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Originally published In Press as doi:10.1074/jbc.M408470200 on November 12, 2004

J. Biol. Chem., Vol. 280, Issue 3, 2065-2077, January 21, 2005
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A Na+/Cl–-coupled GABA Transporter, GAT-1, from Caenorhabditis elegans

STRUCTURAL AND FUNCTIONAL FEATURES, SPECIFIC EXPRESSION IN GABA-ERGIC NEURONS, AND INVOLVEMENT IN MUSCLE FUNCTION*

Guoliang Jiang{ddagger}, Lina Zhuang{ddagger}, Seiji Miyauchi{ddagger}, Katsuya Miyake§, You-Jun Fei{ddagger}, and Vadivel Ganapathy{ddagger}

From the {ddagger}Department of Biochemistry and Molecular Biology and §Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia 30912

Received for publication, July 26, 2004 , and in revised form, November 8, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
GABA functions as an inhibitory neurotransmitter in body muscles and as an excitatory neurotransmitter in enteric muscles in Caenorhabditis elegans. Whereas many of the components of the GABA-ergic neurotransmission in this organism have been identified at the molecular and functional levels, no transporter specific for this neurotransmitter has been identified to date. Here we report on the cloning and functional characterization of a GABA transporter from C. elegans (ceGAT-1) and on the functional relevance of the transporter to the biology of body muscles and enteric muscles. ceGAT-1 is coded by snf-11 gene, a member of the sodium-dependent neurotransmitter symporter gene family in C. elegans. The cloned ceGAT-1 functions as a Na+/Cl–-coupled high-affinity transporter selective for GABA with a Kt of ~15 µM. The Na+:Cl–:GABA stoichiometry for ceGAT-1-mediated transport process is 2:1:1. The transport process is electrogenic as evidenced from GABA-induced inward currents in Xenopus laevis oocytes that express ceGAT-1 heterologously. The transporter is expressed exclusively in GABA-ergic neurons and in two other additional neurons. We also investigated the functional relevance of ceGAT-1 to the biology of body muscles and enteric muscles by ceGAT-1-specific RNA interference (RNAi) in rrf-3 mutant, a strain of C. elegans in which neurons are not refractory to RNAi as in the wild type strain. The down-regulation of ceGAT-1 by RNAi leads to an interesting phenotype associated with altered function of body muscles (as evident from changes in thrashing frequency) and enteric muscles (as evident from the rates of defecation failure) and also with altered sensitivity to aldicarb-induced paralysis. These findings provide unequivocal evidence for a modulatory role of GABA and ceGAT-1 in the biology of cholinergic neurons and in the function of body muscles and enteric muscles in this organism.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
{gamma}-Aminobutyric acid (GABA)1 is the most abundant inhibitory neurotransmitter in vertebrates and invertebrates. GABAergic neurotransmission is used clinically as the target of antiepileptic, anxiolytic, and antispasmodic drugs (1–5). Several molecular components are involved in GABA-ergic neurotransmission. These include glutamic acid decarboxylase (the enzyme needed for the production of GABA from glutamate), vesicular GABA transporter (the transporter in presynaptic neurons that transports GABA into synaptic vesicles), GABA receptors (the receptors present on the postsynaptic membrane to transmit the GABAergic signals), and GABA transporters (the transporters present in presynaptic neurons and glial cells that participate in the clearance of GABA from the synapse). Mammals, including humans, express four different GABA transporters (GATs) of which only two are present specifically in GABA-ergic neurons. These are GAT-1 and GAT-3 (6, 7). GAT-1 is the predominant GABA transporter in the brain, co-localizes with markers of GABAergic neurons, and is expressed in the axons and presynaptic membranes (8–10). GAT-3 is also expressed exclusively in the brain where it is found in GABA-ergic nerve terminals as well as in glial cells (11, 12). The other two GABA transporters (GAT-2 and BGT-1/GAT-4, also known as betaine/GABA transporter) are not related to GABA-ergic neurotransmission. Antiepileptic, anxiolytic, and antispasmodic drugs produce their therapeutic effects by enhancing GABAergic neurotransmission (1–5). The activation of GABAergic neurotransmission can be accomplished by drugs that function either as agonists of GABA receptors or inhibitors of GABA transporters. The latter class of drugs such as tiagabine (an antiepileptic drug) increases the levels of GABA in the synaptic cleft by their ability to block the clearance of the neurotransmitter from the synapse via GABA transporters and thus enhances GABA-ergic neurotransmission (5). Even though GAT-1 and GAT-3 have been identified at the molecular level more than a decade ago and are known to be linked to diseases such as epilepsy and schizophrenia (6, 7), there are no knock-out animal models reported in the literature describing the phenotypic changes that occur as a consequence of specific deletion of either GAT-1 or GAT-3.

GABA-ergic neurotransmission plays a critical role in the function of body muscles and enteric muscles in the nematode Caenorhabditis elegans (13). There are 26 GABA-ergic neurons in this organism as identified by immunocytochemistry using a polyclonal antibody specific for GABA (14). These consist of the 6 DD neurons, 13 VD neurons, 4 RME neurons, 1 AVL neuron, 1 DVB neuron, and 1 RIS neuron. The connectivity of all of these neurons that use GABA as the neurotransmitter has been well established (14). The DD and VD neurons control the function of body muscles that are involved in locomotion. The RME neurons innervate the muscle in the head that participates in foraging. The AVL and DVB neurons control the function of enteric muscles that participate, together with body muscles, in the defecation cycle. The function of the RIS neuron is not well understood. The molecular components of GABAergic neurotransmission in C. elegans are similar to those in mammals. These include glutamic acid decarboxylase (known as unc-25), vesicular GABA transporter (known as unc-47), and a GABA receptor (known as unc-49) (13, 15). Two other proteins have also been shown to be essential for normal GABAergic neurotransmission in C. elegans (i.e. unc-30 that participates in the differentiation of GABA-ergic neurons and unc-46 that is essential for synaptic release of GABA) (13, 15). unc-25, unc-30, unc-46, unc-47, and unc-49 are members of the uncoordinated gene class. All of the members of this gene class, when made nonfunctional, lead to a loss of coordination of muscle function in the organism. The C. elegans counterpart(s) of mammalian GABA transporters has/have not yet been identified at the molecular level.

GABA transporters in mammals belong to the SLC6 gene family, which consists of at least 19 members (7). Most of the transporters in this gene family recognize neurotransmitters/neuromodulators (e.g. GABA, serotonin, norepinephrine, dopamine, glycine, proline, and taurine) as substrates and are energetically coupled to the transmembrane gradients of Na+ and Cl–. Therefore, this family is called Na+/Cl–-dependent neurotransmitter transporter gene family. C. elegans also contains a sodium-dependent neurotransmitter symporter family (snf). A search in the WormBase (Release WS93 at www.wormbase.org) using the mammalian GABA transporter protein sequence as a query has revealed that there are 14 members in this snf gene family (snf-1, snf-2, and so forth) in the worm genome. Only two of them have so far been identified at the functional level. dat-1 (T23G5.5) encodes a dopamine transporter (16) and mod-5 (Y54E10BR.7) encodes a serotonin transporter (17). Recently, we undertook a study to identify the taurine transporter in C. elegans. When we searched the data base for the snf gene family with the mammalian taurine transporter as a query to identify the most likely candidate for the C. elegans taurine transporter, the search yielded snf-11 as the likely candidate based on amino acid sequence. Therefore, we isolated a full-length snf-11 cDNA clone from a C. elegans cDNA library and characterized its transport function, hoping that this clone would function as a Na+/Cl–-coupled transporter for taurine. Quite unexpectedly, these studies showed that snf-11 is not a taurine transporter but a GABA transporter. This represents the first GABA transporter to be identified at the functional level in C. elegans. In this paper, we describe the structural and functional aspects of this GABA transporter (ceGAT-1). In addition, we show here that ceGAT-1 is expressed in all of the GABA-ergic neurons (plus in two non-GABAergic neurons) and that down-regulation of GAT-1 expression by gene-specific RNA interference (RNAi) leads to changes in the function of body muscles and enteric muscles. Furthermore, RNAi-induced down-regulation of GAT-1 also leads to an interesting pharmacologic phenotype with altered cholinergic neurotransmission as evident from enhanced sensitivity to aldicarb (an inhibitor of acetylcholineesterase)-induced paralysis. To our knowledge, this represents the first report on the consequences of down-regulation of a GABA transporter in an animal model.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—[3H]GABA (specific radioactivity, 70 Ci/mmol) was purchased from PerkinElmer Life Sciences. The human retinal pigment epithelial (HRPE) cell line, used routinely in our laboratory for heterologous expression of cloned transporters, was maintained in Dulbecco's minimum essential medium/F-12 medium (1:1) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. Lipofectin was purchased from Invitrogen. Restriction enzymes were obtained from New England Biolabs (Beverly, MA). Magna nylon transfer membranes used in library screening were purchased from Micron Separations (Westboro, MA). Unlabeled GABA and its analogs and other amino acids were obtained from Sigma. A wild type nematode strain, C. elegans N2 (Bristol) and all of the other C. elegans mutant strains such as rrf-3 (NL-2099), unc-25 (e156), unc-47 (e307), and unc-2 (e55) were obtained from the Caenorhabditis Genetics Center (St. Paul, MN).

Nematode Culture and RNA Preparation—Nematode culture was carried out using a standard procedure with a large-scale liquid cultivation protocol (18). The nematodes were cleaned by sedimentation through 15% (w/v) Ficoll 400 in 0.1 M NaCl. The pellet was then used for total RNA preparation. Total RNA was isolated using the TRIzol reagent (Invitrogen). Poly(A)+ mRNA was purified by affinity chromatography using oligo(dT)-cellulose.

Reverse Transcription (RT)-PCR and Hybridization Probe Preparation—Our original aim was to clone the C. elegans taurine transporter. A search in the WormBase using the mammalian taurine transporter protein sequences as queries revealed that the gene T03F7.1 (GenBankTM accession number Z74041 [GenBank] ) located on chromosome V encodes a candidate plasma membrane transporter that may function as the sodium/chloride-coupled taurine transporter. Therefore, a pair of PCR primers specific for this gene was designed based on the sequence of the cosmid T03F7.1, 5'-GCA CTT TTC CTT CTT CTC GTC-3' (forward primer) and 5'-GCA AAA ACC CAG CAA AAC TTC-3' (reverse primer). These primers yielded a single DNA fragment in RT-PCR using the total RNA isolated from C. elegans as the template in the reaction. The size of the product was ~0.9 kb, which agreed with the predicted size based on the distance between the two primers in the theoretically derived gene transcript. The RT-PCR product was gel-purified and subcloned into pGEM-Teasy vector (Promega Madison, WI). The molecular identity of the insert was established by DNA sequencing. This cDNA fragment was used as a probe to screen a C. elegans cDNA library.

Construction of a Directional C. elegans cDNA Library—SuperScript plasmid system from Invitrogen was used to establish the cDNA library using the poly(A)+ RNA from C. elegans. The transformation of the ligated cDNA into Escherichia coli was performed by electroporation using ElectroMAX DH10B-competent cells. The bacteria plating, the filter lifting, the DNA fragment labeling, and the hybridization methods followed the routine procedure (19–24). The DNA sequencing of the full-length ceGAT-1 cDNA clone was performed using an automated PerkinElmer Applied Biosystems 377 Prism DNA sequencer (Foster City, CA) and the Taq DyeDeoxy terminator cycle sequencing protocol.

Functional Expression of ceGAT-1 in Mammalian Cells by Vaccinia Virus Expression System—Functional expression of the ceGAT-1 cDNA in HRPE cells was done using the vaccinia virus expression system as described previously (19–24). HRPE cells grown in 24-well plates were infected with a recombinant vaccinia virus (VTF7–3) at a multiplicity of 10 plaque-forming units/cell. This virus carries the gene for T7 RNA polymerase in its genome. The virus was allowed to adsorb onto the cells for 30 min at 37 °C with gentle shaking of the plate. Cells were then transfected with the plasmid DNA (empty vector pSPORT or ceGAT-1 cDNA construct) using the lipofection procedure (Invitrogen). The cells were incubated at 37 °C for 12 h and then used for the determination of transport activity. The uptake of [3H]GABA was determined at 37 °C. In most experiments, the uptake medium was 25 mM Hepes/Tris, pH 7.5, containing 140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgSO4, and 5 mM glucose. In experiments in which the cation and anion dependence of the transport process was investigated, NaCl was replaced isosmotically by sodium gluconate or N-methyl-D-glucamine (NMDG) chloride. Uptake measurements were routinely made in parallel in control cells transfected with the empty vector alone and in cells transfected with the vector-cDNA construct. The uptake activity in cDNA-transfected cells was adjusted for the endogenous activity measured in control cells to calculate the cDNA-specific activity. Experiments were performed in triplicate, and each experiment was repeated at least three times. Results are presented as means ± S.E.

In Vitro Transcription, Oocyte Expression, and Electrophysiological Studies—ceGAT-1 was expressed heterologously in Xenopus laevis oocytes by microinjection of ceGAT-1 cRNA. The procedures for in vitro transcription, oocyte isolation, and microinjection have been described previously (19–22). The plasmid ceGAT-1 cDNA (constructed in pSPORT vector) was linearized with NotI and transcribed in vitro to cRNA using the mMESSAGE mMACHINE RNA transcription kit (Ambion, Austin, TX). The expression of ceGAT-1 was initially detected by comparing the uptake of [3H]GABA in uninjected oocytes and ceGAT-1 cRNA-injected oocytes. The electrophysiological characteristics of the heterologously expressed ceGAT-1 were then studied as described previously (19–22).

Analysis of Tissue-specific Expression Pattern of ceGAT-1—To study the tissue-specific expression pattern of the cegat-1 gene in C. elegans, a transcriptional cegat-1::gfp fusion gene was constructed and transgenic animals expressing these transgenes were established as described previously (19–22). The GFP expression vector pPD117.01 was kindly provided by Dr. A. Fire (Carnegie Institute, Baltimore, MD). The cosmid T03F7.1 containing the cegat-1 gene and its promoter was obtained from the Sanger Center (Cambridge, United Kingdom). A DNA fragment containing the cegat-1 gene promoter region was generated by PCR using this cosmid DNA as the template. The sense primer (coordinates in cosmid T03F7.1 are 3,896–3,925) with an XbaI adapter attached to the 5'-end was 5'-TCT AGA CAT TCG GCG AAG GAT GGT CGA GAG-3', and the reverse primer (coordinates in cosmid T03F7.1 are 5,609–5,637) with a BamHI adapter attached to the 5'-end was 5'-GGA TCC CCT GCA GGA GAT TTC TGG ATA CAA-3'. A recombinant KlenTaq DNA polymerase (AB Peptides, St. Louis, MO) was used for the long-range PCR. The size of the expected PCR product is ~2.0 kb. The amplified region contains the putative promoter for the cegat-1 gene. The PCR product was digested with XbaI and BamHI and ligated into multiple cloning sites II in the pPD117.01 vector. Bacterial transformation and plasmid preparation followed a standard protocol (25, 26). The minigene fusion constructs were verified by DNA sequence analysis. Transgenic lines were established using a standard germ line transformation protocol (25, 26). A cloned mutant collagen gene containing the rol-6 (plasmid pRF4) was used as a dominant genetic marker for DNA transformation. The F1 rollers were picked up according to their characteristic rolling behavior and cultured individually to establish transformed lines. GFP expression pattern was determined by fluorescence microscopy (19–22). F2 rollers with extrachromosome arrays were selected for fluorescence microscopy to determine the GFP expression pattern. Cell identification was validated by their morphology in combination with relative positions of the GFP-positive cells in the same animals using differential interference contrast image.

Bacteria-mediated RNAi—A fragment of the coding region of ceGAT-1 cDNA was generated by EcoRI digestion and subcloned into a "double T7" plasmid pPD129.36 (kindly provided by Dr. A. Fire, Carnegie Institute), which was subsequently transformed into HT115(DE3) cells. The induction of HT115 cells harboring the double T7 plasmid to express dsRNA and the bacteria-mediated RNAi procedure were carried out as described previously (21, 22). To serve as a negative control for the bacteria-mediated RNAi in assessment of the influence of ceGAT-1 on animal behavior, we included an HT115 strain containing the empty vector alone. RNAi experiments were carried out using the normal strain N2 as well as the RNAi-hypersensitive rrf-3 strain. Three mutant strains, unc-25 (e156), unc-47 (e307), and unc-2 (e55) (15, 27–29) were also used as references for animal phenotypes associated with defective GABA-ergic neurotransmission.

Assay of Aldicarb-induced Paralysis—This was done according to the procedure described by Mathews et al. (30). Control and experimental (RNAi) worms (N2 and rrf-3 strains) (20–25 worms/experiment) were exposed to 0.5 mM aldicarb. Paralyzed worms were identified by a lack of response in terms of movement upon poking with a metal wire. The number of paralyzed worms was counted at different time periods following exposure to aldicarb. The experiment was repeated three times, and the data were analyzed as the percent of animals that were paralyzed at each time point. In the experiment in which the relevance of the abolition of GABA-ergic neurotransmission to aldicarb-induced paralysis was investigated using mutant strains, the worms (control strain N2 and the mutant strains unc-25, unc-47, and unc-2) were exposed to 0.5 mM aldicarb without the bacteria-mediated RNAi and the percent of paralyzed worms was determined at different time points following the exposure to aldicarb. This experiment was repeated twice.

Thrashing Assay—This assay was used to investigate the frequency of contraction of body muscles in worms subjected to RNAi. These experiments were done using the RNAi-hypersensitive rrf-3 strain. Control and experimental (RNAi) worms were synchronously grown to the L4 stage. Individual worms were transferred into a microtiter well containing 60 µl of M9 buffer on top of agar. After a 2-min recovery period from the picking procedure, thrashes were counted for 2 min. A thrash is defined as a change in the direction of bending in the middle of the body (26, 30, 31). Eight worms were examined in each group. The experiment was repeated twice. Data are given as frequency of thrashing/2 min.

Defecation Failure Assay—This assay was used to investigate the function of enteric muscles. These experiments were also done using the RNAi-hypersensitive rrf-3 strain. For the analysis of expulsion of the intestinal contents during the defecation cycle, synchronously grown control and experimental (RNAi) worms were examined with a dissecting microscope (x50 magnification) for the presence or absence of an expulsion of intestinal contents through the anus after each contraction of the posterior muscles of the body (26, 30–32). 10 worms were examined in each group, and each animal was observed for 12 consecutive defecation cycles. The experiment was repeated twice. Data are given as the percent of events in which there was no discharge of intestinal contents, but defecation cycle occurred as determined by posterior muscle contraction.

Analysis of Steady-state Levels of ceGAT-1 mRNA in Control and RNAi Worms—The efficiency of RNAi-induced down-regulation of ceGAT-1 expression was evaluated by monitoring the steady-state levels of ceGAT-1 mRNA in control worms and in worms subjected to ceGAT1-specific RNAi. The RNAi-hypersensitive rrf-3 strain was used in these experiments. Total RNA was isolated from control and experimental (RNAi) worms, and ceGAT-1 mRNA levels were measured by semiquantitative RT-PCR. Because RNAi was induced with a dsRNA representing the 5'-half of ceGAT-1 cDNA, RT-PCR primers were designed based on the sequence in the 3'-half of the cDNA. The sense and antisense primers were 5'-CAAGAAAAAAGTGTAGCCGAAG-3' and 5'-GCCATAAAAGTGCCCATCC-3', respectively. Semiquantitative RT-PCR was carried out as described previously (33) using a commercially available kit (Ambion) following manufacturer's protocol. Universal 18 S RNA primers were used as an internal control. The RT-PCR products were separated on an agarose gel, and the bands were visualized with ethidium bromide. The signals were quantified using STORM phosphorimaging system (Molecular Dynamics, Sunnyvale, CA) and processed using ImageQuant (version 4.2a) software application. The experiment was repeated twice with separate sets of control, and RNAi worms and RT-PCR experiments were repeated three times with each set of RNA samples. Data are expressed as ceGAT-1 mRNA/18 S RNA ratio.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Molecular Cloning and Structural Characterization of ceGAT-1—We isolated a full-length cDNA clone coded by the gene T03F7.1, which we thought of as a likely candidate for the Na+/Cl–-coupled taurine transporter in C. elegans based on the similarity in amino acid sequence. However, functional studies with this cDNA clone in heterologous expression systems revealed that it was a Na+/Cl–-coupled transporter for GABA rather than taurine (see below). Therefore, we named this transporter ceGAT-1 because it represented the first GABA transporter to be identified at the molecular and functional levels in C. elegans. The gene coding for this transporter is localized on chromosome V. The cegat-1 gene was originally designated as snf-11 (sodium-dependent neurotransmitter symporter family member 11). Its size is at least ~3.1 kb, excluding the putative promoter region. The gene consists of six exons as deduced by a comparison between the sequence of the cloned cDNA and that of the GenBankTM accession number Z74041 [GenBank] for T03F7.1 from the nematode genome sequence project (WormBase release WS93). The structural organization of the gene is shown in Fig. 1A. The ceGAT-1 cDNA is 2,162-bp long and contains a poly(A) tail. The 5'- and the 3'-untranslated regions are 27- and 302-bp long, respectively. The ceGAT-1 protein deduced from the cDNA sequence contains 610 amino acids with an estimated molecular size of 69.3 kDa and an isoelectric point of 6.38 (Fig. 1B). According to the Kyte-Doolittle hydropathy plot, the ceGAT-1 protein possesses 12 putative transmembrane domains. The sequences of ceGAT-1 cDNA and its encoded protein have been deposited into the GenBankTM (accession number AY529124 [GenBank] ). A pairwise comparison analysis of the transporter protein sequences between ceGAT-1 and its functional counterparts in humans, GAT-1, GAT-2, GAT-3, and GAT-4 (BGT-1) using the BESTFIT algorithm in the GCG package (version 10.2) has shown that ceGAT-1 is most closely related to GAT-1 (66.3% similarity and 55.5% identity). The similarity/identity ratios between ceGAT-1 and GAT-2/GAT-3/GAT-4 are 63.4/53.7, 63.2/52.5, and 62.4/52.7%, respectively. A putative snf signature domain (pfam00209.11, www.ncbi.nlm.nih.gov/BLAST/Blast.cgi) is present in the highly conserved region of the ceGAT-1 protein sequence that spans the C-terminal part of the first transmembrane domain, the first extracellular loop, and the N-terminal part of the second transmembrane domain. The signature domain has the following sequence: WRF(G/P)YX4NGGGX(F/Y). Trp34 and Arg35 in this domain are conserved in human GAT-1 (Trp68 and Arg69) (Fig. 2) where these residues have been shown to be involved in the binding of Na+ and Cl–, respectively (7). The position of the tyrosine residue that is known to be involved in the binding of GABA in human GAT-1 (Tyr140) (7) is also conserved in ceGAT-1 exactly at the same location (Tyr106) with reference to the Na+- and Cl–-binding residues in their respective proteins (Fig. 2).



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  		FIG. 1.
Organization of cegat-1 gene (A) and primary structure of ceGAT-1 (B). A, exons are indicated by filled boxes and numbered, and introns are indicated by solid lines. The untranslated regions in exons 1 and 6 are indicated by blank boxes. The consensus polyadenylation signal AATAAA is also shown. Sizes and positions of the exons and the introns are drawn to the exact scale. B, amino acid sequence of ceGAT-1. Putative transmembrane domains are underlined and numbered.

 



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  		FIG. 2.
Comparison of amino acid sequence between ceGAT-1 and human GAT-1. Identical amino acid residues are indicated by a vertical line, conservative substitutions are indicated by two dots, and non-conservative substitutions are indicated by a single dot. The amino acid residues in human GAT-1 involved in the binding of Na+ (Trp68), Cl– (Arg69), and GABA (Tyr140) are boxed. These residues are conserved in ceGAT-1. The locations of these residues are identical in both proteins.

 
Functional Characterization of ceGAT-1 Using a Heterologous Expression System in Mammalian Cells—The functional analysis of the cloned ceGAT-1 was carried out by heterologous expression of the cDNA in HRPE cells using the vaccinia virus expression system. GABA uptake (2.5 µM) in the assay medium containing 140 mM NaCl, pH 7.5, increased severalfold in cDNA-transfected cells compared with control cells transfected with vector alone (Fig. 3A). There was little time-dependent uptake of GABA in control cells, suggesting that these cells may not possess any constitutive GABA transporter activity. The uptake in cDNA-transfected cells was linear during the time period examined (up to 30 min). The initial uptake rates in cDNA-transfected cells were ~18-fold higher than in control cells (27.1 ± 0.6 versus 1.5 ± 0.1 pmol/106 cells/min) (Fig. 3B). The cDNA-induced uptake was obligatorily dependent on the presence of Na+ and Cl– since the uptake was abolished completely when measured in the assay medium containing sodium gluconate (absence of chloride) or NMDG chloride (absence of sodium) in place of NaCl (Fig. 3B). There was negligible Na+- and Cl–-dependent GABA uptake in control cells, again indicating that there was no or little constitutive expression of GABA transporter in HRPE cells.



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  		FIG. 3.
Time course (A) and ion dependence (B) of GABA uptake via ceGAT-1 in HRPE cells. A, cells were transfected with either vector alone (pSPORT) (•) or vector carrying ceGAT-1 cDNA ({circ}). Uptake of [3H]GABA (2.5 µM) was determined in these transfected cells for different time periods in uptake medium containing 140 mM NaCl. B, cells were transfected with either vector alone (pSPORT) or vector carrying the ceGAT-1 cDNA insert. Uptake of GABA (2.5 µM) was determined for 15 min in buffers with or without Na+/Cl– ions (NaGLU, a chloride-free buffer in which NaCl was replaced isosmotically with sodium gluconate; NMDG.Cl, Na+-free buffer in which NaCl was replaced isosmotically with NMDG chloride).

 
The substrate specificity of ceGAT-1 was studied using a competition analysis by monitoring the ability of unlabeled GABA and various structurally related compounds at a concentration of 2.5 mM to compete with [3H]GABA (2.5 µM) uptake via ceGAT-1 (Fig. 4A). Uptake measurements were made in parallel in vector-transfected cells and in cDNA-transfected cells, and the cDNA-specific uptake was calculated by subtracting the uptake in vector-transfected cells from the uptake in cDNA-transfected cells. Only the cDNA-specific uptake was used in the analysis. Among the compounds tested, only unlabeled GABA and {beta}-aminobutyric acid were able to compete with [3H]GABA to a marked extent. However, the affinity of the transporter for the latter compound was lower than that for GABA as evident from the dose-response relationship for the inhibition of [3H]GABA by unlabeled GABA and {beta}-aminobutyric acid (data not shown). The IC50 values (concentration necessary to cause 50% inhibition) were 13 ± 2 and 233 ± 30 µM for GABA and {beta}-aminobutyric acid, respectively. These data suggest that the shifting of the amino group from the {gamma}-carbon to {beta}-carbon interferes significantly with recognition by the transporter. If the amino group was moved even further as in {alpha}-aminobutyric acid, it completely abolished the interaction with the transporter. {beta}-Alanine, glutamate, and carnitine showed very little but significant ability to compete with GABA. Other compounds such as taurine, creatine, choline, betaine, and glycine had no effect on GABA uptake. It is of interest to note that betaine, a substrate for mammalian BGT-1/GAT-4, was not recognized by ceGAT-1. The ceGAT-1-mediated GABA uptake was saturable with a Michaelis-Menten constant (Kt) of 17 ± 4 µM (Fig. 4B). These data show that ceGAT-1 is a Na+/Cl–coupled high-affinity GABA transporter.



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  		FIG. 4.
Substrate specificity (A) and saturation kinetics (B) of the transport process mediated by ceGAT-1 in HRPE cells. A, uptake of 2.5 µM [3H]GABA was measured in the absence or presence of potential inhibitors (2.5 mM) in cells transfected with vector alone (pSPORT) or ceGAT-1 cDNA. Uptake was measured for 15 min in the presence of NaCl. The cDNA-specific uptake was calculated by adjusting for the uptake in vector-transfected cells. The cDNA-specific uptake in the absence of inhibitors was taken as the control (100%), and the uptake in the presence of inhibitors is given as a percent of this control value. {beta}-Amino BA, {beta}-aminobutyric acid; {alpha}-Amino BA, {alpha}-aminobutyric acid. B, uptake of GABA was measured for 15 min in the presence of NaCl over a substrate concentration range of 1–100 µM in cells transfected with vector alone (pSPORT) or ceGAT-1 cDNA. The cDNA-specific uptake was calculated by adjusting for the endogenous uptake measured in vector-transfected cells.

 
The effect of Na+ or Cl– on the ceGAT-1-mediated uptake of GABA was then investigated by measuring the uptake in the presence of varying concentrations of extracellular Na+ (with a fixed concentration of Cl–) or Cl– (with a fixed concentration of Na+). These experiments were done only with cells transfected with ceGAT-1 cDNA. The uptake measured in the absence of either Na+ or Cl– was subtracted from the uptake measured in the presence of varying concentrations of Na+ or Cl– to calculate cDNA-specific uptake. For Na+ activation kinetics, the concentration of Na+ in the uptake medium was varied from 0 to 140 mM. The osmolality of the medium was maintained by adding the appropriate concentrations of NMDG chloride as a substitute for NaCl. The relationship between the cDNA-specific uptake and Na+ concentration was sigmoidal with a Hill coefficient of 1.7 ± 0.2, suggesting the involvement of more than one Na+ per GABA molecule transported (Fig. 5A). For Cl– activation kinetics, the concentration of Cl– in the uptake medium was varied from 0 to 140 mM. The osmolality of the medium was maintained by adding appropriate concentrations of sodium gluconate as a substitute for NaCl. The relationship between the cDNA-specific uptake and Cl– concentration was hyperbolic with a Hill coefficient of 0.8 ± 0.1, suggesting the involvement of one Cl– per GABA molecule transported (Fig. 5B). Thus, the Na+:Cl–:GABA stoichiometry for ceGAT-1-mediated uptake is 2:1:1.



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  		FIG. 5.
Na+ activation kinetics (A) and Cl– activation kinetics (B) of GABA uptake via ceGAT-1 in HRPE cells. A, HRPE cells were transfected with vector carrying ceGAT-1 cDNA. The uptake of [3H]GABA (2.5 µM) was determined in these transfected cells with a 15-min incubation in uptake medium containing various concentrations of Na+. The concentration of Cl– was kept constant at 140 mM. Inset, Hill plot. B, HRPE cells were transfected with vector carrying ceGAT-1 cDNA. The uptake of [3H]GABA (2.5 µM) was determined in these transfected cells with a 15-min incubation in uptake medium containing various concentrations of Cl–. Concentration of Na+ was kept constant at 140 mM. Inset, Hill plot. In these experiments, endogenous GABA transport activity was not taken into account because the Na+- and Cl–-dependent uptake activities were calculated by subtracting uptake values obtained at zero concentrations of respective ions. Because HRPE cells do not express Na+/Cl–-coupled GABA transporter endogenously, the uptake measured in cDNA-transfected cells in the absence of Na+ or Cl– represented the endogenous GABA uptake activity.

 
Functional Characterization of ceGAT-1 Using the X. laevis Oocyte Expression System—GABA exists as a zwitterion with one positive charge and one negative charge on the molecule under the experimental conditions. Therefore, the predicted Na+:Cl–:GABA stoichiometry of 2:1:1 would suggest that the transport process mediated by ceGAT-1 should be electrogenic. To determine whether this is true, we used the X. laevis oocyte expression system to express ceGAT-1 heterologously. First, we tested whether the transporter is expressed in oocytes injected with ceGAT-1 cRNA by measuring the uptake of [3H]GABA (1 µM) in the presence of NaCl in cRNA-injected oocytes and in uninjected control oocytes. The uptake in cRNA-injected oocytes was 1.45 ± 0.04 pmol/oocyte/h (Fig. 6A). This value was 4-fold higher than the uptake measured under identical conditions in control oocytes (0.38 ± 0.02 pmol/oocyte/h). Second, we examined the electrogenic nature of the ceGAT-1-mediated transport process by using the two-microelectrode voltage-clamp technique. Perfusion of cRNA-injected oocytes with GABA (1 mM) in the presence of NaCl induced inward currents (~15 nA) at a holding membrane potential of –50 mV (Fig. 6B). These data show unequivocally that ceGAT-1-mediated GABA transport is electrogenic, associated with transfer of positive charge into the oocytes. The GABA-induced current was obligatorily dependent on the presence of both Na+ and Cl–. In the absence of Na+ or Cl– (NaCl was substituted by NMDG chloride or by sodium gluconate, respectively) in the perfusion buffer, GABA did not induce any detectable current in cRNA-injected oocytes (Fig. 6B). The GABA-induced currents of ceGAT-1 were further analyzed in terms of their dependence on membrane potential. Steady-state currents induced by 1 mM GABA were determined at different testing membrane potentials (from +10 to –150 mV) using a jumping protocol (Software Clampex, version 10.0, Axon Instruments, Inc., Union City, CA). The GABA-induced currents increased with rising testing membrane potentials (i.e. more negative membrane potentials) (Fig. 6C). These data show that the transport rate increases with increasing membrane potentials, suggesting a role for membrane potential as a driving force for ceGAT-1. Thus, the transport process mediated by ceGAT-1 derives its driving force from the electrochemical Na+ and Cl– gradients as well as from membrane potential. We did not attempt to investigate the kinetics of the transport process (substrate saturation kinetics, Na+ activation kinetics, and Cl– activation kinetics) using the oocyte expression system because of the small magnitude of the currents induced by the substrate.



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  		FIG. 6.
Functional expression of ceGAT-1 in X. laevis oocytes. A, uptake of [3H]GABA (1 µM) was measured in the presence of NaCl for 1 h in control (uninjected oocytes) oocytes and in oocytes injected with ceGAT-1 cRNA. The horizontal bars indicate the mean value for each group. B, oocytes injected with ceGAT-1 cRNA were sequentially perfused with 1 mM GABA in a NaCl-containing buffer, a chloride-free buffer in which NaCl was replaced isosmotically with sodium gluconate (NaGLU), or a Na+-free buffer in which NaCl was replaced isosmotically with NMDG chloride (NMDG.Cl). C, I-V relationship for GABA-induced currents in oocytes injected with ceGAT-1 cRNA examined in the presence of Na+ and Cl–, in the presence of Na+ but in the absence of Cl–, or in the presence of Cl– but in the absence of Na+.

 
Analysis of Tissue Expression Pattern of the cegat-1 Gene—We investigated the tissue expression pattern of the cegat-1 gene in C. elegans using transgenic GFP fusion technique in which the transgene consisted of cegat-1 gene-specific promoter fused with GFP cDNA. The expression of GFP in this fusion gene is controlled by the cegat-1 gene-specific promoter. Therefore, the expression pattern of GFP in transgenic C. elegans expressing the fusion gene is likely to match the expression pattern of the native cegat-1 gene. With this technique, we determined that ceGAT-1 is expressed in all of the GABA-ergic neurons whose identity has been previously established by immunocytochemistry by McIntire et al. (14) using polyclonal antibody against GABA. These GFP-positive neurons include the VD and DD neurons in the ventral cord, the RMED, RMEV, RMEL, RMER, AVL, and RIS neurons in the head area and the DVB neuron in the tail region (Fig. 7). There are two additional GFP-positive neurons in the tail region that have not been classified as GABAergic neurons based on lack of staining for GABA by immunocytochemistry (14). These two neurons are PVQR and PVQL, which are the anterior interneurons in the right and left lumbar ganglia. These two neurons innervate the ventral cord motor neurons and may also participate in the regulation of the coordinated body muscle movement (www.wormatlas.org) (34). We confirmed the identity of these GFP-positive neurons by epifluorescence microscopy and by the location of the neurons as revealed by a combination of Nomarski-differential interference contrast microscopic observation and 4',6-diamidino-2-phenylindole nuclei-staining method. This expression pattern is evident from the early larva stage through the adult stage (data not shown). We observed an identical expression pattern with at least 10 transgenic animals.



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  		FIG. 7.
Identity of the neurons expressing cegat-1 gene in C. elegans. Expression of the cegat-1 gene in C. elegans was determined by a GFP reporter fusion construct. The construct was designed such that the expression of GFP was driven by the cegat-1 gene-specific promoter. This construct was then used to generate GFP-expressing transgenic worms. The top panel identifies the GFP-positive DD and VD neurons in the ventral cord (10x), the middle panel identifies the GFP-positive neurons in the tail region (20x), and the bottom panel identifies the GFP-positive neurons in the head region (40x).

 
Consequences of RNAi-mediated Down-regulation of cegat-1 in C. elegans—GABA-ergic neurons are involved in the function of body muscles and enteric muscles, and ablation of specific GABA-ergic neurons by laser microsurgery leads to specific phenotypes as a consequence of a loss of function of body muscles and enteric muscles (13, 14). These phenotypes include three distinct types: 1) a "shrinker" phenotype related to body muscles; 2) a "loopy" phenotype related to muscles in the head involved in foraging; and 3) "defecation defect" phenotype related to enteric muscles (14). Because ceGAT-1 is expressed specifically in GABA-ergic neurons, we hypothesized that RNAi-mediated down-regulation of this transporter would influence the function of body muscles and enteric muscles and hence produce specific phenotypes. First, we evaluated the function of body muscles by analyzing the influence of RNAi-mediated down-regulation of ceGAT-1 on aldicarb-induced paralysis. Aldicarb is an inhibitor of acetylcholinesterase, and exposure of the worms to this compound leads to paralysis due to enhanced cholinergic signals at the motor endplate (30). When we used the normal strain N2, exposure the worms to aldicarb (0.5 mM) induced paralysis in a time-dependent manner; however, this phenotype was not altered by feeding the worms with bacteria harboring dsRNA specific for ceGAT1 (Fig. 8). It is known that neurons are refractory to RNAi effects compared with other cell types in C. elegans (35, 36). Because ceGAT-1 is expressed exclusively in neurons, it is likely that the apparent lack of any effect on aldicarb-induced paralysis by RNAi in N2 worms is due to the refractory nature of the neurons. Therefore, we repeated the RNAi experiments using the RNAi-hypersensitive strain rrf-3. In this strain, even neurons are sensitive to RNAi (35, 36). With this strain, we found that RNAi-mediated down-regulation of ceGAT-1 enhanced the sensitivity of the worms to aldicarb-induced paralysis (Fig. 8). The time needed for aldicarb exposure to induce paralysis in 50% of the worms doubled in worms subjected to RNAi compared with controls. Both with normal strain and rrf-3 strain, control experiments consisted of feeding the worms with bacteria harboring the pPD129 vector alone without the ceGAT-1-specific cDNA insert. These data show that ceGAT-1-specific RNAi renders the rrf-3 worms, but not the N2 worms, hypersensitive to aldicarb-induced paralysis. Thus, there is clearly an interaction between cholinergic neurotransmission and GABA-ergic neurotransmission.



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  		FIG. 8.
Influence of bacteria-mediated cegat-1 gene-specific RNAi on aldicarb-induced paralysis in wild type strain (N2) and in RNAi-hypersensitive rrf-3 strain. Control worms (•, N2 strain; {blacksquare}, rrf-3 strain) were fed bacteria carrying the empty vector pPD129, and experimental (RNAi) worms ({circ}, N2-RNAi; {square}, rrf-3-RNAi) were fed bacteria harboring the plasmid with the ceGAT-1-specific cDNA insert. The control and experimental worms were exposed to aldicarb (0.5 mM), and the number of paralyzed worms was counted as described under "Experimental Procedures." Each group had 20–40 worms, and the experiments were repeated three times. Data are given as the percent of worms that went into paralysis.

 
We also evaluated the role of GABA-ergic neurotransmission in cholinergic signaling by using unc-25 (a mutant lacking glutamic acid decarboxylase) and unc-47 (a mutant lacking the vesicular GABA transporter). We compared the rate of aldicarb-induced paralysis between control N2 worms and the two mutant worms (Fig. 9). These experiments did not involve RNAi. Control N2 worms exhibited normal GABAergic neurotransmission, whereas unc-25 and unc-47 exhibited a loss of GABA-ergic neurotransmission. Therefore, a comparison among these worms in terms of their response to aldicarb-induced paralysis would directly provide information on the role of GABA-ergic neurotransmission in cholinergic signaling. These studies showed that both mutants that lack GABA-ergic neurotransmission exhibited markedly enhanced sensitivity to aldicarb-induced paralysis. As an internal control for these experiments, we used the unc-2 mutant that lacks a major calcium channel and hence refractory to cholinergic signaling (29). As predicted, exposure to aldicarb, which is expected to enhance cholinergic signaling by increasing the levels of acetylcholine at the motor endplates, did not have any effect on the worms (Fig. 9). In other words, there was no paralysis in CD55 mutant worms in response to aldicarb exposure.



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  		FIG. 9.
Aldicarb-induced paralysis in control N2 strain and in unc-25 (defective in glutamic acid decarboxylase), unc-47 (defective in vesicular GABA transporter), and unc-2 (defective in a calcium channel) mutant strains. In this experiment, there was no RNAi. Wild type and mutant strains were exposed to aldicarb (0.5 mM), and the number of paralyzed worms was counted as described under "Experimental Procedures." Each group had 25 worms, and the experiments were repeated twice. Data are given as the percent of worms that went into paralysis. •, N2 strain; {circ}, unc-25; {blacksquare}, unc-47; {square}, unc-2.

 
Aldicarb functions similar to nerve gases such as sarin and tabun, which increase the levels of acetylcholine at nerve terminals and at motor endplates and induce paralysis. The experiments described thus far show that down-regulation of ceGAT-1 in the presence of aldicarb sensitizes the worms to aldicarb-induced paralysis. What happens to the function of body muscles and enteric muscles when ceGAT-1 is down-regulated in the absence of aldicarb? To answer this question, we subjected the RNAi-hypersensitive rrf-3 worms to ceGAT-1-specific RNAi and quantified the changes in the function of body muscles and enteric muscles by the thrashing assay and the defecation failure assay, respectively. GABA acts as an inhibitory neurotransmitter in body muscles (13, 14), and congruent with this function of GABA-ergic neurons, the rates of body movement as assessed by the thrashing assay were significantly increased by RNAi-induced down-regulation of ceGAT-1 (thrashing rate: 152 ± 8 times/2 min in control worms and 188 ± 9 times/2 min in RNAi worms; p < 0.005) (Fig. 10A). In contrast, GABA acts as an excitatory neurotransmitter in enteric muscles (13, 14) and hence GABA-ergic neurons are involved in the maintenance of normal defecation cycle. In agreement with this function, the rates of defecation failure increased significantly in worms subjected to RNAi-induced down-regulation of ceGAT-1 (Fig. 10B).



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  		FIG. 10.
Phenotypes induced by bacteria-mediated cegat-1 gene-specific RNAi in rrf-3 strain. Control worms were fed bacteria carrying the empty vector pPD129, and experimental (RNAi) worms were fed bacteria harboring the plasmid with the ceGAT-1-specific cDNA insert. A, thrashing frequency was counted for 2 min in control and experimental (RNAi) worms. B, defecation failure (i.e. absence of discharge of intestinal contents after posterior body contraction) was determined in control and experimental (RNAi) worms. Statistical test was performed using the Microsoft Excel data analysis software. Values are reported as the mean ± S.E. Comparison between experimental worms and control worms was made using a two-sample Student's t test. *, statistically different from control (p < 0.005).

 
To determine the effectiveness of RNAi-induced down-regulation of ceGAT-1 expression, we compared the steady-state levels of ceGAT-1 mRNA in control rrf-3 strain worms and in rrf-3 worms subjected to RNAi. Semiquantitative RT-PCR was used to monitor the levels of ceGAT-1 mRNA. The levels of 18 S RNA were measured simultaneously as an internal control. These studies showed that RNAi led to a 40% decrease in ceGAT-1 mRNA levels (Fig. 11).



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  		FIG. 11.
Analysis of ceGAT-1 mRNA levels by semiquantitative RT-PCR in control rrf-3 worms and in rrf-3 worms subjected to RNAi. A, a representative gel showing the relative signals for RT-PCR products for ceGAT-1 mRNA and 18 S RNA in control (pPD129) worms and in experimental (RNAi) worms. B, data from two independent experiments expressed as the ceGAT-1 mRNA/18 S RNA ratio. *, statistically different from control (p < 0.005).

 
Taken collectively, these functional studies show that GABA-ergic neurotransmission is affected by ceGAT-1-specific RNAi and that GABA plays a critical role in the function of body muscles and enteric muscles through interaction with cholinergic signaling.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We have cloned and functionally identified a GABA transporter from C. elegans. The transporter is a member of the snf family identified in this organism. The cloned transporter is snf-11 and functions as a Na+/Cl–-coupled transporter for the neurotransmitter GABA. It exhibits high-affinity for GABA (Kt ~15 µM) and is highly selective for GABA. The only other structurally related compound with the ability to interact with the transporter to a significant extent is {beta}-aminobutyric acid. Other neurotransmitters/neuromodulators such as taurine, glycine, and glutamate do not interact with the transporter. The function of this transporter is demonstrable in two different heterologous expression systems: 1) mammalian cells and 2) X. laevis oocytes. The transport process exhibits a Na+:Cl–: GABA stoichiometry of 2:1:1 and is electrogenic. Because this is the first GABA transporter identified at the molecular and functional levels in C. elegans, we named this transporter ceGAT-1.

Mammals express four different GABA transporters known as GAT-1, GAT-2, GAT-3, and BGT-1/GAT-4 (6, 7). Based on the functional characteristics and expression pattern, the cloned ceGAT-1 is distinct from BGT-1/GAT-4 because ceGAT-1 does not interact with betaine. Whereas ceGAT-1 is expressed exclusively in GABA-ergic neurons, there is no overlap between the markers of GABAergic neurons and BGT-1/GAT-4 in mammalian brain (6, 7). Furthermore, we have cloned snf-3, another member of snf family from C. elegans, which we have shown to function as a Na+/Cl–-coupled transporter for betaine.2 Whether C. elegans expresses additional GABA transporters is not known at present. We have cloned three other members of the C. elegans snf family (snf-5, snf-6, and snf-12), and functional analysis with heterologous expression in mammalian cells has shown that none of these cells possesses the ability to transport GABA.2 Nonetheless, because ceGAT-1 is selectively associated with GABA-ergic neurons, we postulate that this transporter is likely to be the C. elegans ortholog of either GAT-1 or GAT-3 in mammals. GAT-1 and GAT-3 are closely associated with GABA-ergic neurons in mammalian brain. The identification of GAT-1 in C. elegans suggests that the role of the transporter in the modulation of GABA-ergic neurotransmission is likely to be similar to that of GAT-1 and/or GAT-3 in mammalian GABA-ergic neurons. The functional characteristics of ceGAT-1 identifying it as a Na+/Cl–-coupled, high-affinity, and selective transporter for GABA support the hypothesis that the transporter functions in the clearance of GABA from the synaptic cleft or motor endplate. These findings are of significance with respect to the biology of GABA-ergic neurotransmission in C. elegans. Previous studies have identified at the molecular and functional levels the enzyme responsible for GABA synthesis (unc-25), the transporter responsible for packaging GABA in synaptic vesicles (unc-47), and the receptor responsible for the transmission of GABA signals (unc-49) (15), but there are no reports in the literature on the molecular and functional identity of any transporter that is responsible for the clearance of GABA from the synaptic cleft or motor endplate. Thus, the findings presented in this paper fill an important gap in our knowledge in terms of GABA-ergic neurotransmission in this organism.

Even though all of the currently known GABA transporters in mammals have been cloned and functionally characterized several years ago, there are no reports to date in the literature on the generation of knock-out animal models for any of these transporters. There is strong evidence from clinical and pharmacological studies that GABA-ergic neurons are involved in muscle function as evidenced from a convincing relationship between epilepsy and GABA-ergic neuronal dysfunction (37–39). The data presented in this paper provide evidence for the first time for the involvement of a GABA transporter in the maintenance of muscle function in an animal model by characterizing the phenotype associated with the down-regulation of the transporter.

The phenotype resulting from the RNAi-mediated down-regulation of ceGAT-1 is seen only with the RNAi-hypersensitive rrf-3 strain but not with the normal N2 strain. This is expected because neurons in normal worms are comparatively more refractive to RNAi than non-neuronal cells (35, 36). The rrf-3 strain exhibits hypersensitivity to RNAi, and neuron-specific genes can be down-regulated by RNAi in this strain. RNAi is made possible in C. elegans because of the expression of Sid-1, a transporter for dsRNA in cells (40, 41). However, Sid-1 is expressed at very low levels in neurons in this organism (40) and that is why neurons in C. elegans are resistant to RNAi-induced effects. Recently, this problem was solved by the identification of a C. elegans strain (rrf-3) in which neurons became sensitive to RNAi (35, 36). The hypersensitivity of the rrf-3 strain to RNAi despite the low levels of expression of Sid-1 is attributed to the loss of function of a putative RNA-directed RNA polymerase. The neurons that are generally refractory to RNAi in the wild type strain respond effectively to RNAi in the rrf-3 mutant strain. It was necessary to use the rrf-3 strain for our studies because ceGAT-1 is expressed exclusively in neurons.

The RNAi-mediated down-regulation of ceGAT-1 in the rrf-3 mutant strain produces a specific phenotype associated with changes in the function of body muscles and enteric muscles. The phenotype includes increased rates of body movement that is detectable in the frequency of thrashing, decreased function of enteric muscles that is detectable in the frequency of defecation failure, and increased sensitivity to aldicarb-induced paralysis. GABA has a differential function in body muscles and in enteric muscles. In the control of the function of body muscles, GABA serves as an inhibitory neurotransmitter, whereas in the control of the function of enteric muscles, it serves as an excitatory neurotransmitter (13, 14). This differential function is made possible because of the distinct GABA-related receptors associated with these functions. The unc-49 region contains three genes coding for different isoforms of GABA receptors, which function as anion channels (42–44). GABA-induced influx of chloride via these receptors leads to hyperpolarization of muscle membrane and thus causes relaxation of the muscles. The body muscles are associated with unc-49 gene products, and thus GABA functions as an inhibitory neurotransmitter in these muscles. In contrast, enteric muscles are associated with exp-1 gene product that codes for a GABA-gated cation-selective channel (45). Therefore, GABA-induced influx of cations via this receptor in enteric muscles leads to depolarization and hence contraction of the muscles. There is precedence for such a differential function of GABA. Whereas GABA acts predominantly as an inhibitory neurotransmitter in adult mammalian brain, it serves as an excitatory neurotransmitter in immature mammalian brain (46). In contrast to C. elegans, however, the differential functions of GABA in the mammalian brain are not mediated by different GABA receptors but rather by the varied functions of the same receptors (chloride influx versus chloride efflux due to changes in transmembrane chloride gradients in immature brain versus adult brain). In the rrf-3 mutant strain, ceGAT-1-specific RNAi leads to increased frequency of thrashing, which suggests that the body muscles contract at a higher frequency. This was an unexpected result. Because ceGAT-1 is expected to function in the clearance of GABA from the synaptic cleft or motor endplates, we thought that RNAi-mediated down-regulation of ceGAT-1 would lead to increased levels of GABA signaling. GABA is an inhibitory neurotransmitter in the function of body muscles, and therefore, increased GABA signaling resulting from the down-regulation of ceGAT-1 is expected to increase the inhibitory tone in the function of body muscles. Instead, what we found was that the down-regulation of ceGAT-1 led to a decrease in the inhibitory tone of these muscles as evidenced from increased body movement. We speculate that chronic up-regulation of GABA signaling due to continued loss of the GABA clearance mechanism leads to either desensitization or down-regulation of GABA receptors. Such an effect would explain the increased frequency of body movement in the rrf-3 mutant strain subjected to ceGAT-1-specific RNAi. This would also provide the molecular basis for the increased defecation failure in these worms. Because GABA normally functions as an excitatory neurotransmitter in the function of enteric muscles, down-regulation of GABA signaling would decrease the contraction frequency of enteric muscles that are intimately involved in the defecation process. This would result in increased defecation failure, as it has been indeed observed in our studies.

The phenotype associated with increased sensitivity to aldicarb-induced paralysis in the rrf-3 mutant strain subjected to RNAi-mediated down-regulation of ceGAT-1 is also interesting. Cholinergic neurons function in the contraction of body muscles, and aldicarb, an inhibitor of acetylcholineesterase, is expected to increase cholinergic neurotransmission by interfering with the breakdown of acetylcholine. However, aldicarb exposure does not result in increased muscle function but rather causes paralysis. This is again most likely due to desensitization or down-regulation of cholinergic receptors following continued elevation of acetylcholine in the synaptic cleft or motor endplates. Alternatively, aldicarb may lead to hypercontractile paralysis. A loss of GABA-ergic neurotransmission in unc-25 and unc-47 mutants enhances the sensitivity of the worms to aldicarb-induced paralysis. The molecular mechanisms responsible for these effects are not clear, but the findings show convincingly that GABA-ergic neurons modulate the function of cholinergic neurotransmission. RNAi-mediated down-regulation of ceGAT-1 in rrf-3 mutant strain also enhances the sensitivity of the organism to aldicarb-induced paralysis, an effect similar to that resulting from the loss of GABA-ergic neurotransmission in unc-25 and unc-47 mutants. These findings again support our hypothesis that chronic RNAi-mediated down-regulation of ceGAT-1 actually leads to a loss of GABAergic neurotransmission rather than an increase in GABA signaling.

In summary, we have shown here that snf-11, a member of the sodium-dependent neurotransmitter symporter gene family in C. elegans, codes for a Na+/Cl–-coupled high-affinity GABA transporter (ceGAT-1) and that this transporter plays a critical role in GABA-ergic neurotransmission. Down-regulation of ceGAT-1 by RNAi leads to an interesting phenotype associated with altered function of body muscles and enteric muscles and also with altered sensitivity to aldicarb-induced paralysis. These findings provide unequivocal evidence for a modulatory role of GABA in the biology of cholinergic neurons and in the function of body muscles and enteric muscles in this organism.


   FOOTNOTES
 
* This work was supported in part by the National Institutes of Health Grants HD44404, HL64196, AI49849 (to V. G.), and AG22468 (to Y.-J. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY529124 [GenBank] . Back

To whom correspondence should be addressed. E-mail: vganapat{at}mail.mcg.edu.

1 The abbreviations used are: GABA, {gamma}-aminobutyric acid; ceGAT-1, C. elegans GABA transporter-1; RNAi, RNA interference; RT, reverse transcription; GFP, green fluorescent protein; HRPE, human retinal pigment epithelial; NMDG, N-methyl-D-glucamine; dsRNA, double-stranded RNA; cRNA, complementary RNA; snf, sodium-dependent neurotransmitter symporter family; BGT, betaine/GABA transporter; unc, uncoordinated. Back

2 G. Jiang, Y. J. Fei, and V. Ganapathy, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Michael Krause (Laboratory of Molecular Biology/NIDDK, National Institutes of Health, Bethesda, MD) for assistance in confirming, by fluorescence microscopy, the identity of the GFP-positive neurons in transgenic C. elegans expressing the transcriptional cegat-1:gfp fusion constructs.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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