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J Biol Chem, Vol. 273, Issue 18, 11056-11061, May 1, 1998


Functional Modulation of Human Recombinant gamma -Aminobutyric Acid Type A Receptor by Docosahexaenoic Acid*

Junichi NabekuraDagger §, Kazuo NoguchiDagger , Michael-Robin Witt, Mogens Nielsen, and Norio AkaikeDagger

From the Dagger  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

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Human gamma -aminobutyric acid type A (GABAA) receptors were expressed in the baculovirus/Sf-9 insect cell expression system using recombinant cDNA of alpha 1beta 2gamma 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 alpha 1beta 2gamma 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 alpha 1 and beta 2 subunit combinations. The data show that the gamma  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.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

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 gamma -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 alpha 1beta 2gamma 2s subunits expressed in the Sf-9 insect cell system. GABAA receptor complexes composed of alpha 1, beta 2, and gamma 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 alpha 1beta 2gamma 2s subunits expressed in the Sf-9 insect cell system.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

The construction of expression vectors for the human GABAA receptors subunits alpha 1, beta 2, and gamma 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 alpha 1 and beta 2 subunits were identical to those previously reported (19, 20) and that the gamma 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 alpha 1, beta 2, and gamma 2s subunits at a multiplicity of infection of 3:1:2 or alpha 1 and beta 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
I=I<SUB><UP>max</UP></SUB> · C<SUP>n</SUP>/(<UP>EC</UP><SUB>50</SUB><SUP>n</SUP>+C<SUP>n</SUP>) (Eq. 1)
where I is the normalized value of the current, Imax the maximal response, C the GABA concentration, EC50 the concentration corresponding to the half-maximal response, and n the apparent Hill coefficient. The concentration-inhibition curves were drawn using a mirror image of the modified Michael-Menten equation in combination with a least-square fitting routine after normalizing the amplitudes of the responses unless otherwise stated.
I/I<SUB><UP>control</UP></SUB> ≃ 1−C<SUP>n</SUP>/(<UP>EC</UP><SUB>50</SUB><SUP>n</SUP>+C<SUP>n</SUP>) (Eq. 2)
I indicates the normalized value of the current obtained with the concentration (C) of the drug, where the control response is defined as 1. Then,
I=1−C<SUP>n</SUP>/(<UP>IC</UP><SUB>50</SUB><SUP>n</SUP>+C<SUP>n</SUP>) (Eq. 3)
IC50 and n denote the concentration giving half-maximal response and the Hill coefficient, respectively.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

In Sf-9 cells transfected with homomeric alpha 1, beta 2, or gamma 2s or dimeric alpha 1gamma 2s or beta 2gamma 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 alpha 1 and beta 2, or with trimeric alpha 1, beta 2, and gamma 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 alpha 1beta 2, and 10-6 M and 4.7 × 10-5 M in alpha 1beta 2gamma 2s receptor subunit combinations.


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  		Fig. 1.   Effect of DHA on the IGABA in alpha 1beta 2gamma 2s GABAA receptor complex subunit combinations. A, effects of 10-6 M (a) and 3 × 10-6 M DHA (b) on the response induced by 3 × 10-5 M GABA. a) 10-6 M DHA potentiated the peak amplitude of IGABA soon after the addition of DHA (shaded bar). The potentiation by DHA (10-6 M) disappeared immediately after the removal of DHA from the perfusate. GABA was applied at an interval of 2 min. b, DHA (3 × 10-6 M) gradually suppressed the peak amplitude of IGABA. The most right current traces in a and b were obtained 4 min after the removal of DHA from the perfusate. Treatment with either concentration of DHA (c, 10-6 M; d, 3 × 10-6 M) accelerated the desensitization of IGABA after the peak. The current traces numbered in a (1 and 2) and b (3 and 4) correspond to those in c and d, respectively. Peaks of GABA responses in the presence and absence of DHA were related to each other in c and d. B, concentration- and time-dependent effect of DHA on 3 × 10-5 M GABA response. The relative amplitudes of IGABA, normalized to the IGABA prior to the addition of DHA (*) in each cell were plotted as a function of time. Each symbol and bar indicates the mean ± S.D. of 6-10 cells.

Although Zn2+ suppressed the response to 3 × 10-5 M GABA in a concentration-dependent manner in both alpha 1beta 2 and alpha 1beta 2gamma 2 s receptor subunit combinations, the threshold and IC50 values (alpha 1beta 2; 10-8 M and 4.6 × 10-7 M, alpha 1beta 2gamma 2 s; 10-5 M and 5.7 × 10-4 M) show that alpha 1beta 2 combinations are three orders of potency more sensitive to the effects of Zn2+ as compared with alpha 1beta 2gamma 2s receptor subunit combinations.

Effects of DHA on GABA Responses of Sf-9 Cells Expressing alpha 1beta 2gamma 2s Receptor Subunit Combinations-- In alpha 1beta 2gamma 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.

The effect of DHA on the peak amplitude induced by GABA in alpha 1beta 2gamma 2s receptor subunit combinations was concentration dependent. The peak IGABA was potentiated by 10-6 M DHA (n = 8) (Fig. 1A, a). This potentiation was apparent even if 10-6 M DHA was applied simultaneously with GABA and faded rapidly upon removal of DHA from the perfusate. The effect of 10-6 M DHA on the GABA response was also dependent on the concentration of GABA used, since 10-6 M DHA significantly potentiated the current responses to 3 × 10-5 M GABA or lower. However, at concentrations of 10-4 M or higher, the potentiation was not evident (Fig. 2B, bar graph inset).


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  		Fig. 2.   Concentration-response relationship of GABA in the presence and absence of DHA in alpha 1beta 2gamma 2s GABAA receptor complex subunit combinations. A, the current responses to different concentrations of GABA in the presence of 10-6 M (a) and 3 × 10-6 M (b) DHA. The traces in a and b were obtained from the same cells. The holding potential was -40 mV. Dotted lines indicate the peak level of the GABA response in the absence of DHA. B, concentration-response relationships for GABA without addition of DHA (open circles), with 10-6 M (closed triangles), 3 × 10-6 M (closed circles) and 10-5 M DHA (closed squares). The peak amplitudes of IGABA were normalized to the peak IGABA induced by 3 × 10-5 M GABA in the absence of DHA in each cell (*). Each symbol and vertical line indicates mean ± S.D. value of six to nine cells. The bar graph inset shows the potentiating ratios of the peak IGABA by 10-6 M DHA. The potentiating ratio was calculated as the ratio of the peak amplitude of IGABA in the presence of 10-6 M DHA to that in the absence of DHA at each concentration of GABA in each cell. #p < 0.05 (Student's t test). Vertical bars and lines indicate the mean ± S.D. of six to nine cells.

DHA, at concentrations higher than 3 × 10-6 M, invariably suppressed the peak IGABA in a concentration and time-dependent manner (Fig. 1A, b, and B). The time interval to the maximal suppression of IGABA was dependent on the DHA concentration applied (Fig. 1B). The peak IGABA suppressed by DHA gradually returned to the original value after the washing out of DHA (Fig. 1A, b, and B). The concentration-response relationships for GABA in the absence or presence of DHA demonstrate that DHA affects the peak IGABA without affecting the threshold concentration and EC50 value; respective values were 10-6 M and 5.3 × 10-5 M in the absence of DHA, 10-6 M and 4.1 × 10-5 M in the presence of 10-6 M DHA, and 10-6 M and 4.8 × 10-5 M in the presence of 3 × 10-6 M DHA (Fig. 2). These results indicate that DHA at a concentration higher than 10-6 M inhibits the GABA response in alpha 1beta 2gamma 2s receptor subunit combinations in a noncompetitive manner.

DHA and other fatty acids affect the mobilization of intracellular effector molecules such as protein kinase C (23) and intracellular Ca2+ (24, 25). Since several different putative phosphorylation sites have been suggested in GABAA receptor complexes consisting of alpha 1beta 2gamma 2s subunit combinations (26), we investigated the possible involvement of several intracellular modulators in the effects of DHA on the GABA response of alpha 1beta 2gamma 2s receptor subunit combinations. Addition of H-89, a protein kinase A inhibitor, chelerythrine, a protein kinase C inhibitor and intracellular application of BAPTA, a Ca2+ chelator, did neither affect the potentiation of the 3 × 10-5 M GABA response by 10-6 M DHA nor the inhibition of the GABA responses induced by 3 × 10-6 M DHA (Fig. 3). Furthermore, the effects of DHA on the desensitization of IGABA persisted in the presence of these inhibitors. The T0.8 values for the experiments with the inhibitors in the presence of 3 × 10-6 M DHA in all experiments were control (DHA only); 0.35 ± 0.08 s (n = 6) H-89 (10-6 M); 0.32 ± 0.05 s (n = 4), chelerythrine (3 × 10-6 M); 0.38 ± 0.06 s (n = 4), BAPTA (10-3 M); 0.41 ± 0.09 s (n = 4). These results suggest that neither protein kinase A or C or intracellular Ca2+ are involved in the modulatory actions of DHA on the GABA response of alpha 1beta 2gamma 2 s receptor subunit combinations.


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  		Fig. 3.   No effect of protein kinase C and A inhibitors and intracellular BAPTA on the potentiation of IGABA by DHA in alpha 1beta 2gamma 2 s GABAA receptor subunit combinations. The potentiating effect of 10-6 M DHA (left) and the suppressive action of 3 × 10-6 M DHA (right) on 10-5 M GABA response persisted in the presence of either chelerythrine, a protein kinase C inhibitor (3 × 10-6 M, hatched bars), H-89, a protein kinase A inhibitor (10-6 M, dotted bars), or intracellular application of BAPTA (3 × 10-3 M, filled bars). NS, no significant difference (p > 0.1, Student's t test). Chelerythrine and H-89 were dissolved in the perfusate. BAPTA was dissolved in the pipette solution.

Surprisingly, the facilitatory effect of DHA (10-6 M) on the peak IGABA was blocked by adding Zn2+ (10-5 M) (Fig. 4A), a concentration at which Zn2+ only minimally suppressed the peak of GABA response in the alpha 1beta 2gamma 2s GABAA receptor subunit combinations (Fig. 4A). The DHA concentrations for threshold and IC50 values for the response induced by GABA (3 × 10-5 M) were 3 × 10-7 M and 3 × 10-6 M with 10-5 M Zn2+, respectively. Other divalent cations such as Ni2+ (Fig. 4C, n = 4) and Cu2+ (n = 4, data not shown) mimicked the antagonistic effect of Zn2+ on the potentiation of the GABA response by DHA. Removal of both Ca2+ and Mg2+ from the perfusate did not affect the potentiating ratio of the response induced by 10-6 M DHA (19.2 ± 4.8%, n = 4). However, the changes in the desensitization kinetics of IGABA induced by DHA were unaffected by these divalent cations (Fig. 4, A and C).


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  		Fig. 4.   Blockade of the potentiating effect of DHA on the GABA response by Zn2+ in alpha 1beta 2gamma 2s GABAA receptor subunit combinations. A, the potentiation of GABA (3 × 10-5 M) response by DHA (10-6 M) was blocked by Zn2+ (10-5 M). Each series of current traces was obtained from the same cell. Zn2+ and DHA were applied 30 s and 1 min before the GABA application, respectively. The holding potential was -40 mV. Note that the desensitization of IGABA affected by DHA is not restored with Zn2+. B, concentration-response relationship for DHA on GABA (3 × 10-5 M) response in the presence (closed circles) or absence of 10-5 M Zn2+ (open circles). The peak amplitude of 3 × 10-5 M GABA response 2.5 min after the start of the perfusion with DHA were normalized to that immediately prior to DHA in each cell. Symbols and vertical lines indicate mean ± S.D. of six to nine cells. C, Ni2+ mimicked the blocking action of Zn2+ on the potentiation of the GABA response by DHA (10-6 M). Dashed lines indicate the peak current levels prior to the perfusion with DHA in the presence or absence of Ni2+ (10-5 M).

Unsaturated fatty acids other than DHA, e.g. AA have been reported to inhibit the GABAA response in rat substantia nigra neurons (7). Therefore, we examined the effects of various unsaturated fatty acids on GABAA alpha 1beta 2gamma 2s receptor subunit combinations with the surprising result that only AA was able to mimic the effects of DHA while compounds structurally closely related to DHA, e.g. docosapentaenoic acid or an endogenous, relatively common unsaturated fatty acid, oleic acid, were inactive (Fig. 5).


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  		Fig. 5.   Effects of various unsaturated fatty acids on the GABA (3 × 10-5M) response in alpha 1beta 2gamma 2s GABAA receptor subunit combinations. Horizontal bars indicate the mean ± S.D. of 6-10 cells. *p < 0.05 (paired t test) compared with the effect of GABA (3 × 10-5 M) in the absence of fatty acid. The relative IGABA in the presence of fatty acid was compared with that obtained in the absence of fatty acid in each cell. DPA, docosapentaenoic acid; DTtA, docosatetraenoic acid; DTrA, docosatrienoic acid; DDA, docosadienoic acid; OA, oleic acid.

Effect of DHA on GABAA Receptors Composed of alpha 1beta 2 Subunit Combinations-- In Sf-9 cells expressing GABAA receptors composed of alpha 1beta 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 alpha 1beta 2gamma 2s receptor subunit combinations could be observed in alpha 1beta 2 subunit combinations.


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  		Fig. 6.   Effect of DHA on the GABA response in alpha 1beta 2 GABAA receptor subunit combinations. A, concentration-inhibition relationship of DHA on GABA (3 × 10-5 M) response. Current traces; DHA was continuously applied starting 2.5 min prior to GABA. The peak amplitudes of IGABA 2.5 min after the addition of DHA were normalized to the IGABA immediately prior to the addition of DHA in each cell. B, the concentration-response relationship for the IGABA in the presence (closed circles) or absence of 10-6 M DHA (open circles) in the perfusate. The current traces was obtained from the same cell. The peak of the GABA response obtained 2.5 min after the addition of DHA was normalized to that of GABA (3 × 10-5 M) response in the absence of DHA (*) in each cell. Each symbol and vertical line indicates the mean ± S.D. of 6-10 cells. C, no effect of 10-6 M DHA on the desensitization of IGABA. Current traces were obtained prior to (left trace) and 4 min after the addition of DHA (right trace) in the same cell. The peak amplitudes were arbitrary manipulated for a convenience to compare the decays of GABA responses in the presence and absence of DHA. The holding potential was -40 mV.

The concentration-response relationship for GABA indicates that GABA concentrations for threshold and EC50 values were 3 × 10-6 M and 4.35 × 10-5 M in the absence, and 3 × 10-6 M and 1.15 × 10-5 M in the presence of DHA (10-6 M) (Fig. 6B). These data suggest that DHA is a noncompetitive inhibitor of GABA in alpha 1beta 2 subunit combinations.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

In the present study it was shown that 1) in GABAA receptor complexes consisting of alpha 1beta 2gamma 2s and alpha 1beta 2 receptor subunit combinations, Zn2+ blocks the GABA-induced Cl- channel activity. The presence of a gamma 2s subunit in the GABAA receptor complexes markedly diminished the inhibitory effect of Zn2+. 2) The presence of the gamma 2s subunit in the receptor complex is essential for DHA to potentiate the GABA response of alpha 1beta 2gamma 2s receptor subunit combinations, this response is blocked by Zn2+, Cu2+ and Ni2+. Furthermore, DHA accelerates the desensitization of IGABA in alpha 1beta 2gamma 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 alpha 1beta 2gamma 2s and alpha 1beta 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 gamma 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 × 10-6 M or higher, DHA is a noncompetitive antagonist of GABAA receptor responses in alpha 1beta 2gamma 2s (Fig. 1) and alpha 1beta 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.

The inhibitory action of DHA is time- and concentration-dependent (Figs. 1 and 6). In addition, the DHA-induced inhibition of IGABA ceases only slowly after the removal of DHA from the extracellular solution. There are several possible explanations for the gradual action of DHA on the GABAA receptor-channel complex. First, the suppression of the GABA response by fatty acids might be mediated through an intracellular second messenger system, since fatty acids have been shown to enhance the diacylglycerol-dependent activation of protein kinase C (23) and to increase the level of free intracellular Ca2+ (24). Furthermore, the GABAA receptor-Cl- channel complex has been reported to be modulated by intracellular Ca2+ (27), by protein kinase A (28) or by protein kinase C (13, 26). However, the present results show that neither chelerythrine, a protein kinase C inhibitor, H-89, a protein kinase A inhibitor, nor intracellular BAPTA interfered with the effects of DHA on the GABA response (Fig. 3), suggesting that these intracellular signaling pathways are not involved in the effects of DHA on the GABAA receptor complex.

Second, since fatty acids have been shown to be able to modify the membrane fluidity and function of integral membrane proteins (1), the slow onset of the action of DHA might be explained by changes induced in the lipid microenvironment of the GABAA receptor-chloride ion channel complex. However, this explanations seem improbable since among various structurally closely related fatty acids examined, only DHA and AA affect the GABA responses (Fig. 5). AA and DHA have been shown to inhibit muscimol-induced Cl- uptake in rat cerebral cortical synaptosomes (29). Likewise, free fatty acids have been shown to decrease the binding of tert-butylbicyclophosphorothionate, which has been proposed to bind at the Cl- channel domain (10, 30). Therefore, it might be concluded that DHA and AA bind to a site close to the Cl- channel, acting as a Cl- channel blocker.

Potentiation of the Peak Amplitude and Acceleration of the Desensitization of the GABA Response in the alpha 1beta 2gamma 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 alpha 1beta 2gamma 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 alpha 1beta 2 GABAA receptor subunit combinations (Fig. 6).

A striking action of DHA on the GABAA response is that DHA (10-6 M) potentiated the peak IGABA in alpha 1beta 2gamma 2s GABAA receptor combinations, this effect was absent in alpha 1beta 2 GABAA receptor combinations. The potentiating effect of DHA is obvious when GABA concentrations lower than 10-4 M was applied, no potentiation was observed at GABA concentrations of 10-4 M or higher (Fig. 2B). Although this might be due to the limitation of the Y-tube application system (22), another plausible explanation might be a "ceiling effect," since the GABA concentration of 10-4 M to 10-3 M induces the maximal current response achievable in a given cell and therefore masks potentiating effects of DHA. The very rapid desensitization of IGABA induced by higher concentrations of GABA itself in addition to the accelerated desensitization induced by DHA might reduce the true peak current. Unlike the slow inhibition of GABA response by DHA (Fig. 1), the potentiation of IGABA was apparent even if DHA (10-6 M) was applied simultaneously with GABA.

The high affinity benzodiazepine binding within the GABAA receptor complex is dependent on the presence of the gamma 2 (or gamma 3) subunit (31). Unsaturated free fatty acids increase the benzodiazepine binding on the GABAA receptors in native (11) and recominant (12) GABAA receptor chloride channel complexes. Free fatty acids interact with a GABAA receptor subunit configuration that requires the presence of a gamma 2 subunit. The potentiation of the GABA response on one hand and the increased desensitization of GABA responses on the other seem to be independent of each other since: 1) the development of the acceleration of desensitization is a slow, while the potentiation of the GABA response by DHA is a rapid phenomenon (Fig. 1A, a); and 2) divalent cations blocked the potentiation of GABA response by DHA, but the desensitization was not affected (Fig. 4).

The existence of a fatty acid binding domain has been reported for the N-methyl-D-aspartic acid receptor channel complex (32) and the binding of fatty acids to the specific binding domain induces a conformational change of the protein (33). The present data suggest that the binding of unsaturated fatty acids to a specific binding site near or at the gamma 2 subunit results in a conformational change of the GABAA receptor complex which leads to an alteration of the response to GABA, e.g. the potentiation of the IGABA and the acceleration of the desensitization of the GABAA receptor composed of alpha 1beta 2gamma 2s subunit combinations.

The Effect of Zn2+ on the GABA Response of alpha 1beta 2gamma 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 alpha 1beta 2 GABAA receptor complexes were 3 orders of magnitude more sensitive to the inhibitory effect of Zn2+ than at alpha 1beta 2gamma 2 GABAA receptor subunit combinations. This result indicates that the presence of gamma 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 alpha 1beta 2gamma 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 alpha 1beta 2gamma 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 gamma 2s subunit. 4) Zn2+ could interfere with the interaction between gamma  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

* 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, gamma -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.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Stubbs, C. D., and Smith, A. D. (1984) Biochim. Biophys. Acta 779, 89-137[Medline] [Order article via Infotrieve]
  2. Piomelli, D., and Greengard, P. (1990) Trends Pharmacol. Sci. 11, 367-373[CrossRef][Medline] [Order article via Infotrieve]
  3. Miller, B., Sarantis, M., Traynelis, S. F., and Attwell, D. (1992) Nature 355, 722-725[CrossRef][Medline] [Order article via Infotrieve]
  4. Nishikawa, M., Kimura, S., and Akaike, N. (1994) J. Physiol. 475, 83-93[Abstract/Free Full Text]
  5. Schmid, A., and Schulz, I. (1995) J. Physiol. 484, 661-676[Medline] [Order article via Infotrieve]
  6. Horimoto, N., Nabekura, J., and Ogawa, T. (1997) Neuroscience 77, 661-671[CrossRef][Medline] [Order article via Infotrieve]
  7. Hamano, H., Nabekura, J., and Ogawa, T. (1996) J. Neurophysiol. 75, 1264-1270[Abstract/Free Full Text]
  8. Nielsen, M., Witt, M. R., and Thogersen, H. (1988) Eur. J. Pharmacol. 146, 349-353[CrossRef][Medline] [Order article via Infotrieve]
  9. Ueno, E., and Kuriyama, K. (1981) Neuropharmacology 20, 1169-1176[CrossRef][Medline] [Order article via Infotrieve]
  10. Koenig, J. A., and Martin, I. L. (1992) Biochem. Pharmacol. 44, 11-15[CrossRef][Medline] [Order article via Infotrieve]
  11. Witt, M. R., and Nielsen, M. (1994) J. Neurochem. 62, 1432-1439[Medline] [Order article via Infotrieve]
  12. Witt, M. R., Westh-Hansen, S. E., Rasmussen, P. B., Hastrup, S., and Nielsen, M. (1996) J. Neurochem. 67, 2141-2145[Medline] [Order article via Infotrieve]
  13. McKernan, R. M., and Whiting, P. J. (1996) Trends Neurosci. 19, 139-143[CrossRef][Medline] [Order article via Infotrieve]
  14. Wisden, W., Laurie, D. J., Monyer, H., and Seeburg, P. H. (1992) J. Neurosci. 12, 1040-1062[Abstract]
  15. Fritschy, J. M., Benke, D., Mertens, S., Oertel, W. H., Bachi, T., and Mohler, H. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 6726-6730[Abstract/Free Full Text]
  16. Gao, B., and Fritschy, J. M. (1994) Eur. J. Neurosci. 6, 837-853[CrossRef][Medline] [Order article via Infotrieve]
  17. Quirk, K., Gillard, N. P., Rogan, C. I., Whiting, P. J., and McKernan, R. M. (1994) J. Biol. Chem. 269, 16020-16028[Abstract/Free Full Text]
  18. Westh-Hansen, S. E., Rasmussen, P. B., Hastrup, S., Nabekura, J., Noguchi, K., Akaike, N., Witt, M. R., and Nielsen, M. (1997) Eur. J. Pharmacol. 329, 253-257[Medline] [Order article via Infotrieve]
  19. Schofield, P. R., Pritchett, D. B., Sontheimer, H., Kettenmann, H., and Seeburg, P. H. (1989) FEBS Lett. 244, 361-364[CrossRef][Medline] [Order article via Infotrieve]
  20. Hadingham, K. L., Wingrove, P. B., Wafford, K. A., Baink, C., Kemp, J. A., Palmer, K. J., Wilson, A. W., Wilcox, A. S., Sikela, J. M., Ragan, C. I., and Whiting, P. J. (1993) Mol. Pharmacol. 44, 1211-1218[Abstract]
  21. Mihic, S. J., Whiting, P. J., Klein, R. L., Wafford, K. A., and Harrisk, R. A. (1994) J. Biol. Chem. 269, 32768-32773[Abstract/Free Full Text]
  22. Murase, K., Randic, M., Shirasaki, T., Nakagawa, T., and Akaike, N. (1990) Brain Res. 525, 84-91[CrossRef][Medline] [Order article via Infotrieve]
  23. McPhail, L. C., Clayton, C. C., and Snyderman, R. (1984) Science 224, 622-625[Abstract/Free Full Text]
  24. Calderaro, V., Parrillo, C., Balestrieri, M. L., Giovane, A., Filippelli, A., and Rossi, F. (1994) Mol. Pharmacol. 45, 737-746[Abstract]
  25. Packham, D. E., Jiang, L., and Conigrave, A. D. (1995) Cell Calcium 17, 399-408[CrossRef][Medline] [Order article via Infotrieve]
  26. Kellenberger, S., Malherbe, P., and Sigel, E. (1992) J. Biol. Chem. 267, 25660-25663[Abstract/Free Full Text]
  27. Inoue, M., Oomura, Y., Yakushiji, T, and Akaike, N. (1986) Nature 324, 156-158[CrossRef][Medline] [Order article via Infotrieve]
  28. Porter, N. M., Twyman, R. E., Uhler, M. D., and MacDonald, R. L. (1990) Neuron 5, 789-796[CrossRef][Medline] [Order article via Infotrieve]
  29. Schwartz, R. D., and Yu, X. (1992) Brain Res. 585, 405-410[CrossRef][Medline] [Order article via Infotrieve]
  30. Samochocki, M., and Strosznajder, J. (1993) Neurochem. Int. 23, 261-267[CrossRef][Medline] [Order article via Infotrieve]
  31. McKernan, R. M., and Whiting, P. J. (1996) Trends Neurosci. 19, 139-143
  32. Petrou, S., Ordway, R. W., Singer, J. J., and Walsh, J. V. (1993) Trends Biochem. Sci. 18, 41-42[CrossRef][Medline] [Order article via Infotrieve]
  33. Honma, Y., Niimi, M., Uchiumi, T., Takahashi, Y., and Odani, S. (1994) J. Biochem. Tokyo 116, 1025-1029[Abstract/Free Full Text]
  34. Draguhn, A., Verdorn, T. A., Eweri, M., Seeburg, P. H., and Sakmann, B. (1990) Neuron 5, 781-788[CrossRef][Medline] [Order article via Infotrieve]
  35. Kilic, G., Moran, O., and Cherubini, E. (1993) Eur. J. Neurosci. 5, 65-72[CrossRef][Medline] [Order article via Infotrieve]
  36. Dhopeshwarkar, G. A., and Subramanian, C. (1975) Lipids 10, 238-241[CrossRef]
  37. Piomelli, D. (1994) Crit. Rev. Neurobiol. 8, 65-83[Medline] [Order article via Infotrieve]
  38. Donaldson, J., Pierre, T. S., Minnich, J. L., and Barbeau, A. (1973) Can. J. Biochem. 51, 87-92[CrossRef][Medline] [Order article via Infotrieve]
  39. Huag, F. S. (1967) Histochemie 8, 355-368[CrossRef][Medline] [Order article via Infotrieve]
  40. Xie, X., and Smart, T. G. (1991) Nature 349, 521-524[CrossRef][Medline] [Order article via Infotrieve]

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