psychiatry, St.
Hans Hospital, DK-4000 Roskilde, Denmark
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ABSTRACT |
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Human -aminobutyric acid type A
(GABAA) receptors were expressed in the
baculovirus/Sf-9 insect cell expression system using recombinant
cDNA of
1
2
2s subunits.
The effect of unsaturated fatty acids on GABAA receptor
complexes was investigated electrophysiologically using conventional
whole cell recording under voltage clamp. Three distinct effects of
docosahexaenoic acid (DHA) on the GABA responses were observed. First,
DHA, at a concentration of 10
7 M or greater,
accelerated the desensitization after the peak of the GABA-induced
current. Second, DHA (10
6 M) potentiated the
peak amplitude of GABA response. This potentiation by DHA was inhibited
in the presence of Zn2+ (10
5 M);
Cu2+ and Ni2+ mimicked the action of
Zn2+. Zn2+ (10
5 M)
did not block the GABA response on
1
2
2s receptor complexes. Third, DHA, at a concentration of 3 × 10
6
M or higher, gradually suppressed the peak amplitude of
GABA response. A protein kinase A inhibitor, a protein kinase C
inhibitor, and a Ca2+ chelator did not modify the effects
of DHA on GABA-induced chloride ion current. Six unsaturated fatty
acids other than DHA were examined. Arachidonic acid mimicked the
effect of DHA while e.g. oleic acid had no effect.
The inhibition of the GABA response in the presence of DHA was also
observed in cells expressing GABAA receptors of
1 and
2 subunit combinations. The data
show that the
subunit is essential for DHA and arachidonic acid to
potentiate the GABA-induced Cl
channel activity and to
affect the desensitization kinetics of the GABAA
receptor.
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INTRODUCTION |
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Polyunsaturated fatty acids are essential fatty acids that are abundantly bound in the phospholipids of brain membranes. The fatty acids influence the membrane fluidity of the neuronal plasma membrane as well as the functional properties of integral membrane protein (1). Arachidonic acid (AA)1 and docosahexaenoic acid (DHA) are polyunsaturated fatty acids localized in the 2-position of membrane phospholipids and are liberated by various intracellular enzymes such as phospholipase A2 and diacylglycerol lipase (2).
Recently, various actions of these free fatty acids on the functional
properties of neuronal plasma membrane neurotransmitter receptors have
been reported, such as N-methyl-D-aspartic acid receptor (3, 4), kainate receptor (4), and potassium channel receptor
(5, 6). As for the effect of unsaturated fatty acids on
-aminobutyric acid type A (GABAA) receptors, an electrophysiological study demonstrated that AA and DHA potently suppressed the GABAA receptor-mediated current response in
the rat substantia nigra neurons (7). Receptor binding studies have
shown marked effects of unsaturated fatty acids on brain GABAA receptor complexes in vitro (8-11), as
well as on human recombinant GABAA receptors expressed in
Sf-9 insect cells (12). These findings suggest a variety of actions of
fatty acids on the GABAA receptor-channel complex. The
diversity of effects induced by free fatty acids might be due to
ontogenetic, phylogenetic, and regional differences of the
GABAA receptor subunit composition in the brain regions
studied (13). Furthermore, a recent study on recombinant human
GABAA receptors (12) reported that the effect of
unsaturated fatty acids was dependent on the subunit composition of the
GABAA receptor complexes. In addition, various combinations
of GABAA receptor subunits might be expressed in the same
neuron, complicating the interpretation of data on the effect of free
fatty acids on the GABAA receptor complex obtained at the
level of single cells, as, for example, by whole cell patch clamp
techniques.
The insect cell Sf-9/baculovirus expression system in combination with
electrophysiological techniques provide the means for the investigation
of functional difference between GABAA receptors consisting
of various subunits. For this purpose, we employed recombinant human
GABA receptor-channel complexes composed of 1
2
2s subunits expressed in
the Sf-9 insect cell system. GABAA receptor complexes
composed of
1,
2, and
2s
subunits have been suggested to be highly abundant in the vertebrate
brain (13, 14) and in most brain areas, e.g. hippocampus
(15), cortex (16), and cerebellum (17).
In this study, the effects of DHA and other fatty acids on GABA-induced
chloride currents were investigated in recombinant human
GABAA receptor-channel complexes composed of
1
2
2s subunits expressed in
the Sf-9 insect cell system.
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EXPERIMENTAL PROCEDURES |
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The construction of expression vectors for the human
GABAA receptors subunits 1,
2, and
2s has been described previously (18). Solid-phase DNA sequencing (Dynal®) combined with sequenase version 2.0 DNA sequencing kit (U. S. Biochemical Corp.) of each insert DNA verified that the amino acid sequences of the
1 and
2 subunits were identical to those
previously reported (19, 20) and that the
2s subunit
varied at amino acid residue 81 (threonine instead of serine) and amino
acid residue 142 (threonine instead of serine) compared with the
reported amino acid sequences (21).
Expression of GABAA Receptor in Sf-9 Cells--
Sf-9
insect cells were grown in spinner flask cultures at 27 °C in
serum-free medium (Sf900-II-SFM, Life Technologies, Inc.). Insect cells at a density of 7.5 × 105 cells/35-mm
Petri dish (Falcon) were infected with baculovirus containing human
cDNA of 1,
2, and
2s
subunits at a multiplicity of infection of 3:1:2 or
1
and
2 subunits at a multiplicitity of infection of 3:1.
The infected cells were incubated at 27 °C for 48 h.
Electrophysiological Experiments-- At approximately 48 h postinfection, Sf-9 cells were constantly perfused with extracellular solution at a flow rate of 3-4 ml/min. The composition of the standard external solution was (in mM): NaCl 150, KCl 5, MgCl2 1, CaCl2 2, glucose 10, HEPES 10. The pH was adjusted to 7.4 by Tris base. Patch pipettes contained a solution of (in mM): Cs2SO4 50, CsCl 78, MgCl2 6, EGTA 5, ATP 5, HEPES 10 (pH 7.2 by Tris base). Series resistances in whole cell voltage clamp experiments were calculated from the capacitative current peak in a 10 mV voltage step and were in range of 10-25 megohms.
Electrical recordings were carried out using conventional whole cell patch recording. Patch pipettes were made of glass capillaries with an outer diameter of 1.5 mm using a vertical puller (Narishige, PB-7, Japan). The cells were voltage-clamped using a voltage-clamp amplifier (Nihon Koden, CEZ-2300, Japan). All signals were filtered with a low pass filter with a cut-off frequency of 1 KHz, monitored on a syncroscope (Iwatsu, MS-SlOOA, Japan) and a pen recorder (Sanei, Recti Horiz 8K, Japan), then digitized at a rate of 44 KHz (Sony, PCM 501 ESN, Japan). The data were stored on video tape (Mitsubishi, HV-F73, Japan). All experiments were carried out at room temperature (23-25 °C).Drug Application-- Rapid application of drugs was achieved by the "Y tube" method as described previously (22). In the present study, each drug was applied at an interval of more than 2 min unless otherwise stated. The drugs used for current recording were GABA, H-89, chelerythrine, and BAPTA (Sigma). DHA, AA, docosahexapentaenoic acid, docosatetraenoic acid, docosatrienoic acid, docosadienoic acid, and oleic acid were from Funakoshi, Tokyo, Japan. Free fatty acids were dissolved in dimethyl sulfoxide and diluted into the extracellular solution just before use. The final concentration of dimethyl sulfoxide was less than 0.1% (v/v).
Statistical Analysis-- The experimental values are presented as mean ± S.D. Student's t test was used when two groups were compared. When relationships between the peak current amplitude and the GABA concentration were examined, continuous lines were fitted according to the following equation
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(Eq. 1) |
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(Eq. 2) |
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(Eq. 3) |
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RESULTS |
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In Sf-9 cells transfected with homomeric 1,
2, or
2s or dimeric
1
2s or
2
2s
receptor subunit combinations, GABA (up to 10
3
M) did not induce any detectable current (less than 5 pA,
n = 10-12 in each group) at a holding potential
(VH) of
40 mV (data not shown). On the other hand,
in cells transfected either with dimeric
1 and
2, or with trimeric
1,
2,
and
2s receptor subunit combinations, 3 × 10
5 M GABA produced an initial peak inward
current followed by a gradual desensitization at a
VH of
40 mV (Figs. 1
and 6). Immediately after the removal of GABA from the perfusate, the
inward current returned to the current level prior to the application
of GABA. The respective concentrations of GABA for threshold and
EC50 were 10
6 M and 3.1 × 10
5 M in
1
2,
and 10
6 M and 4.7 × 10
5
M in
1
2
2s
receptor subunit combinations.
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Although Zn2+ suppressed the response to 3 × 105 M GABA in a
concentration-dependent manner in both
1
2 and
1
2
2 s receptor subunit
combinations, the threshold and IC50 values
(
1
2; 10
8 M and
4.6 × 10
7 M,
1
2
2 s; 10
5
M and 5.7 × 10
4 M) show
that
1
2 combinations are three orders of potency more sensitive
to the effects of Zn2+ as compared with
1
2
2s receptor subunit
combinations.
Effects of DHA on GABA Responses of Sf-9 Cells Expressing
1
2
2s Receptor Subunit
Combinations--
In
1
2
2s
receptor subunit combinations, three distinct time and
concentration-dependent effects of DHA were observed.
First, DHA (10
6 M) gradually accelerated the
decay of IGABA following the peak amplitude as
shown in the Fig. 1A, a. In the absence of DHA,
the decay time to 80% of the initial peak amplitude
(T0.8) was 2.17 ± 0.09 s (mean ± S.D., n = 7). After continuous application of DHA
(10
6 M) for 3 and 10 min, the
T0.8 values were reduced to 1.26 ± 0.08 s
(n = 7) and 0.34 ± 0.06 s (n = 6), respectively. Both T0.8 values obtained
with DHA are significantly different from the control T0.8 value (p < 0.05, t test). After the removal of DHA from the perfusate, the
T0.8 values returned gradually to the control
value. This process usually required more than 20 min.
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Effect of DHA on GABAA Receptors Composed of
1
2 Subunit Combinations--
In Sf-9
cells expressing GABAA receptors composed of
1
2 subunit combinations, DHA, in a
concentration-dependent manner, suppressed the response to
GABA (3 × 10
5 M) (Fig.
6A). The effect of DHA was
restricted to the inhibitory effect, since neither a potentiation of
the GABA response (Fig. 6A) or an alteration of the
desensitization of IGABA (Fig. 6C) as
in the case of
1
2
2s
receptor subunit combinations could be observed in
1
2 subunit
combinations.
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DISCUSSION |
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In the present study it was shown that 1) in GABAA
receptor complexes consisting of
1
2
2s and
1
2 receptor subunit combinations, Zn2+ blocks the GABA-induced Cl
channel
activity. The presence of a
2s subunit in the
GABAA receptor complexes markedly diminished the inhibitory
effect of Zn2+. 2) The presence of the
2s
subunit in the receptor complex is essential for DHA to potentiate the
GABA response of
1
2
2s
receptor subunit combinations, this response is blocked by
Zn2+, Cu2+ and Ni2+. Furthermore,
DHA accelerates the desensitization of IGABA in
1
2
2s combinations, this
effect was not blocked by the divalent cations. 3) DHA and AA, at
concentrations higher than 10
6 M, block the
GABA-induced Cl
channel activity in
1
2
2s and
1
2 receptor subunit combinations, while
other unsaturated fatty acids, e.g. oleic acid, had no
effect. These result suggest that DHA has a number of molecular target sites within the GABAA receptor channel complex. One site
might be related to the Cl
channel domain. Two additional
sites relate functionally to the presence of the
2
subunit in the receptor complex. One site is involved in the
potentiation of the GABA response and is sensitive to antagonism by
Zn2+. The other site may participate in the acceleration of
the desensitization of the GABA-gated current, an effect insensitive to
Zn2+.
Inhibitory Effect of DHA on the GABA-induced Chloride Ion Current
of GABAA Receptor Complexes--
At a concentration of
3 × 106 M or higher, DHA is a
noncompetitive antagonist of GABAA receptor responses in
1
2
2s (Fig. 1) and
1
2 (Fig. 6) receptor subunit
combinations. A similar inhibitory effect of DHA has been reported on
native GABAA receptor complexes of acutely dissociated
neurons from rat substantia nigra (7). On the other hand, DHA has been
shown to potentiate the N-methyl-D-aspartic acid
responses of dissociated rat nigra neurons (7) as well as cortical
neurons (4). The present results suggest that DHA selectively inhibits
the function of the GABAA receptor-chloride ion channel
complexes.
Potentiation of the Peak Amplitude and Acceleration of the
Desensitization of the GABA Response in the
1
2
2s GABAA
Receptor Subunit Combinations by DHA--
A distinct action of DHA on
the GABA response is to accelerate the desensitization of
IGABA after the peak response in
1
2
2s GABAA
receptor subunit combinations. This change in the desensitization kinetics was observed at DHA concentration as low as 10
7
M, and showed both slow development and recovery (Fig. 1).
In contrast, DHA did not change the desensitization of
1
2 GABAA receptor subunit
combinations (Fig. 6).
The Effect of Zn2+ on the GABA Response of
1
2
2s GABAA
Receptor Subunit Combinations and Their Modulation by DHA--
In
accordance with previous findings (34), Zn2+ blocked the
GABAA receptor response. Our experiment demonstrated that
1
2 GABAA receptor complexes
were 3 orders of magnitude more sensitive to the inhibitory effect of
Zn2+ than at
1
2
2 GABAA
receptor subunit combinations. This result indicates that the presence
of
2s subunits render the GABAA
receptor-channel complex less sensitive to Zn2+. The
binding site for Zn2+ on the GABAA
receptor-channel complex has been reported to be located at the
extracellular part of the Cl
channel complex, resulting
in a reduction of the opening frequency of the Cl
channel
(35). Interestingly, Zn2+ (10
5 M)
inhibited the potentiation of the GABAA receptor response induced by 10
6 M DHA in
1
2
2s GABAA
receptor subunit combinations (Fig. 4). At least four possible
mechanisms of action for this effect can be suggested. 1)
Zn2+ could inhibit the binding of GABA to the receptor in
1
2
2s receptor
combinations. However, since a similar concentration-response relationship for GABA in the absence or presence of 10
5
M Zn2+ is
observed2 this explanation is
unlikely. 2) Zn2+ could reduce the bioactivity of DHA.
However, since the desensitization of IGABA is
equally affected by DHA (10
6 M) in the
absence or presence of 10
5 M Zn2+
(Fig. 4A), this explanation is improbable. 3)
Zn2+ could influence the binding of DHA to a site at the
2s subunit. 4) Zn2+ could interfere with the
interaction between
subunits and other subunits of the
GABAA receptor.
Possible Involvement of DHA in Neuronal Function-- DHA and AA are abundant fatty acids in the brain tissue. The respective concentration of DHA and AA are 17 and 12% (by weight) of total fatty acids in the adult rat brain (36). These fatty acids can be released from membrane phospholipids by the action of phospholipase A2 (2). The physiological relevant concentration of these fatty acids in the neuron has been estimated to be 1-10 µM (37). An increase in phospholipase activity occurring in, e.g. ischemia and epilepsy, will cause a transient rise of the concentration of free fatty acids by more than several times (37). Zn2+ is found throughout the brain, being concentrated in the particular areas such as cortex and hippocampus (38). It is of particular interest that Zn2+ is concentrated in the synaptosomes (39) and Zn2+ has been shown to be released from the nerve terminal (40).
The present result suggest a variety of actions of DHA and AA on the activity of GABAA receptor channel complex and emphasize the importance of the lipid microenvironment for the activity of ligand-gated ionic channels. The combination of three factors, namely, the concentration of DHA and AA, the subunit composition of the GABAA receptor complexes and the presence of Zn2+ induce a whole spectrum of potential modulatory mechanisms affecting GABAA response complexes in the central nervous system. ![]() |
FOOTNOTES |
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* This work was supported by Grant-in-Aid for Scientific Research of The Ministry of Education, Science and Culture, Japan Nos. 09260225 and 09670046 (to J. N.), Nos. 07407002 and 07276101 (to N. A.), and by the Novo-Nordisk Foundation (to M.-R.W.) and Beckett Foundation (to M. N.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom all correspondence should be addressed. Tel.: 81-92-642-6090; Fax: 81-92-633-6748; E-mail: nabekura{at}mailserver.med.kyushu-u.ac.jp.
1
The abbreviations used are: AA, arachidonic
acid; DHA, docosahexaenoic acid; GABAA, -aminobutyric
acid type A; BAPTA,
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid.
2 J. Nabekura, K. Noguchi, M.-R. Witt, M. Nielsen, and N. Akaike, unpublished data.
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REFERENCES |
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