Neurochem. Int. Vol. 22, No. 3, pp. 263-270, 1993 Printedin Great Britain.All rights reserved
0197-0186/93$6.00+0.00 Copyright© 1993PergamonPress Ltd
GABA TRANSPORTER m R N A : IN VITRO EXPRESSION A N D QUANTITATION IN NEONATAL RAT A N D POSTMORTEM H U M A N BRAIN YUE XIA, 1 MICHAEL S. POOSCH,1 CHRISTOPHERJ. WHITTY, I GREGORY KAPATOS1 and MICHAEL J. BANNON1'2. ~Cellularand Clinical Neurobiology Program, Department of Psychiatry 2Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan, U.S.A. (Received 6 May 1992; accepted 16 July 1992)
Abstract--A previously isolated rat eDNA clone encoding the membrane transporter for the neurotransmitter y-aminobutyric acid was expressed in transfected COS cells. The resultant transporter protein was characterized kinetically and pharmacologically, The apparent Kt (6.1/zM) and the pharmacological profile of a neuronal-type transporter observed in these mammalian cells were consistent with previous data obtained in Xenopus laevis oocytes. Post-natal levels of 7-aminobutyric acid transporter mRNA in rat cerebellum,cerebral cortex and striatum (as measured by nucleaseprotection assay) transiently exceeded levels present in the adult brain. Human y-aminobutyric acid transporter mRNA also was measured by nuclease protection assay using as probe a human transporter eDNA homolog obtained by polymerase chain reaction. These studies suggest that quantitation of rat and human ~,-aminobutyricacid transporter mRNAs may provide a useful index of transporter gene expression.
Amino acid and amine neurotransmitters are inactivated largely by reuptake into presynaptic nerve terminals or, in some cases, nearby glia. Recapture of released neurotransmitter serves to effectively limit the extent, duration and area of receptor activation. Sodium-dependent plasma membrane transporters selective for the various neurotransmitters have been identified, and in several cases, eDNA clones isolated (for a review, see Amara and Pacholczyk, 1991). The transporter for the amino acid y-aminobutyric acid (GABA) is of particular interest for several reasons. First, the GABA transporter is quantitatively important, inasmuch as GABA is thought to be a transmitter at 20% of all mammalian synapses (Iversen and Bloom, 1972). Secondly, neuronal and glial forms of GABA transporters with distinct pharmacological profiles have been described (Iversen and Kelly, 1975; Schon and Kelly, 1975; Bowery et al., 1976; Krogsgaard-Larsen, 1980). In addition, recent evidence suggests that under some conditions the direction of GABA transport may be reversed, result-
ing in a transporter-mediated, calcium-independent release of GABA (Bernath and Zigrnond, 1990; OsetGasque et al., 1990; Meyer, 1991). The isolation and characterization of a rat GABA transporter eDNA clone (GAT-1) recently has been described (Guastella et al., 1990). The experiments described here sought to further characterize the kinetic and pharmacological properties of the transporter encoded by this clone and expressed in mammalian cells in vitro, to quantitate GABA transporter mRNA levels in different rat brain regions during development and to demonstrate the presence of the human homolog in post-mortem tissues.
Rat and human tissues Sprague-Dawley rats (Hilltop Lab Animals) were killed by decapitation on post-natal days (P) 1~11 and brain regions were dissected according to Glowinski and Iversen (1966). Human brain tissues from two subjects (a 37 y old black female and 47 y old white female) were obtained at autopsy *To whom correspondence should be addressed : Center for (with post-mortem intervals of approx 16 h) from the Wayne Cell Biology,Sinai Hospital, 6767 W. Outer Dr., Detroit, County Medical Examiner's Office and dissected according MI 48235, U.S.A. to the atlas of DeArmond et al. (1976), as previously Abbreviations : GABA, y-aminobutyric acid. described (Bannon et al., 1990, 1991, 1992). 263
YUE X1A el al.
COS-7 cell transfection and kinetic' and pharmacological analyses COS-7 cells obtained from American Type Tissue Culture were plated at a density of 2 × 105 cells/60 mm plate in Dulbeceo's modified Eagle medium containing 10% fetal calf serum, 2 mM glutamine, penicillin (50 U/ml) and streptomycin (50/~g/ml). Twenty-four h later, cells were transfected by the calcium phosphate technique (Ausubel et al., 1989) with 0.5 #g of the rat GABA transporter clone GAT1 in a pRC/CMV vector (Guastella et al., 1990; kindly provided by Dr John Guastella) plus 4.5 /~g of carrier plasmid. After an additional 24 h, cells were rinsed with phosphate-buffered saline before addition of fresh media. [3H]GABA accumulation was quantitated 20 24 h later as follows : cells were rinsed twice with Krebs-Ringer-HEPES (KRH) buffer (140 mM NaCI, 5 mM KC1, 2.5 mM CaC12, 1.25 mM MgSO4, 25 mM HEPES and 10 mM glucose, pH 7.4), preincubated for 30 min at 3T'C with 100/tM aminooxyacetic acid (AOAA; a GABA transaminase inhibitor), and then incubated for 60 min at 3TC in 1.9 ml KRH with 19/~Ci [3H]GABA (39.9 Ci/mmol) in the presence of various concentrations of unlabelled GABA and 100 #M AOAA. Since a low affinity GABA accumulation was observed at high substrate concentrations ( > 4 0 #M, data not shown), the kinetics of high affinity GABA transport were selectively studied using substrate concentrations of 0.625-40 #M unlabelled GABA. Following three rinses with KRH buffer, cells were disrupted with 1% sodium dodecyl sulfate, and [3H]GABA was quantitated by liquid scintillation spectroscopy. Specific [3H]GABA accumulation was defined as the difference between GABA accumulation in GAT- l-transfected COS-7 cells and the accumulation seen in COS cells mock-transfected with 5 #g carrier plasmid DNA and assayed in parallel. Initial experiments demonstrated that specific accumulation was temperature-dependent and linear over the 60 min incubation period (data not shown). Each experiment was conducted twice (using triplicate determinations for each data point) with very similar results ; one experiment is shown. Kinetic parameters were calculated using Enzfitter software (Elsevier Biosoft, version 1.03). For pharmacological analyses, experiments were conducted exactly as described above, except that 5 #M unlabelled GABA was included in the incubation, as were various concentrations (0.01-1000/tM) of GABA transporter inhibitors. GABA transporter rnRNA nuclease protection assay Rat and human GABA transporter clones for use in nuclease protection assays were obtained using standard techniques (Ausubel et al., 1989). For a rat probe, the 3' portion of the rat clone GAT-I was removed by digestion with the restriction endonuclease BstXl. The 5' 333 bp of the clone (RGAT-BstXI; Fig. 1) remaining in the Bluescript vector was religated, amplified in transformed Escherichia coli and the sequence verified by the dideoxy method. In order to obtain a human GABA transporter clone (HGAT-PCR; Fig. I), 1 ttg human putamen RNA, extracted by the method of Chomczynski and Sacchi (1987), was reverse transcribed using random hexamer primers, eDNA amplification was accomplished by polymerase chain reaction (94°C, 2 min; 55"C, 3 min; 72°C, 3 min, for 25 cycles), using primers corresponding to human GABA transporter sequences (Nelson et al., 1990) and constructed to contain adjacent restriction endonuclease sites (sense primer : 5'-TAATACG-
RGAT-BstXI (333 bp)
HGAT PCR fragment (276 bp) -
Fig. l. Schematic representation of the 7-aminobutyric acid transporter mRNA and eDNA clones. The coding and noncoding regions of GABA transporter mRNA are shown as boxes and lines, respectively. Within the coding region, putative transmembrane domains are indicated by stippling. The RGAT-BstXI probe was subcloned from GAT-I eDNA, while the HGAT-PCR probe was obtained by polymerase chain reaction as described in Experimental Procedures. These clones were used for nuclease protection analysis of rat and human mRNA, respectively. ACTCACTATAGG GAATTC CCTATGTTCAAGGGCG TGGGCCTTGCGC~-3' ; antisense primer : 5'-GATTTAGGTGACACTATAGTCTAGAGCGGATCTGACCTGGCTTATCCAGCCCG-3': underlined sequences corresponding to EcoRI and Xbal sites, respectively). A prominent DNA band of approx 324 bp visualized after agarose gel electrophoresis was excised, extracted, digested with EcoRl and XbaI, ligated into pGEM-3Z plasmid, amplified in E. coli and sequenced by the dideoxy method. HGAT-PCR nucleotide sequence was an exact match with the previously published sequence (not shown). Rat and human GABA transporter mRNAs were quantitated using a variation of a previously described nuclease protection protocol (Haverstick et al., 1990). RGAT-BstXIderived or HGAT PCR-derived 32p-labelled antisense RNAs (300-800 pg) were used as probes. Clone-derived sense RNA standards (0-100 pg) were assayed in parallel with 15-25 #g tissue RNA. After overnight hybridization at 55°C, samples were digested with SI nuclease (400 U, 73°C, 60 rain) prior to polyacrylamide/urea gel electrophoresis. Analyses were conducted using 2 sets of post-natal rat or post-mortem human brain tissues, each assayed twice in separate hybridizations. Signal intensities oftbe resulting bands were quantitated by densitometric analysis of autoradiograms.
RESULTS G a t - l - t r a n s f e c t e d COS-7 cells exhibited high affinity G A B A a c c u m u l a t i o n with a n a p p a r e n t Kt o f 6.t /~M (Fig. 2), similar to that seen in Xenopus oocytes injected with G A T - I m R N A (Guastella et al., 1990). The pharmacological sensitivity of this high affinity G A B A accumulation was assessed with various G A B A t r a n s p o r t inhibitors. The p o t e n t G A B A t r a n s p o r t inhibitor nipecotic acid, which is nonselective for n e u r o n a l vs glial forms o f G A B A transport, blocked
GABA transporter mRNA in rat and human brain
5 Total . - - - "
0.3 0.2 0.1 I I
.¢ .¢ m .¢
~ ' " ~
GABA ( ~ M )
Fig. 2. Kinetics of the rat ~-aminobutyric acid transporter GAT-1 expressed in COS-7 cells. [3H]GABA accumulation was determined over a range of GABA concentrations, as described in Experimental Procedures. Specific accumulation was the difference between total accumulation in GAT-1-transfected cells and the nonspecific accumulation seen in mock-transfected cells. Scatchard plot analysis of a typical GABA accumulation experiment is shown in the inset.
G A B A accumulation in GAT-l-transfected COS cells with a Ki of 5.4 #M (Fig. 3). 2,4:Diaminobutyric acid (DABA), a G A B A transport inhibitor reportedly selective for neuronal G A B A uptake, also inhibited G A B A accumulaion in transfected cells with an apparent K~ of 172/~M (Fig. 3). In contrast, no significant inhibition of G A B A accumulation was seen in the presence of0.01-100 #M 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol (THPO) or fl-alanine, agents that are believed to be selective for glial G A B A uptake (Fig. 3). Thus the pharmacological properties of GAT-l-induced G A B A accumulation in mammalian COS cells are very similar to the data obtained in amphibian Xenopus laevis oocytes (Guastella et al., 1990). Since the completion of these studies, similar kinetic and pharmacological data following GAT-I expression in other mammalian cell lines have been published (Keynan et al., 1992). The developmental profile of rat G A B A transporter m R N A expression in different brain regions was determined by nuclease protection assays using the GAT-1 subclone RGAT-BstXI (Fig. 1). A typical experiment is presented in Fig. 4. The G A B A transporter m R N A levels in the cerebral cortex were only 28% of adult levels at post-natal day 1 (P1), then rapidly increased to levels 25-40% above the adult
between P10-30 before finally declining to adult levels. In the cerebellum, G A B A transporter m R N A levels were equivalent to adult levels from birth until P10, then increased 3-fold between P21-30 (Fig. 4). Striatal G A B A transporter m R N A levels also approximated adult levels at birth, transiently increased to twice adult levels between P6-10, then rapidly returned to adult levels by P21 (Fig. 4). In the adult, the rank order of G A B A transporter m R N A levels is striatum > cerebral cortex > cerebellum (Fig. 4). In agreement with the finding that only a single band was protected in a nuclease protection assay throughout post-natal development, a single band of approx. 4.5 kb was visualized by Northern blot analysis of R N A from various brain regions of both P6 pups and adult rats (data not shown). A human G A B A transporter eDNA clone was obtained by polymerase chain reaction (Fig. 1), using primers derived from the published human G A B A transporter sequence (Nelson et al., 1990). As the previously isolated human clone had been used to detect rat, but not human, G A B A transporter m R N A (Nelson et al., 1990), nuclease protection assays were utilized in the present study to investigate whether human G A B A transporter m R N A complementary to the cloned sequence is in fact present to any extent in
YUE XIA et al. ""7
100 80 0
THPO o I
tO0 80 60 40 20
IO-B 10-7 10-o 10-5 10"4' lO-a 10-o 10-7 lO-e lO-O 10-4 lO-a Inhibitors
Fig. 3. Pharmacological inhibition of GAT-l-mediated [3H]7-aminobutyric acid accumulation in COS-7 cells. The concentration-dependent effects of nipecotic acid (NIP), 2,4-diaminobutyric acid (DABA), 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol (THPO), and beta-alanine (fl-ALA) on specific GABA accumulation were determined as described in Experimental Procedures.
human tissues. A number of regions from 2 human brains were examined. A typical experiment, illustrated in Fig. 5, shows that human G A B A transporter m R N A is abundantly expressed in various human brain regions. The absence of smaller bands indicative of extensive R N A degradation is consistent with the idea that human G A B A transporter m R N A may be stable post-mortem. DISCUSSION
The high affinity G A B A transporter expressed in mammalian COS-7 cells as a result of GAT-1 transfection (Fig. 2) exhibited the same apparent Kt (approx 6 #M) as that observed in rat brain synaptosomes (Coyle and Enna, 1976 ; Wong and McGeer, 1981) and after GAT-1 expression in Xenopus laevis oocytes or in other mammalian cell lines (Guastella et al., 1990; Keynan et al., 1992). This concordance contrasts with data on the cloned dopamine transporter, which when expressed in vitro (Kilty et al.,
1991 ; Shimada et al., 1991 ; Usdin et al., 1991) exhibits a Kt 7-250 times higher than that of the native dopamine transporter (approx. 0.13 # M ; Holz and Coyle, 1974; Near et al., 1988). It is conceivable that the more faithful in vitro expression of the G A B A transporter may be related to its normally lower affinity for substrate or to a lesser dependence on some accessory protein(s) for optimal functioning. These issues warrant further investigation. A great deal of data supports the contention that there are neuronal and glial forms of the GABA transporter (Iversen and Kelly, 1975; Schon and Kelly, 1975 ; Bowery et al., 1976 ; Krogsgaard-Larsen, 1980). Guastella et al. (1990) reported that a single concentration of several uptake inhibitors partially attenuated [3H]GABA accumulation in GAT-I mRNAinjected Xenopus oocytes with the apparent potency rank order of nipecotic acid > DABA > THPO, /3alanine. In the present experiments, high affinity G A B A accumulation in transfeeted COS ceils was significantly attenuated by the potent but nonselective
GABA transporter mRNA in rat and human brain
PIO P2I P30 P41
0 CB •
/ F-¢ 0
\ \ \ \ \
. . " " " / "/"
" " .......... . . . . . . . .
Fig. 4. Developmental profile of ?-aminobutyric acid transporter mRNA content in several rat brain regions. GABA transporter mRNA levels were determined by nuclease proteetion assay as described in Experimental Procedures. Inset : A typical autoradiogram illustrating the developmental changes in GABA transporter mRNA levels in rat cerebral cortex, striatum and cerebellum at the post-natal days (Pl~L1) indicated. Fifteen #g RNA aliquots were loaded in each lane. The post-natal levels of GABA transporter mRNA (as seen in the inset) are expressed as a percentage of the post-natal day 41 content in cerebral cortex (squal "s; 20.6 attomol//zg RNA), striatum (triangles; 31.0 attomol/#g RNA) and cerebellum (circles ; 8.32 attomol//zg RNA).
Fig. 5. Analysis of human postmortem ?-aminobutyric acid transporter mRNA. Samples were assayed by nuclease protection as described in Experimental Procedures. The upper band in each lane represents remaining undigested probe, while the lower band represents GABA transporter mRNA. Standard lanes (on the left): 0, 2.5, 5.0, 10 and 20 pg GABA transporter RNA standard. Sample lanes on the fight contained 25 #g RNA aliquots from (left to fight) : cerebellum (I .9 attomol GABA transporter mRNA/#g), cerebral cortex (2.1 attomol/#g), substantia nigra (0.9 attomol//zg), caudate (1.4 attomol//~g), putamen (1.2 attomol/#g) and nucleus accumbens (1.7 attomol//~g).
YUE X~A et al.
inhibitor nipecotic acid (K~ 5.4 #M) and by the neuronal transporter-selective inhibitor DABA (K~ 172 #M) but not by glial transporter-selective agents THPO or fl-alanine (Fig. 3). Similar findings using different cell lines and drugs were reported recently by Keynan et al. (1992). Thus, these data are consistent with the notion that GAT- 1 encodes a neuronal GABA transporter. On the other hand, immunocytochemical studies have yielded conflicting data. Using polyclonal antibodies raised against the purified GABA transporter protein (whose partial sequence was exploited for GAT-1 cloning), GABA transporter protein was visualized in both neurons and gila throughout the brain (Radian et al., 1990). These data suggested that these antibodies might be recognizing common or multiple antigenic determinants on 2 distinct transporters or, alternatively, neuronal and glial transporters might be the same protein (encoded by the same mRNA) whose differences arise from distinct post-translational modifications. In a more recent study by the same group (Mabjeesh et al., 1992), however, the same antibodies, as well as polyclonal antisera directed against various GABA transporter fragments, did not recognize the GABA transporter expressed in cultured astroglia. In the present study, hybridization analyses using GAT-l and RGAT-BstXI as probes are in agreement with previous reports (Guastella et al., 1990; Nelson et al., 1990) visualizing a single GABA transporter mRNA band of approx. 4.2-4.4 kb in various brain regions. In contrast, oocyte expression of size-fractionated RNA has suggested there are tissue-specific differences in the size of GABA transporter-encoding messages (Blakely et aL, 1988, 1990). Thus, the issuc of GABA transporter gene, mRNA and protein multiplicity remains unresolved at the present time. In all three rat brain regions examined in detail, post-natal levels of GABA transporter mRNA exceeded the adult levels eventually attained (Fig. 4). This observation agrees with previous reports that GABA uptake in post-natally-derived synaptosomes is greater than in the adult (Coyle and Enna, 1976; Wong and McGeer, 1981), as is the level of GABA transporter expression in X e n o p u s laevis oocytes injected with post-natally-derived mRNAs (Blakely et al., 1990). In the cerebral cortex, the post-natal levels of GABA transporter m R N A began significantly lower than adult levels at birth, but achieved a plateau 25-40% above adult levels between P10-30 (Fig. 4). Previous reports have shown that cortical GABA uptake is also well below adult levels at birth, then plateaus 30-60% above adult levels over the same
time period (Coyle and Enna, 1976; Wong and McGeer, 1981). In the cerebellum, GABA transporter mRNA levels approximated adult levels for the first 10 post-natal days, followed by a rapid 3-fold increase in message between P21-30 (Fig. 4). Over this same time period, cerebellar GABA uptake increases to 230% of adult levels (Coyle and Enna, 1976). Nearadult levels of GABA transporter mRNA also were present in the striatum at birth, followed by an early (P6-PI0), transient 2-fold increase in transporter message and a rapid return to adult levels by P21 (Fig. 4). The reported changes in post-natal striatal GABA uptake (Wong and McGeer, 1981) are very similar in magnitude, although delayed by approx 1 week relative to changes in GABA transporter message. Thus, the GABA transporter mRNA data presented strongly support the contention (Blakely et al., 1990) that transporter mRNA abundance is a primary determinant of transporter expression during development. During ontogenesis, there is no simple relationship among the various GABAergic markers. Adult or supra-adult levels of GABA transporter activity are evident several weeks before adult levels of GABA are achieved. In turn, increases in GABA content precede maximal expression of the GABA synthetic enzyme glutamic acid decarboxylase and GABA receptor binding (Coyle and Enna, 1976 ; Wong and McGeer, 1981). Glutamic acid decarboxylase may best reflect the development of the GABAergic phenotype, as it has been suggested that GABA may also serve as a metabolic intermediate in the developing brain (Coyle and Enna, 1976). Whether localized to GABAergic neurons or neighbouring gila, the high level of postnatal GABA transporter expression relative to GABA biosynthesis might effectively limit the extent of functional GABAergic neurotransmission during development. To the best of our knowledge, Fig. 5 represents the first quantitation of post-mortem human GABA transporter mRNA. The abundance of human GABA transporter mRNA in cerebral cortex, cerebellum and caudate-putamen was approx 5-25% of rat transporter mRNA levels in the corresponding tissues (compare Figs 4 and 5). Although the influence of post-mortem interval and other variables on GABA transporter mRNA levels was not addressed in these pilot experiments, GABA transporter message appeared to be relatively stable post-mortem, consistent with previous data from our lab on the postmortem stability of other human brain mRNAs encoding transporters, neurotransmitters and G-proteins (Bannon et al., 1990, 1991, 1992; Granneman and
GABA transporter mRNA in rat and human brain Bannon, 1991). Thus it should be possible to quantitate human G A B A transporter m R N A by nuclease protection and in situ hybridization studies as a postm o r t e m index of G A B A transporter gene expression. The efficacy of synaptic transmission at GABAergic synapses is dependent upon numerous factors, including the biosynthesis, release, receptor interaction and degradation or reuptake of G A B A . Inasmuch as it has been estimated that G A B A may be a transmitter at 20% of all synapses in the mammalian brain (Iversen and Bloom, 1972), elucidating the molecular basis of G A B A transporter heterogeneity and regulation of gene expression will further our understanding of brain physiology and pathophysiology. Acknowledgements--The authors thank Dr John Guastella
for the gift of the cDNA clone GAT-1 and acknowledge the skilled technical assistance of Mr Rolf Hanson. These studies were supported in part by USPHS grants DA06470 (to MB and GK), MH43026 (to MB) and NS26081 (to GK).
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