Molecular Cloning and Functional Characterization of a High-A.fl%ty GABA Transporter from the Lepidopteran Central Nervous System

Xiujuan Gao

Depanment of Zoology I # Submitted in partial filfilment of the requirements for the degree of Master of Science

Faculty of Graduate Studies The University of Western Ontario London, Ontario September, 1998

Q Xiujuan Gao 1998 National Library Bibliothèque nationale If1 ofCanada du Canada Aquisitions and Acquisitions et Bibliographie Services services bibliographiques 395 Wellington Street 395, nie Wellington Ottawa ON K1A ON4 ûttawaON K1AON4 Canada Canada

The author has granted a non- L'auteur a accorde une licence non exclusive licence dowing the exclusive permettant à la National Lîbrary of Canada to BIcbliothèque nationale du Canada de reproduce, loan, distrt'bute or sell reproduire, prêter, distribuer ou copies of this thesis in microforrn, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/nlm, de reproduction sur papier ou sur format électronique.

The author retains ornerslip of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fkom it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son penmission. autorisation. ABSTFUCT

This study was designed to isolate a cDNA encoding a GABA transporter fiom the caterpillar Trichoplrisia ni and express the pro tein for functional c haracterization in order to find a new target for pesticide design. The experimental approach involved cDNA library screening and sequencing of positive clones; baculovinis cloning and expression of the cDNA in cultured cells; and pharmacological analysis of the protein. The isolated cDNA contains an open reading frame (ORF) encoding a 608-residue protein, designated

TrnGAT-1. The deduced amino acid sequence shows high identity with a known insect

GABA transporter MasGAT. Hydropathy analysis of the deduced sequence suggests the presence of 12 transmembrane domains, a similar structure to al1 the other GABA transporters sequenced so far. Accumulation of the TmGAT-1 mRNA was detected only in samples derived from the caterpillar brain. Cells infected with TrnGAT-1-recombinant baculovinis showed a 20- to 30-fold increase in Na--dependent [3~~~~~uptake compared to control-infected cells. Various GABA uptake inhibitors were used to outline the pharmacological properties of the cloned transporter. Although TmGAT-1 is most similar to marnmalian GABA transporter GAT-L in kinetic properties, it is pharmacologicalIy distinct fiom mammalian GAT-1 and other cloned GABA transporters.

The unique pharmacology of TmGAT-1 suggeas that a GABA transport system in the lepidopteran central nervous system (CNS)may be a useful target in the rational design of rapidly-acting neuroactive agents to control Pest insects.

Keywords: GABA transporter, Trfchopltrsiani; ùisect; cloning; baculovinis.

.** III First, [ would like to express my sincerest gratitude to my supervisors Dr. Stanley

Caveney and Dr. B. Carneron Donly for the opportunity to study in the field of molecular and cellular biology. Their instructions, encouragement, support and substantive assistance were generously provided throughout my study. Their constructive coments, patient proof- reading and edting led to completion of the thesis.

1 wish to extend my deep appreciation to Heather and Rod for enormous help in the pharmacological experiments, Alex Richman and John Jevnikar for assistance in molecular biological experiments and ce11 culture. Heather also spent time on proog reading of the thesis. 1 would especially like to thank Tabita Malutan for the help in computer treatment of the molecular biological data and beyond. Chantel, Niki and

Richard are acknoledged for their friendship dun'ng my study.

Gratefùl appreciations are conveyed to Mary Martin for the work facilitating my study program. My advisory cornmittee, Dr. Burr Atkinson and Dr. G. Kelly are acknowledged for their thoughtfùi comrnents and suggestions.

My special thanks go to my sisters, brothers and my parents, their encouragement and love are always with me.

I also thank my son Ningning for his help in collecting the references during the thesis writing.

Finally, 1 would iike to thank my husband Jingtai Han for his understanding, support and help, Without him the completion of this study would have been impossible.

iv TABLE OF CONTENTS .. CERTIFICATE OF EXAMINATIO N ...... -11... ABSTRACT...... 11s ACKNOWLEDGEMENTS ...... iv TABLE OF CONTENTS ...... v. LIST OF FIGURES...... WI.. LIST OF TABLES ...... vu... LIST OF ABBREVLATIONS...... vIII

CHAPTER 1 IUUTRO DUCTION ...... 1

CKAPTER 2 MATERIALS AND METHODS...... ,., ...... 7

2.1 Isolation and Expression of TrnGAT- 1 cDNA...... 7 1.1.1 cDNA Library Screening...... 7 2.1.2 Restriction Mapping and Sequencing...... 10 2.1.3 Nonhem Blot Hybridization Analysis ...... 11 2.1.4 Southem Blot Hybndization Analysis...... 12 2.1.5 Baculovinis CIoning and Expression ...... 13 2.1.6 Virus Amplification and Plaque Assays ...... 14 P hannacological Characteriaion of TmGAT- 1...... 15 1.2.1 Cells and Viral Infection ...... 15 2.2.2 Transporter Assays ...... 16 2.2.3 Chernicals Used in Kinetic and Pharmacological Studies... 18

RESULTS ...... 19

Molecular Cloning and Expression of TmGAT-1 ...... 19 3.1.1 Isolation of cDNA Clone ...... 19 3.1.2 TrnGAT-I Structure...... 19 3-1.3 Transcript Size and Tissue Distribution ...... 23 3 .1.4 Gene Copy Nurnber of TmGAT- 1...... 23 . * 3.1.5 Cloninç in Baculovirus...... 27 Pharmacology of TrnGAT- 1...... 27 3 2.1 Time Dependence and Kinetic Analysis...... 27 3 2.2 Ion Dependence...... 31 . ** 3 .2.3 Inh~bstlonS tudies ...... 34

CHAPTER 4 DISCUSSION ...... 40

4.1 4.2 Phosphorylation... sites ...... 4 1 4.3 Tissue Dtstnbut~on...... 42 4.4 Transporter. Kinetics ...... 43 4.5 Ionic S toichiometry ...... 45 4.6 Pharmacologicai Characterization...... 46

CURFUCUL UM VITA ...... -57 LIST OF FtGURES

Figure 1. Molecular mechanisrns of GAB Aergic synapse ...... -6

Figure 2 . Restriction analysis of TmGAT- 1 cDNA ...... -20

Figure 3 . Sequencing strategies of TmGAT- l cDNA ...... 21

Figure 4 . The nucleotide sequence and deduced amino acid sequence of TrnGAT- 1...... --37

Figure 5 . Aiignment of the amino acid sequences of TmGAT.1. MasGAT. rGAT- 1 and hGAT 1,...... 24

Fiyre 6 . Hydropathy plot and hypothetical secondary structure of the TrnGAT- i ...... 25

Figure 7. Northern blot hybn'dization analysis of TmGAT- 1 mRNA in vanous caterpillar tissues...... 26

Figure 8. Southem blot hybridization analysis of' T. ~i caterpillar genomic DNA...... 28

Figure 9 Time course of ['HIGABA uptake by TrnGAT-1 ...... 29

Figure 10 . Transporter kinetics of GABA uptake by TmGAT- 1...... 30

Figure 1 1. Effect of Na- and Cl* on ['H]GABA transport...... 32

Figure 12. Na- dependence of GABA uptake by TmGAT- 1...... 33

Figure 13 . Cl-dependence of GABA uptake by TmGAT- 1...... 35

Figure 14. Inhibition of TrnGAT-1 activity by selected GABA inhibitors...... 37

Figure 15. Inhibition of TmGAT-1 activity by the inhibitor riluzole ...... 38

LIST OF TABLES

Table 1. Inhibition of ['H]GABA uptake by TrnGAT-1 by GABA and by selectedblockers of GABA transport in mammaiian cells...... 36+

Table 2. Na.. dependent GABA transporters in the insect CNS ...... 44 viî LIST OF ABBREVLATIONS

ACKC Amino cyclohexanecarboxylic acid p GPA B-Guanidinopropionic acid bp Base pair OC Degrees Celsius cDNA Complementary DNA CNS Central nervous system DABA Diamino butyric acid Dig Digoxigenin DNA Deoxyribonucleic acid EDTA Ethylene diamine tetraacetic acid GABA ./-Amino butyric acid h Hour Go Inhibitor concentration blocking 50% of substrate uptake kb Kilobase Km Substrate concentration at half the maximum uptake velocity min Minute M Molar rnM Millimolar w Micromolar MOI Multiplicity of infection mRNA Messenger RNA ORF Open reading fiame PCR Polymerase chah reaction RNA Ribonucleic acid SDS Sodium dodecyi sulfate SSC Standard saline citrate THfP Tetrahydroisoxazole pyridin Tris Tris (hydroxymethyl) aminomethane vm. Maximum uptake velocity Chapter L INTRODUCTION

Chemical signaling at the neuronal synapse is accomplished by a series of cellular processes, starting with the triggered release OP neurotransmitter into the synaptic space

Rom presynaptic vesicles, neurotransrnitter binding to specific receptors on the postsynaptic membrane of target neurons, followed by neurotransmitter re-uptake by membrane transporrers on the surface of the neuron from which the neurotransmitter was released, or on adjacent glial cells (Borden, 1996; Suzdak, 1993) (Fig. 1). This releaseke- uptake cycle forms pan of the basic synaptic rnechanism in both the venebrate and invertebrate CNS.

Neurotransmitter receptors and transporters are integral membrane proteins. The recepton are activated by neurotransmitter molecules, while the transporters are involved in tenninating the neuronal signaling cycle by taking up and stonng neurotransmitter in the nervous system. Two neurotransmitter transport systems are essential to the finctioning OF the venebrate nervous system (Attwell and Mobbs, 1994). The first system includes uptake carriers in the plasma membrane of neurons and glial cells which pump neurotransmitter From the extracellular space into the cell. These carriers help to teminate the postsynaptic action of neurotransmitters released from neurons, and to replenish the neurotransmitter supply of the neurons. Once inside the cell, neurotransmitters are further transponed into synaptic vesicles by the second system, an apparently independent transport system which is located in the vesicle membrane (Fig. 1). These vesicle- membrane carriers are powered by a pH or voltage gradient across the vesicle membrane. They have structures distinct frorn those of the plasma-membrane carriers (Worrall and

Williams, 1994; Attwell and Mobbs, 1994; Schuldiner, 1997).

GABA (parnino butyric acid) is the predominant inhibitory neurotransmitter in the mammalian central nervous system (CNS)(Guastella et al., 1990), and has been estimated to be present in 60-70% of al1 synapses within the CNS (Suzdak,1993). In insects, it is found in both the central and peripheral nervous systems associated with inhibitory motoneurons (Mbungu, 1995; Sattelle, 1992). Unfike rnarnmals, insects use GABA as an inhibitory neuromuscular transmitter (Van Marle, 1985), suggesting an insect-specific target for the rational design of neuroactive agents to control pest insects. GABA receptors are widely distributed in the insect nervous system (Sieghart, 1995; Hosie et al.,

1997) and have been the target of commercial insecticides such as dieldrin (Tanaka et al.,

1984). However, structural mutation of these GABA receptors as a response of resistance to such insecticides has been revealed (ffrench-Constant and Roush, 1991), and such resistance is cornmonly exhibited in insects (Georghiou, 1986). Since insectiside resististance poses a serious problem in pest management, the structure-function relationship of GABA recepton has been extensively studied in order to find the mechanisrns behind resistance (Sattelle, 1992; Hosie et al., 1997). Consequently this study is part of an ongoing search for proteins other than GABA receptors that are present in the GABAergic pathways (Fig. 1) in agiculturally important insects that may be blocked by new-generation insecticides.

Dismption of neurotransrnitter transport rnechanisrns? in this panicular instance

GABA transport, provides an alternate way of rnodulating synaptic signalling. This has been shown to be significant in clinical medicine. GXBAergic transmission is teninated

by GABA uptake into the presynaptic renninals and/or the surrounding glia by high-

affinity transporters specific for this neurotransmitter. Reduction in GABA

neurotransmission ha been implicated in the etiology of a variety of neurological

disorders, in panicular epilepsy (Sudzak, 1993). Phannacological inhibition of GABA

transport represents a novel approach to increasing GABAergic activity, and such agents

may represent novel therapeutic agents (Borden, 1996). Thus, GABA transport systems

represent critical targets for therapeutic and pathological alterations of synaptic function.

Studies of the molecular and pharnacological propenies, locaiization, and structure-

function relationships of neurotransmitter transporters are essential to our understanding

of synaptic siçnaling and the potential role of transporter defects in neurologie and

psychiatnc disorders, and particularly for the development of novel therapeutic agents

(Shafqat, 1993).

In recent years, molecular cloning studies have revealed that two distinct gene

families code for plasma membrane neurotransmitter transporter proteins. One family

codes for a set of Na--(and Cr)-dependent proteins including transporters for GABA, glycine, proline, taurine, betaine, and creatine; and the other family codes for a set of Na'-

(and Qdependent, but CI--independent, transporters that include transporters for the excitatory amino acids L-glutamate and L-aspartate. These two sets of plasma membrane transporters exhibit siçnificant differences in their ionic requirements and pro posed structures which are likely to reflect underlying differences in transport mechanisms (Shafqat et al., 1993). This study is focused on the plasma membrane GABA transporter proteins.

Giü3A transporters belong to the Na'- and CLcoupled transporter farnily

(Wright, et al., 1992; Borden, 1996). They CO-transportthe neurotransmitter with Na' and

Cl- in an electrogenic fashion (Kanner, 1997) with a stoichiornetry of 2-3 Na' and 1 Cl- for

I GABA zwitterion (Radian and Kanner, 1983; Suzdak, 1993). Ail GABA transporter subtypes have absolute requirement for extracellular Na- (Miller, 1997). The requirement for extracellular Cl- is less specific, however, wit h significant uptake activity retained when

Cr is replaced by cenain anions (Goncalves et al., 1994). GABA trahsporters have been well studied with respect to the stoichiometry of CO-transponed ions, and to their substrate and inhibitodmodulator specificities. Recent progress has centred on the elucidation of the structure of these transporters through molecular cloning, sequencing and mode1 building studies. Some progress has been made on the elucidation of tùnctional domains within the transporters and on the regulation and mechanisms of action.

Molecular cloning has revealed the existence of a greater GABA transporter heterogeneity than previously suspected. Since 1990, four different rnammalian GABA transporters

(termed GAT-1, GAT-2, GAT-3 and BGT-1) have been cloned from mouse, rat, canine and human which exhibit 50-69% amino acid sequence identity. They have different anities for GABA, different substrate and blocker pharmacologies, and different tissue localizations (Guastella et al., 1990; Liu et al., 1993; Lopez-Corcuera et al., 1992; Schloss et al., 1994; Brecha and Weiçmann, 1994; Johnson et al., 1996). Recently, a GABA transporter fromhIa~~dircasexta embryos (MasGAT) has also been cloned (Mbungu et al., 1995). It shows 50-58% amino acid sequence identity with the known mammalian GABA transporters, and pharmacologically most resembles the mammalian GAT-1 transporter.

However, MasGAT is apparently insensitive to cis-3 aminocyclohexanecarboxylic acid

(ACHC), which distinguishes it from mammalian GAT- 1. Therefore, MasGAT potentially represents a fifth GAT subtype (Miller, 1997).

It is my belief that the insect GAi3A transporter deserves consideration as a potential target for insect control. For this to be assessed by industrial mass screening protocols, the protein target first must be isolated, expressed and charactenzed CI vitro.

Consequently my thesis describes the isolation of a cDNA encoding a GABA transport protein fiom the caterpillar Trichopitisia tli and its pharmacological characterization in a ce11 expression system. The thesis consists of four chapters. Chapter 1 has described the objective of the research project and bnefly introduced the work involved in the thesis.

~Matenalsand Methods employed in the experiments are shown in Chapter 2. Chapter 3 presents the results and Chapter 4 discusses the related issues and highlights the major conclusions obtained through this study. Figure 1. Molecular mechanisms of GABAergic synapse. Glutamic acid decarboxylase (GAD)convens glutarnic acid to GABA. A GABA camer coupled to a proton pump mediates the uptake of GABA into synaptic vesicles. GABA is released by synaptic vesicle exocytosis and acts on post-synaptic GABA-A receptorhon channels or the GABA-B (G- protein-coupled) receptors. A plasma membrane GABA transporter is shown retneving GABA Born the extracellular (synaptic) space. GABA taken up into the presynaptic terminai is transported into synaptic vesicles andfor catabolized by GABA transaminase (GABA-T) into succinic semi-aldehyde. Chrpter 2 MATERIALS AND METKODS

2.1 Isolation and Expression of TrnGAT-1 cDNA

2.1.1 cDNA Library Screening

A Trichophrsia rii head cDNA library, cloned into EcoR LXho I site of the ZAP

Express vector (Stratagene) (Donly et al., 1997), and a probe were obtained from B.C.

Donly. The probe was amplified by PCR using degenerate primers GABA-1 and GABA-2 designed based on known venebrate GABA transporter sequences with 7: tri head cDNA as the target. Primers GABA- 1 (5'-GGN AAY GTN TGG MGN TTY CCN TA-3 ') and

GABA-2 (5'-ACN CCY TTC CAD ATR CAR AAR TA-3') are corresponding to nucleotides 45 1 to 473 and 997 to 10 19 of TrnGAT- 1 cDNA respectively (Fig. 4). To screen the library, the probe was diçoxigenin-labeied by random priming using the DIG

DNA labelinç kit (Boehrinçer Mannheim). From a screeninç of 5 x 10' plaque forming- units, four hybridizing plaques were isolated. The sizes of the inserts were then determined by PCR (with primers T3GABA-2 and T7IGABA-1). Library screening was performed as follows. The host Eschcrichia coli strain XLI-blue was streaked on a LB plate containing

12.5 &ml tetracycline and grown at 37T overnight. A single colony was picked and inoculated into 30 ml of LB medium containing 0.2% maltose (w/v), 10 mM MgSOa and

12.5 pg tetracycline/ml. The bacterial culture was grown ovemight at 37'C with vigorous shaking (5 an ODsaa of LO), then spun down at 2000 rpm for 10 min. The peuet was resuspended in about 15 ml of LO mM MgSOd at an ODtjor of 0.5 for dilution of bacteriophage h in SM baer ( 5.9 g of NaC1,3 g of MgSOa, 50 mi of 1 M Tris-HCI (pH 7.5) and 5ml of 2% (wh) gelatilill). About 5 x 10' plaque-forming units were taken frorn the diluted cDNA library (L: 100 in SM) and added to 6 ml of the bacterial suspension. The mixture was divided into 10 aliquots of 0.6 ml and then incubated at 37OC for 15 min. At the same time, 0.7% agarose in NZY medium was melted by microwave and set in a

49.S°C water bath. The 150 x 15 mm MY-agar plates were dkd at 37OC for 2 h before plating. 6.5 ml of 0.7% agarose-NZY medium was added to each of the 10 culture tubes and immediately placed on the NZY-agar plates. Mer solidification the plates were incubated at 37OC in the invened position ovemight until clear plaques appeared. The plates were stored at 4°C ovemisht, after which 2 Hybond-N nylon membranes (L37 mm,

0.45 micron pore size) were laid on the plates seperately, the first for 1 min and the second for 4 min. After lifting, the membranes were dried for about 10 min, and the denaturation and neutralization were camed out by soaking the tilters in 0.5 M NaOY 1.5 M NaCl for

5 min, transferrinç the filters in order to 0.5 M Tris-HCI (pH 7.9, 1.5 LM NaCl twice for 5 min each and then 2 x SSC (0.3 M NaCl in 30 rnM sodium citrate, pH 7.0) nvice for 5 min each, The DNA was cross-linked to the membranes with a CL-1000 uhraviofet crosslinker (setting 1200) for both sides. The membranes were wet by 2 x SSC and then put one by one into a glass bottle containing 25 mi of prehybridization solution (5 x SSC,

2% blocking reagent, 0.5% SDS) and prehybridized at 6S°C for 4 h. Mer prehybridization, a deoatured Dig-labeled DNA probe (600 bp) was added to the prehybndization solution (12 nghi) and hybridized at 60°C ovemight. Washing of the membranes was perfomed at high strhçency. The membranes were fint washed with 2 x

SSC, 0.1% SDS at room temperature hvïce for LS min each, then with 0.2 x SSC, 0.1% SDS at 60°C twice for 30 min each. The detection of substrate was performed by rinsing the membranes bnefly in buffer 1 (0.15 M NaCI, 0.1 M maleic acid, pH 7.9, incubating the membranes 30 min in buffer 2 (buKer I containing 2% blocking reagent) with gentle shaking (75 rpm on platform shaker), incubating the membranes for 30 min in diluted antibody solution (1:lO 000 in buffer 2) with gentle shaking, washing the membranes in buffer 1 six times for 10 min each, incubating the membranes 5 min in buffer 3 (0.1 M Tris

(pH 93,0.1 M NaCI, 50 mM MgCl$ and transfemng the membranes to diluted "CSPD solution (1: 10 in buffer 3). The membranes were then placed between two acetate sheets supported by a used X-ray film for autoradiography. The positive plaques were picked up by using a sterile Pasteur pipette. The agar plugs were added to 1 ml of SM with one drop of chloroform in an Eppendorftube which was then stored at 4'C ovemight. Five p1 of the phage suspension from the stock of the primaiy screening was diluted 100, 1000, 10 000 times with SAM,from which 2 pl was used to infect the E. coli host cells. The secondary and tertiary screening was perfomed as the primary screening except 100 x 15-mm NZY- agar plates and 82-mm Hybond-N nylon membranes (0.45 micron pore size) were used.

From secondary and teniary screening four single positive plaques were picked up and added to 500 pl of SM buffer with one drop of chloroform. The phage plugs were stored at 4'C overnight to release the phage.

The four positive cDNA insens contained within the ZAP express vector were excised and recircularized in pBK-CMV phagemid iiz vivo (Stratagene). The phagemids were then transformed into E. cdi XLOLR cells and extracted with a commercially available kit (QWprep-8 PIasmid Kit). The excision of pBK-CMV phagemid was performed as follows. Two overnight ce11 cultures of ?a 1-blue and XLOLR were grown in LB medium containing 50 pg/ml kanamycin to an ODsoa of 0.5 and 1.0 respectively.

The XL1-blue cell culture was spun down at 1000 x g for 10 min and resuspended at an

ODsao of 1.0 in 10 mM MgSO4. 200 pl of the XI-blue ce11 suspension was mixed with

250 pl of phage stock and 1 pi of ExAssist helper phage (>1 x 106 pfdmi) and incubated at 37 OC for L5 min, Three ml of LB medium were added to the above mixture and incubated at 37 OC with shaking until Iysis occurred (about 2-3 h). The phage lysate was heated at 70 OC for 15 min and ce11 debris was spun down at 4000 x g for 15 min. Two aliquots of 10 and 100 pl of supernatant were added to two tubes containing 200 pl of

XLOLR cell culture seperately and incubated at 37 OC for 15 min. 300 pl of LB medium was added to the above mixture and incubated at 37 OC for 45 min, from which 700 pl was spread on LB-agar plates containing 50 &ml kanamycin. The plates ware incubated at 37 OC ovemight. A single colony of each of four positive clones was picked up and added to LOO pl of LB medium. The picked colonies were analysed by PCR (with

T3/GABA-2 primers) and electrophoresis. The pBK-CMV phagemids with positive cDNA inserts were isolated using a QIAprep-8 Plasmid Kit (Qiagen) following the manufacturer's instructions.

2.1.2 Restriction Mapping and Sequencing

A single clone containing the most extensive 5' structure was subjected to restriction analysis and sequenced on both strands, and the other three clones were sequenced fiorn the 5' end (T3 primer) by automated dideoxynucleotide chah termination sequencinç. The sequence was analysed usinç Laser Gene software. The strategies employed to obtain the complete sequence €rom both strands of the clone includes 1) recirculanzing of plasmid: the pBK-CMV plasmids were separately digested with EcoR 1,

Pst 1, Sca 1 and BstX I, and separated on 1% low melting point agarose (SeaPlaque GTG) gel. Mer a restriction fragment was excised the plasmids were rec overed with P-agarase 1 as described by the manufacturer (New England Biolabs, Inc.) and recircularized with Ta

DNA ligase, or the plasmids were digested with BamH VBgl II and then randornly recircularized; and 2) subcloning of restriction fragments: the insert contained within the pBK-CMV phagemid was excised by BamH VMio I digestion and digested with Bgl II.

The resulting restriction Fragments were subcloned into the pBluescript plasmid at BadI site. To set any remaining regions, four primers were designed based on the partial sequence obtained by using the above strategies. They are GABA T3 (5'-AGT GGC MC

AAA ATC AGA TCJ'), G14 T3 (5'-GGC TAC TAC CCT ACT ATC TG-3'), G1 T7

(5'-TGA ACA CGC TAA TGC AGA TA-3') and GS T7 (5'-GGT CTG KAGGA

CAG CTT TG-3').

2.1.3 Northern Blot Hybrîdizrtion Analysis

Ten pg of total RNA isolated kom each tissue (including epidermis, fat body, silk gland, brain, gonads, midgut, hindgut, rectum and malpighian tubules (MT.)) of late instar T'choplmÏa ni Iarvae was separated on a 0.66 M fonnaldehyde agarose gel and transferred to Hybond W nylon (Arnenham), where it was fixed under ultraviolet tight

@only, 1997). A cDNA fiagrnent cornprising nucleotides 1- 1020 of the TmGAT-I clone (amplified by PCR with TYGABA-2 primers) was "P-labeled by random-primed synthesis, and hybridized to the immobilized RNA at 60 OC in Quikhyb solution

(Stratagene) for 2 h. The membrane was washed once at room temperature in 2 x SSC,

0.1% SDS for 20 min, twice at 60 OC in 2 x SSC, 0.1% SDS for 20 min each (1 x SSC is

0.15 M NaCl, 15mM sodium citrate, pH 7.0). The washed membrane was then exposed to

Cronex X-ray film for autoradiography.

2.1.4 Southern Blot Hybridizrtion Anrlysis

Genomic DNA of caterpillar Trichoplicsia id was digested with six different enzymes (Bad 1, Hind [II, Pst 1, Kpn 1, Xho I and Xba I) and phenol-chlorofom extracted and ethanol precipitated. About 20 pg genomic DNMane was separated on 1% açarose gel mminç at 75 V for 10 h. The DNA gel was stained with ethidium bromide in ddH20 for 10 min and destained in ddH20 for 15 min. The DNA was depurinated by incubating the gel in 0.25 M HC1 for IO min, denatured in 0.5 M Na09 1.5 M NaCl three times for 15 min each, and neutralized in 1.0 LMTris-HCl (pH 73, 1.5 M NaCl three times for 15 min each, der which the DNA was transferred to Hybond W nylon

(Amenham), where it was fixed under ultraviolet light. A LOO0 bp genomic DNA fragment comprising nucleotides i786-2085 of sequenced cDNA clone (amplified by PCR with G14 TYGABA T7 prïrners) was Dig-labeled by random priming, and hybridized to the immobilized genomic DNA. The hybridization and detection were accomplished following the procedures described below. The membrane was prehybridized in 20 mi of prehybiidization solution (0.25 M Na&IP04, (pH 72), 1 mM EDTA, 20% SDS, 0.5% blocking reagent) at 68'C for 3 h and hybridized in 10 ml of hybridization solution

(prehybridization solution containing 2.5 nglml denatured probe) at 6jaC overnight.

Washing of the membrane was conducted at high stringency. The membrane was first washed with stringency solution (30 mM NazHPOl (pH 7.2), 1 M EDTq 1% SDS) at

65Tthree times for 20 min each, and then with the wash buffer (3 M NaCI, 0.1 M maleic acid, 0.3% Tween20, pH 8.0) at room temperature for 5 min. After washing, detection was exercised at room temperature by incubating the membrane in antibody blocking buffer (wash buffer containing 1% blocking reagent) for 60 min, in diluted anti-DIG-AP solution (k15 000 in antibody blocking buffer) for 30 min, in wash buffer four times for

15 min each, and in buffer 3 (0.1 M Tris (pH 9.9, 0.1 M NaCl, 50 mM MgCl*) for 5 min.

The membrane was placed between two acetate sheets and 1 ml of diluted Lumigen (1: 100 in buffer 3) was dispensed on to the membrane. Hybridized genomic DNA fragments were cherniluminescence-deteaed by exposure to Cronex x-ray film.

2.1.5 Baculovirus Cloning and Expression

The 2.3-kb insert of sequenced clone TrnGAT-1 was excised fiom the pBK-CMV phagernid with BamH 1/ Xho I and ligated into the Bac-to-Bac baculovims expression syaem (Life Technologies) transfer vector pFastBac1 at the same restriction sites. The resuiting plasmid was then transformed into Exoli XL1-blue and then isolated using a

QIAprep-8 Plasmid Kit (Qiagen) following the manufacturer's instructions. The plasmid pFastBacl containing the TmGAT-1 insen was transposed to bacmid as directed by the supplier (Life Technologies). Recombinant bacmid was then transfected into Sf9 cells. The transfection was performed using the €oIlowinç procedures. 1 x 106 SB cells lwell were seeded in 2 ml of serum and antibiotic-containing TC-LOO medium (Life Technologies) using a 6-well plate, and incubated in a 27 OC ovemight. Transfection solution (1 ml of semm and antibiotic-free TC-100 containing 5 pl of recombinant bacmid DNA and 6 pi of

CelECTIN Reagent) was prepared following the protocol (Life Technologies). The medium was removed from each well, and the cells were washed once with 2 ml of serum and antibiotic-free TC-100, and incubated in L ml of transfection solution at 27 OC for 5 h.

Subsequently, 0.5 ml of transfection solution was removed and 1.5 ml of semm and antibiotic-containing TC-100 were added to each well. The plates were then placed at

27OC incubator For 3 days. Recombinant baculovims was harvested by centrifuging the viral- containing medium at 500 x g for 5 min and transferring the viral containing supernatant to a sterile tube which was then stored at 4°C.

2.1.6 Virus Amplification and Plaque Assays

Viral stocks were arnplified and titered in SB cells. T-25 angle neck flasks were used for viral amplification. 2 x 106 cells lflask were seeded in 5 ml of semm and antibiotic-containing TC-LOO medium and incubated at 27OC for 48 h until the cells reached near 50% confluence. The medium was repiaced with 2 ml of viral supernatant

(hm transfection) to produce a multiplicity of infection (MOI) of about 0.1. The virus was left on the cells for 2 h wirh mixing every 15 min, fier which 8 ml of medium was added to the flask and the Bask was incubated at 27 OC for 4 days. Recombinant baculovinis was harvested as above (see Baculovirus Cloning and Expression section). Plaque assays for viral stock titer were conducted with 6-well plates. 4 x 105 cells/well

were seeded in 2 ml of TC-100 and incubated at 27 OC ovemight. The medium was replaced with I ml of TC-100 containing 200 pl of different dilutions of viral stock to make a serial dilution of 10-~, IO", 104, IO", 104 for each well and incubated for 1 h with rocking every 15 min. Subsequently, the virus-containing medium was quickly removed from al1 wells and 3 ml of plaquing overlay (3 ml of TC-100:l ml of 2% agarose) equilibrated at 42 OC in a water bath was çently dispensed to each well. The plates were then placed on a flat table to set and transferred to a tuppenvare box lined with wet paper towels at 27°C for 7 days until plaques appeared. The plaques were counted daily until the numben did not change for 2 consecutive days. The titer of the viral stock was calculated to be 2.5 x 10' viral particles/ml.

2.2 Phrrmacological Characterizrtion of TrnGAT-1

2.2.1 Cells and Viral Infection

In order to optimize the subsequent experiments of pharmacological assays, three lepidopteran ce11 lines (High Five, SB and SEI) were tested in the preliminary transport assays. Sf21 cells were found to have more adherence to the plate and gave higher signahoise ratio 48 h post-infection. Consequently, SE1 cells were selected for the subsequent experiments. SB1 cells were maintained in monolayer culture at 27OC in TC-

100 medium. In order to determine the optimal MOI for the expression of GABA transporter, the cells were infected with different amounts of virus in 12-well mîcrotiter plates and assayed (see next section). Based on the transport activity of the cells, a MOI of 0.6 was to be used for the subsequent experiments. The infection was performed as follows. 4x10' cells/well were seeded in TC-100 medium and incubated at 27°C ovemight. The medium was replaced with 10 pl of recombinant baculovirus (2.5 x 10' viral particles/ml) in 500 pl of TC-100, and the plates were incubated for 1-2 h with rocking every 15 min. Afterwards, 1 ml of TC- 100 was added to each well and the plates were placed back in the 27°C incubator. Cells were assayed for GABA transporter activity afier 48 h. A similar recombinant construct containing the gene for P-glucuronidase

@/SA), a control supplied with the Bac-to-Bac expression system (Life Technologies), was used in parallel experiments to correct for any background transport activity.

2.2.2 Transport Assays

Growth medium was removed from individual wells in the 12-well culture plate at

Cmin intervals. The cells were rinsed immediately with 1 ml physiological saline containing approxirnately 66 mOsm Na' (54 mM NaCI, 7.3 mM NaH2POc 55 mM KCI,

11.2 mM MgCI2, 11.2 mM MgSOc and 76.8 rnM sucrose, adjusted to pH 7.0 with 1 M

NaOH). A second voiume of saline was added and the cells placed back in a 27°C incubator for L h. At this time, the cells received a third saline rinse and were then exposed for 5 min to 500 pl saline containing 5 pl ['KI GABA (0.05 nmol GABA at a specific activity 86-95 CYmol). Unlabeled GABA was also added to the above 500 pl saline in some experiments (tirnedependence, dose-dependence, Na* and Cl=dependence) to give a final concentration of 10 p.M for time, Na- and Cl'-dependence assays, 0.1-99 pM for dose-dependence assay. Unlabeled competitive transport substrate substituted for GABA in the cornpetitive inhibition studies. Room temperature was 26-28°C during these experiments. Exposure to CH] GABA was terminated by removing the radiolabeled solution and washing with 2 ml of Na'-free physiological saline f~urtimes. The last rinse solution was removed and the cells allowed to air dry. This procedure was repeated on successive wells until the cells in al1 12 wells had been exposed to radiolabel. Afier a further drying of 10 min, the cells were rreated with 500 pi 70% ethanol for 20 min with shaking to extract the accumulated radiolabel. An aliquot of 300 pl was removed from each well, added to scintillation solution, and the radioactivity measured.

To determine the Na'-dependence of GABA uptake, a NaXee saline (in which the two sodium salts above were substituted by equimolar choline chloride and WPQ, pH adjusted to 7.0 with 1 M KOK) and a 100 mM sodium-saline (NaCl raised to 93 mM, other salts as above, but with sucrose deleted, pH adjusted to 7.0 with L M KOH) were combined to give twelve different ma1 in the range 0-!O0 mM. The Cls-dependence of

GABA uptake was detemined by combining a CIXee saline (in which the three chloride salts were substituted by equimolar Mg (a~etate)~,Na gluconate and K gluconate) with

170 mM chloride-saline (the same as 100 mM sodium-saline) to give twelve different [CI'] in the range 0- 170 mM. Radiolabeled GABA was added to yield a final concentration of

10 pM. The Na- and Cl'-dependence of GABA uprake was also determined by ion substitution experiments in which the Na* and Cl- in incubation solutions were replaced by a vanety of cations and anions respectively.

The afinity (Km)of the transporter for GABA and maximum uptake of GABA

(Kt=) per well was obtained through Eadie-Hofstee analysis of the data. The inhibitor concentration blocking 50% of the accumulation of radiolabeled GABA (ICjo) was determined by dose-response plot analysis.

2.2.3 Chemicals Used in Kinetic and Pharrnacological S tud ies

Chemicals were obtained from (supplier: chernical [catalogue #]): Research

Biochemicals hc.. Natick. M O01 760-2447: 1-Amino- 1-cyclohexane carboxyiic acid

(ACHC) [A-L62], guvacine [G-0071, (+)-cis-4 hydroxynipecotic acid [H-1481, 4,5,6,7- tetrahydroisoxazole [5,4-cl pyridin-3-0 1 (THIP) [T-10 11; Sigma-Aldrich, Si. Loiris, MO

631 78-99 16: Arecaidine [A-52591, p-alanine [A-99201, betaine [B-350 11, P- guanidinopropionic acid (PGPA) [G-68781,L-2,4-diamino-n-butyric acid (DABA) [D-

83761, y-amino-n-butyric acid (GABA), hypotaurine [H-13841, (F) nipecotic acid IN-

73791, phloretin p-79 121, taurine [T-06251, veraparnil [V-46291; Tocris Cookson,

BalIwin, MO 6301 1: Muscimol [0289], riluzole [0768], SKF 89976-A [IO8 11; Amersharn,

Bzrckr, UK HP7 9NA: 4-amino-n-[2,3-jH] butyrk acid (GABA) [TRK527] at a radioactive concentration of 1.0 mCVrnl. Batch specific activity ranged between 86 and 95

C'ummol- Chapter 3 RESULTS

3.1 Molecular CIoning and Expression of TrnGAT- t

3.1.1 Isolation of cDNA Clone

Using a PCR product obtained from B.C.Donly (see Matenals and Methods section) as a probe, four clones were isolated from a caterpillar head cDNA library. The

Iengths of the inserts were determined by PCR to be approximately 4.5, 1.9, 2.3 and 2.2 kb, with each containing a common region encompassing the transporter probe fragment.

Restriction analysis revealed that the insen contains restriction sites for several enzymes including EcoR 1, Pst 1, Sca 1, Bgl II and BstX 1 (Fig. 2). The restriction sites for each enzymes are unique for religation to the sites for other enzymes, a charactenstic very usehl for a successive piece-by-piece sequencing. On the basis of restriction analysis, two arategies were designed for full length sequencing of the TrnGAT-L cDNA clone, as shown in Fig. 3. Sequence analysis indicated that al1 the four isolated clones are consistent.

The Fully sequenced cDNA is 2301 bp, with a 5' untranslated reçion of 285 bp, a 3' untranslated region of 192 bp, and a predicted open reading fkame of 1824 bp, encoding a

608-residue protein I designated TmGAT-1 (Fig. 4).

3.1.2 TrnGAT-1 Structure

Upstream of the aaning methionine is an in-frame stop codon located near the 5' end of the cDNAThe region flanking the putative start condon contains adenine and guanine at -3 and +4 positions (Fig 4). It represents a resonabie Kozak consensus sequence of venebrate (S'ACCAUGG, Kozak, 1987). Upstream of ATG condon EcoR I LbVI\ (2296) (1 (892)(1151) h-A I I j SC~I 1 sca I Pst l I 1-

l I Bgl II Bgl II Bgl II 1 (375)

Figure 2. Restriction rnrlysis of TrnGAT-1 cDNA. A single clone containing the TrnGAT-L cDNA was analysed by restriction digestion. It contains restriction sites for the enzymes Pst 1, EcoR 1, BstX 1, Sca 1 and Bgl II. 1393) b 89

GABA T3 (496) +

Figure 3. Sequencing strategies of TrnGAT-1 &NA. According to restriction analysis, the sequencing strategies for TmGAT-1 cDNA employed involved recircularinng plasmids and subcloning restriction Fragments. The remaining regions, represented by dotted lines, were sequenced using newly designed pnmers. These primers are GABA T3, G14 T3, GL T7 and GS T7, FLIPYFLTLFtAYITXTPMELAMC CAG ATG TG;\CC ÀTC rG5T r5d TGGGA riTG TTMG ATT GCG ZT AïA ZTC AU -53 ATC rJCjC TAC GCG f%T

~H:;J:=NEL~CTLLLYM'~LLZ'~~C

.-x ;,rz TGï i\cC 7, .Ai& (XC TAC ?TC ACî LXCEG XC CCA CAC TZC 7TA TT5 ACT

..~L-!?.~~~"?~;XH~~~~IKF.<'~M?~M . . T'CC XG ,=TG XTG GAG TCA *îW .=A %& ATC ,;AC T;tt EC ACA ZAG ATC T?C XC ?CA TAC rîC?G GGA TG

~=.r.t.~.rxà~~t;.;~~n--r5A.Z ZCCAC -75 (CTA #>3C *Xî-TG -TA 7-& TC(= ?TjC CC ?TC ?TC TC &TG TACiG *ZC&TC rXïï fTG fAC AC L2L?;A?SXSSLFFFXLLL:G2DS :AG =Lf T'h .;Cr ATG *fM ZG-5 'ZC ;iTC AC5 rKï GTC ATT CZC ZAG Tri SCCC ÀiG CC CTC Ar& AGAG AAG zF*ZT!4f ï?lTA.JI3SX?SLLI?.ttK ;AG ATC =TC A:C iZI .:?Y XC TZ-X ;TC :C'CG TAI: LTG GT 'XA ?T5 rtî TGT R'ïC 'X7 3G Gûï rZ XTG

Figure 4. The nucleotide sequenee and deduced amino aeid sequenee of TrnGAT-1. The 12 putative transmembrane sequences are underlined. Potential N-glycosylation sites are double underlined and bolded, and potential protein kinase C sites are bracketed and bolded. The six potential casein kinase II sites and eight potential N-rnyristoylation sites are not shown, represents the consensus sequence of Drosophilcr (5' CIWCAUG; Cavener, 1987).

Cornparison of TmGAT-1 with predicted amino acid sequences of other GABA transporters showed 95% identity w*th MasGAT (Mbungu et ni., 1995), 54% identity with rat GAT-I (Radian et al., 1986, Guastella, et at., 1990), and 54% identity with human GAT-1 (Nelson et al., 1990) (Fig. S), 54% identity with mouse GAT-1 (Liu et at.,

1992), 46% identity with rnouse GAT-2(Lopez-Corcuera et al., 1992), 49% identity with both rnouse GAT-3 and mouse GAT-4 (Liu er al., 1993). Hydropathy plot suggests the presence of 12 transmembrane regions, cytoplasmic amino and carboxyl temini, and a large extracellular loop between transmembrane domains 3 and 4 (Fig. 6). The deduced protein contains five potential N-glycosylation sites, four potential protein kinase C phosphorylation sites, eiçht potential N-rnyristoylation sites and six potential casein kinase

II phosphorylation sites.

3.1.3 Transcript Size and Tissue Distribution

Expression of the TrnGAT- 1 mRNA was assessed by blotting total RNA extracted fiom various caterpillar tissues and hybndizing with a '*P-labeled Fragment of the

TmGAT-1 cDNA. Accumulation was detected only in sarnples derived from the brain.

The size of the single transcript was about 4.4 kb (Fig. 7).

3.1.4 Gene Copy Nurnber ofTrnGAT-1

The probe for Southem blot hybridization anaiysis was prepaired fiom T. rii genomic DNA by PCR with primen (CL4 T3lGABA T7) comprising nucleotides 1786-

2085 of sequenced cDNA clone. A 1000 bp product containing about 700 bp of intron D 1KNDGRSD 1 XEL AQCT----- S~Z~~SCVAT~-----;; E 1AQGS-----CH KHDSRSD ftL PSOVAV ----- A -DNSKVA GQS TLVSZAPVASD PKTLVV VQKKAGD A -HCSKVA GQf TBVSeAPVAHD PKTLVV VQKKAAD

TmGAT- l 409 MmGAT 410 SAT-1 397 hG AT- I 397

TWQBKFiiKtVRf TKQtKFKKtVRI SLKQRLQVHfQD SLKQRXQVHVQP

Figure 5. Alignment of the amino rcid sequences of TrnGAT-1, MasGAT, &AT4 and hGAT-1. Deduced amino acid sequences of the Trichophsiu ni, Marduca sextu, rat and human GABA transporters were aligned using the DNAstar program- Identical residues are shaded- Residue number

ExtraceIl ular L

Figure 6. Hydropathy plot and hypothetical secondary structure of the TrnGAT-1. (A) Hydropathy analysis oCTmGAT-1 using the ISREC-Bioinforrnatics TMpred program. Twelve putative transrnembrane regions are numbered. (B) Schematic topologicd mode1 of TmGAT-1. The mode1 is based on hydropathy plot analysis of the sequence of TmGAT-1. Twelve putative transmembrane segments are illustrated as rectangles. The hydrophilic loops are designated EL (extracellular loops), IL (intracellular loops) and are numbered. Each amino acid residue on the loops is shown as a circle. îhree of £ive potential N-glycosyiation sites are Iocated on the extracellular loops and depicted as branched lines; the two remaining putative N-glycosylation sites are located internally and are not shown- Figure 7. Northern blot hybridization anslysis of TrnGAT-1 mRNA in various caterpillar tissues. Total RNA was isolated from vanous tissues (M-T-stands for rnalpighian tubules) dissected fiom late instar T. rli caterpillars and separated on a 0.66 M formaldehyde agarose gel (10 pgllane). The RNA was transferred to a nylon support and hybridked wîth a 32~-labeledfragment of the TrnGAT-1 cDNA before exposure to X-ray film. A single band was visualized in the caterpillar brain. The relative sire of the RNA transcript was detemined fiom the mobility of concurrently separated, ethidiurn bromide- stained DNA standards (bp). was produced. This fragment was subsequently analysed by restnction digestion. Gel electrophoresis showed that none of the restnction sites tested was contained within the fragment. From the enzymes used to digest the genomic DNA fragment, six enzymes were chosen for genomic DNA digestion. The DNA fragment hybridized to a single band in each lane (Fig. 8), indicating that the gene coding for TmGAT-1 is a single copy gene.

3.1.5 Cloning in Baculovirus

The TrnGAT-1 cDNA was cloned into the baculovinis expression vector (Life

Technologies) and the protein was then expressed in cultured cells. The tùnction of

TmGAT-1 protein was analysed using TmGAT-1- expressing SC2 1 cells (see below).

3.2 Pharmacology of TrnGAT-1

3.2.1 Time Dependence and Kinetic Anrlysis

GABA uptake in TmGAT-1 expressing SfLl cells was seen to be time-dependent

(Fig. 9). The accumulation of GABA in infected SB 1 cells increased with respect to tirne over the penod studied. Since it was approximately linear over the first 5 min of incubation, this time interval was used for al1 subsequent uptake studies. The kinetic propenies of TmGAT-1 were detemined from the dose-dependent uptake of ['Hl GABA.

Ceil infection with recombinant virus resulted in a 20- to 30-fold increase in Na'- dependent ['WGABA uptake, compared to control infections (gus-baculovirus), at GABA concentrations below 20 pM (Fig. 10). Mean K.,,, the substrate concentration at half the maximum uptake velocity (KJ, oFGABA uptake was 9.4 + 0.8 pM (n = 3) with a V,, range 85-103 pmols well-' min" at 48 h post-infection. An Eadie-Hofnee plot (slope, -Km; Figure 8. Southern blot hybridization analysis of T. ni caterpillar genomic DNA. T.rii caterpiliar genomic DNA (20 pgflane) was digested with BamH 1 (l), Hind III (2), Pst I (3), Kpn 1(4), Xho 1 (5) and Xba 1 (6). The digested DNAs were separated on a 1% agarose gel, and the fragments were transferred to Hybond N nylon membrane and hybndired to Dig-Iabeled DNA probe prepared from T. genomic DNA comprising nucleotides 1786-2085 of the TrnGAT-L cDNk The relative sizes of the DNA fiagrnents were estimated from concurrently separated DNA standards @ p). O1234567891011 minutes

Figure 9. Time course of ~~GABAuptake by TrnGAT-1. Cells (seeded at 4 x 10' per well) were incubated in 10 pM GABA for the specified length of time. Total accumulation of GABA in the cells increased tinearly within the first 5 min of incubation. Each value is the mean of three experiments. GAT-1 infected cells

Gus-infected cells

O 20 40 60 80 1O0 [GABA]FM

Figure 10. Transport kinetics of GABA uptake by TrnGAT-1. Uptake of GAi3A over a concentration range of 0.1-99 pM was assessed in SfZI cells infected with a TrnGAT-1- expressing baculovinis (W). Values were corrected for the Iow background levels of endogenous uptake seen in cells infected with control (gicr) baculovirus (O) Inset, Eadie- Hofstee analysis of the Na--dependent component of TrnGAT- l-baculovirus elicited GABA uptake. The mean Kmof GABA uptake was 9.4 k 0.8 pM (n = 3) with a V-range of 85- 103 pmols we1la1min-' at a saline [Na] of 66 mM and [Cl'] of 130 mM. y-intercept, Vnw) of a sample expenment is shown in the inset to Fig. 10. Ail data from

GAT-1 infected Sf21 cells show in Fig. LO have been corrected for the low levels of

GABA uptake by an uncharacterized non-saturating NaYndependent transport system, which typically constituted 20% at higher

[GABA] (Fig. 1O, gtcs-infected cells). [A similar endogenous GAB A transport activity has been reported in vertebrate COS-7 cells transfected with insertless vims. Endogenous transport accounted for 10-20% at [GABA] up to 100 rnM (Guimbal et al., 1995)l.

3.2.2 Ion Dependence

GABA uptake by TmGAT- 1 expressing SEt cells is effectively abolished when

Li-, K-, choline and N-methyl-D-glucarnine (NMG) were substituted for Na* in the incubation saline (Fig. 11). The accumulation of GABA is influenced by extracellular

[Na7 (Fig. 12). Very similar to the previousiy reported (Keynan et at., 1992) and described (Matin and Smith, 1972), a siçmoidal shape of the Na- dependence of GABA uptake was displayed in the ma7 range 0-100 mM. According to these experiments, these sodium concentration dependent curves could exhibit a prominent saturable behaviour soon with increase of Pa7, and the velocity of GABA uptake would not have a significant increase. Consequently, the V,, was tentatively chosen to be the value at 100 mM of ma7. Using Michaelis-Menten equation, the affinity K,of the TmGAT-1 CO- transporter for Na' was determined to be 56 rnM at 10 pM GABA (where V/(VmK-V) =

1, see inset in Fig. 12). The residual accumulation of GABA by the cells in the absence of

Na- is typically less than 5% that seen when Na' is present in the saline and is the result of Figure 11. Effect of Na' and Cr substitution on ~~GABAtransport. SfLl cells expressing TmGAT-1 were assayed in 100 mM sodium-saline (Ctrl, see Materials and Methods section) or modifications of this saline. Removal of Na' abolished the GABA transport function effiectively. Replacement of Cl- with Bidid not reduce GABA uptake while replacement with iodide and nitrate ions reduced GABA transport by 35% and 45% respectively. Gluconate, citrate, aspartate and fluoride ions substituted very weakly for the Cl' ion, infected cells

Gus-infected cells

Figure 12. Na'-dependence of GABA uptake by TrnGAT-1. Cells were exposed to 10 pM GABA and uptake mesured at Na- concentrations in the range O to LOO mM. V,, was determined from the uptake at 100 mM Inset, the dependency of GABA uptake on extemal [Nal is show as a Hill plot obtained with Na' concentrations fkom 10 mM to 80 mM. The stoichiornetry of Na' binding was estirnated from the dope to be 2.4. The affinity sf TmGAT-1 for Na* is 56 mM at the saline [Na7 where (VNmax-V) equals 1. The fit through the data points (mean of three experiments) suggests that at an extemai concentration of 1O GAI34 Na- and GABA bind to TrnGAT- 1 in a 2.4: 1 ratio. the activity of an endogenous low-affinity Na'-independent (and presumably also ClS- independent) transport systern that transports a variety of P-and y-amino acids into SBI cells. The Nae-dependent uptake of GABA by cloned GABA transporter is also affected by extracellular [Cl-] (Fig. 13). The afinity of TrnGAT- l for CI- is 10 rnM at 105 mM Na' and 10 pM GABA (Fig. 13, inset). Substitution of Cl- with other anions gave mixed results (Fig. Il). Bi fully substituted for CI-. Gluconate, citrate, aspartate and fluoride ions substituted very weakly for CI'. Replacement with iodide and nitrate ions resulted in

35% and 15% decrease in GABA transport respectively. TmGAT-1 has an ionic dependence of GABA uptake on Na' and Cl-, which has an apparent stoichiometry of between 2 and 3 Na': 1 Ci-: 1 GABA-The Na' and Cl- stoichiometry values, n = 1.4 and

1.3, respectively, are obtained from the exponents of the Hi11 slopes shown in Figs. 12 and

13 (insets). It shoule be pointed that, a more precise estimate for the affinity K., of Na-- dependence and the stoichiometry of Na-GABA cotransport requires additional experiments with higher for obtaining a better value of the Vn,,..

3.2.3 Inhibition Studies

Seventeen compounds reported to block neurotransmitter transport in mammalian cells were tested for their ability to inhibit [.'H]GABA transport in TmGAT-1-baculovirus infected cells (Table 1). in a prelirninary screen, cells were exposed to 0.1 pM [3~GABA in saline containing 1 mM inhibitor. The ICS0values for the five most active cornpounds

(estimated from the concentration that inhibited 50% of control uptake) were then determined (Fig. 14, 15). None of the inhibitors tested were as potent as the natural [CI'] mM

Figure L3. CLdependence of GABA uptnke by TrnGAT-1. Cells were exposed to 10 pM GABA and 105 mM Na', and the uptake was mesured at Cl- concentrations in the range O to 170 mM. V,, was detemined from the uptake at 50 mM [Cl']. Inset, the dependency of GABA uptake on extemal [Cl'] is shown as a Hill plot obtained with Cl* concentrations fiom 3.4 mM to 34 mM. The stoichiometry of Cl- binding was estimated fiom the slope to be 1.3. The affinity of TrnGAT-1 for Cl- is 10 mM at the saline [Cl'] where V/(V,, equals 1. The fit throuçh the data points (mean of three experirnents) suggests that at an extracellular concentration of 10 pM GABA and 105 mM Na*, Cr and GABA bind to TrnGAT-1 in a 1.3 :1 ratio. Table 1. Inhibition of '[HI-GABA uptake by TrnGAT-1 by GABA and by selected blockers of GABA transport in marnmalian cells

% [.Ml control SD

GABA

GABA amiogrm DABA

Muscimol p GPA

Nipecotic acid

Arecaidine

p-alanine ACHC Betaine Taurine Guvacine Hydroxynipicotic Acid Hypotaunne THIP

Othrr irrhibitors Riluzole P hloretin Verapamil

-4bbreviations : DABA diaminobu~ricacid: bGPA. b-guanÏdinc~proprionÏcacid; ACHC. 1-amino- l-cyclohesanc carboqlic acid; THIP. 4.5.6.7. tetrahydroyisosazole- [5,4-CIpyridin-3-01, 40 O-' 1 -- S KF89976A - 30 GAEA + nipemtlc acid + 2o - pGPA + DAM 10 -- - 0 - [inhibitor] (pM)

Figure 14. Inhibition of TrnGAT-1 rctivity by selected GABA inhibitors. The uptake of 0.1 pM [3HJ~~~~by cells expressing TrnGAT-l was assayed in the presence of twelve different concentrations of each compound. The GABA inhibitors tested were DAI34 P-GPA. nipecotic acid and SKF89976A. The data are the means (ISD) of three experiments for each compound. 1 IO 100 concentration (PM)

Figure 15. Inhibition of TrnGAT-1 activity by the inhibitor riluzole. The uptake of 0.1 ['H]GABA by cells expressing TmGAT-1 was assayed in the presence of twelve dEerent concentrations of riluzole. The data are the means (ISD) of three experirnents. transport substrate GABA in inhibitinç ['HIGABA uptake by TrnGAT-1 (Fig. 14). diamino butyric acid (DABA) blocked TrnGAT-1 activity with an ICIo of approximately

430 @A, PGPA had an ICJoof approximately 800 ph4. Nipecotic acid and SKF89976A had ICso values of approximately 880 pM and 940 pM, respectively. Riluzole was the most effective inhibitor of GABA uptake of the non-competitive inhibitors tested (the other two were phloretin and verapamil).

Overall, the ranking order for inhibition of [3~~~~~uptake (expressed as % control at 1 rnM inhibitor concentration) by the six most potent compounds is: riluzole

(16.7%) > DABA (26.1%) > muscimo1 (3 1.6%) > PGPA (40.4%) >SKF89976A (43.3%)

>nipecotic acid (47.8%). By cornparison, uptake of radioiabeled GABA in the presence of

I mM GABA was only 3% of control uptake (Table 1) , corresponding to a ICJ~of 9 pM. (Fig. 14). Chapter 4 DISCUSSION

Most research to date on GABA uptake by cloned neuronal GABA transponers has focussed on the mamrnalian GAT transport systems, because of their clinical importance in ameliorating a variety of nervous disorders. The study of insect neurotransmitter transporters, however, is driven by the need to find new targets for the control of economically important Pest species. For this reason T'op/zîsia rN, an oligophagous caterpillar commonly known as the cabbage looper, was chosen for this study.

4.1 Structure

The GABA transporter cDNAs constitute a family of related yet distinct GABA transporters, as evidenced by their primary sequence homology but dissimilar pharmacoloçical profiles (Swanson et al., 1994). The deduced arnino acid sequence of the

TmGAT-1 shows high homology to that ofknown proteins within a subset of mammalian

Na'/CI--dependent GABA transporters (Guastella et at., 1990; Nelson et al., 1990; Liu et al., 1993) and 95% identity to an insect GABA transporter (MasGAT) isolated fiom the homworm Mm~dticusexta (Mbunçu t.r al., 1995). Hydropathy analysis of the deduced sequence suggests 12 transmembrane domains, a similar structure to al1 the other GABA transporters sequenced so far (Bennett and Kanner, 1997; Borden et at., 1992, 1994;

Nelson et at-, 1990; Clark et ai., 1992; Rasola et al., 1995). Analysis of the prediaed amino acid sequence of TmGAT-1 indicates that there are two potentid N-glycosylation sites within the putative extracellular loop EL2 and one potential N-glycosylation site

located in the EL3 (Fig. 6), which is consistent with MasGAT, but different from the other

cloned GABA transporters. A11 of the other GABA transporters have three N-

çlycosylation sites within EL2 (Miller et al., 1997), except mouse GAT-2 (Lopez-

Corcuera et al., 1992) and canindhuman BGT-1 (Yamauchi et al., 1992; Rosola et al.,

1995), which have only two sites. The functional role of N-glycosylation bas been

preliminanly exarnined in GAT- 1 (Keynan et al., 1992). Incubation of GAT-1 expressing

cells with tunicaniycin, a specific inhibitor of N-glycosylation, caused a decrease in GABA

transport that was dependent on the time of exposure to the inhibitor.

4.2 Phosphorylation sites

Nmost al1 neurotransmitter transporters studied to date are modulated by protein kinase C activaton and by molecu[es that affect protein phosphorylation, such as phosphatases (Beckman and Quick, 1998). This observation, in addition to the presence of multiple consensus phosphorylation sites on transporter proteins, has lead to the suggestion that these eRects are mediated through the direct phosphorylation of the transporters. Sequence analysis showed that TrnGAT-1 has three putative cytoplasmic protein kinase C (PKC) phosphorylation sites, consistent with other GAT subtypes. In an effort to define the role of phosphorylation in the GABA transport process, some researchers have assessed the effects of phosphorylation modulators (Corey et al., 1994a;

Gomeza et ai., 199L; Tian et al., 1994). Cupello et al. (1993) found that the V,, of

GABA uptake into rat brain synaptosomes is increased 58-74% by the PKC activators phorbol 12-myristate 1 3-acetate (PMA) and oleyl-acetyl glycerol (OAG). Modulation of

GABA transport activity by phorbol esters, a phosphorylation activator, was also reported. However, this phenornenon remains unclear because one group reported stimulation by the phorbol ester (Barbour et al., 1988) and the other group obtained conflicting results (e.g., Gomeza et al., 1991). The observed differences between these studies rnay represent differential regdation expressed by the four GAT subtypes. Funher studies are needed to resolve these issues.

4.3 Tissue distribution

GABA is the major inhibitory neurotransmitter in the nervous system of vertebrates (Borden, 1996). There is strong evidence that GABA is an inhibitory neurotransmitter in insect and the arthropod nervous system (Kerkut et al., 1969; Emson et al-, 1974; Chude et al., 1979; Baxter and Torralba, 1975; Fox and Larsen, 1972; Breer and Heilgenberg, L985) and muscle (Bermudez et al., 1988). Van Marle et al. (1985) reported that a hiçh-affinity uptake system for GABA is present in the common inhibitor

(CI) innervated slow muscles 135*! of the locust Schistocerca gregnrin. GABA is also an important neurotransmitter in the CNS of caterpiilan (Maxwell et al., 1978; Kingan and

Hildebrand, 1985). GABA is found throughout the CNS (brain and ventral nerve cord) of mature lamae of the sphinx moth, hIafxi1ica sextu @Gngan and Hildebrand, 1985).

Consequently, I suspect that GABA may serve as a transmitter in inhibitory intemeurons, and possibIy in motoneurons, in the caterpillar Trichopliisia IN. The tissue expression pattern of TmGAT- 1 was examined by probinç total RNA from a variety of caterpillar tissues (Fig. 7). X single band of about 4.4 kb was visualised in the brain sample, matching

the size of the largest clone isolated from the cDNA library. No bands were detectable in

other tissues. This result indicates that the expression of TrnGAT-1 mRNA is mainly

associated with the nervous system. Since it remains to be shown that GABA serves as a

neuromuscular transmitter in caterpillars, GABA receptors and GABA transporters rnay

be absent from the neuromuscular synapse in caterpillars. Consequently, my inability to

detect an accumulation of TmGAT-L mRNA in caterpillar muscles may be biological (the

low [Na7 in lepidopteran blood rnay have led to the loss of NaLdependent transporters in

certain tissues of these insects; a different GABA transporter may occur at the

neuromuscular junction), or technical (there were not enough nerve endings in the

"epidermis" sample extracted for northem blot analysis).

4.4 Transporter kinetics

The activity of the TmGAT- L was demonstrated using SB 1 cells infected with a

recombinant baculovirus carrying the cDNA coding for TrnGAT- 1. Functional analysis

demonstrated that althouçh TmGAT-1 has kinetic properties and stoichiometry of ionic dependence similar to those of the mammalian neuronal GABA transporter GAT-1

(Guastella et al., 1990), it has a unique pharmacological profile that distinguishes it &om

mammalian GAT-1 and other cloned GAB A transporter subtypes. Michaelis-Menten kinetics showed that GABA transport occurs by a saturable process, suggesu'ng that it is associated with the expression of a GABA transporter. Mean Km of GABA uptake was

cdculated to be 9.4 pM, which is within the range of values reponed for high-affinity GABA uptake systems in the mammalian brain (Miller, 1997). A cornparison with Na-- dependent GABA transporters charactenzed from other insects is shown in Table 2. The kinetic data listed suggest that there may be another lepidopteran GABA transponer, of higher afinity for GABA than the two cloned transponers Tm GAT-L and MasGAT, but this remains to be dernonstrated.

Table 2. ~a+-dependentGABA transporten in the insect CNS

High Low affinity affinity

Species source L (W9 K, (mM) wa41 (mM) cw (mM)

e rnb ryonic 0.44 El741 8.7 [174] neurons1

ganglion 1.7 [150] 19 [iSO] synaptosomes'

Manduca cloned from - Sam embryonic cDNA iibrary3

cloned fiom lamai head cDNA librafy4

Re ferences: 1. Bermudez er QI., 1988 2. Breer and Heilgenberg (1985) 3. Mbungu ei al., (1995) 4. This study. 4.5 Ionic stoichiometry

The functioning of al1 rnarnrnalian high-affinity GABA transporters cloned to date is reported to have an absolute requirement for extracellular Na* (e.g. Corey et al., L994b;

Clark and Amara, 1994) as well as for a monovalent inorganic anion, nomally CI*

(Swanson et ai., 1994; Mabjeesh et al., 1992). TmGAT-1 shows strong fùnctional dependence on extracellular Na- concentration. The activity of TmGAT- 1 also depends on extracellular Cl-. The Cf requirement is reported to be Iess ngid than the strict dependence of the transporter on Na*. Siçnificant uptake activity was observed when CI* is replaced by certain anions. The capacity of the anions to substitute for Cf seems to be related to their ionic radii (Goncalves and Carvalho, 1994). Monovalent anions such as Bi and NO; which have relatively srna11 radii are reponed to partially substitute for Cl- in driving

GABA uptake by mamrnalian GATs. My preliminary findings on the anionic dependence of GABA uptake by TmGAT- 1 suggest that monovalent organic anions such as acetate- and gluconate- substitute very weakly for, and mlilti-valent anions such as SO~"and PO:- not at al1 for, CF in the physiological saline. These results differ €rom those on MasGAT

(Mbungu et al., 1995), in which gluconate totally substituted for Cf resulted in only a

50% inhibition of GABA uptake, and 100% activity is retained by MasGAT when acetate has been substituted for Cf These contrasting data rnay result from the differences in the amino acid sequences between the two GABA subtypes, and these differences rnight be exploited to identify the putative CI- bindinç sites in rhese proteins. The &nity of

TrnGAT-L for Cl- (Km= IO mM at L05 mM Na- and 10 pM GABA) is sirnilar to that of mammalian GAT-1 (Km = 19 mM). The slightly higher Cr a~nityof TmGAT-1 compared to mammalian transporters rnay be a consequence of the lower [Cl'] in lepidopteran blood plasma and penneuriai spaces. The reponed apparent stoichiometries of Na: and CI-- dependent GABA transpons are either 2 Na-: I Cl-: 1 GABA (mammalian GAT-1 and

GATJ) or 3 Na': 2 Cr: L GABA (canine GABAhetaine transporter BGT-1 (GAT-2)).

Consequently, GABA uptake is electrogenic at physiological pH, as the net positive charge crossing the plasma membrane would cause the membrane potential of the ceIl to change. This may be of significance in the design of indicator-dye based non-radiometric screening methods for assessing TmGAT-1 activity.

4.6 Pharmacological characterizrtion

Inhibition of GABA transport represents a novel therapeutic approach to increasing GABAerçic activity (Krogsgaard-Larsen et al., 1987; Borden, 1996; Suzdak,

1993; Kanner, 1997). Compounds targetted at the insect GABA transporter may also represent a novel approach to control insect pests. Diaminobutyric acid @ABA), nipecotic acid, P-guanidinopropnonic acid (P-GPA) were show to be moderately active inhibitors of GABA uptake by TmGAT- 1. However, unlike in mammals, where the cyclic

GABA analogue nipecotic acid is a potent inhibitor of Na*-dependent GABA uptake, these compounds are weak inhibitors of GABA uptake by TmGAT-1. A similar poor inhibition of GABA uptake by nipecotic acid is reported for another lepidopteran GABA transporter, MasGAT (Mbungu et al., L995). The lack of inhibition of GABA uptake by betaine or p-alanine suggests that TmGAT-1 does not transport these substrates, similar to mammalian GAT-1, but unlike rnammdian GAT-2 and GAT-3- These inhibitory compounds do not readily cross the blood-brain bai-rier, a property attributable to their high degree of hydrophilicity. In an effort to overcome this problem, Ali et al. (1985) synthesized a number of compounds that nrongly inhibited

GABA uptake, including tiagabine (NO-328) (Nielsen et al., 1991; Roepstorff and

Lambert, 1992; Anderson et al., 1993; Dhar et al., 1994; Hu and Davies, 1997). Tiagibine has served as a mode1 compound in the synthesis of a new generation of potent lipophilic

GABA uptake inhibitors that readily penetrate into the CNS, such as Cl-966 (Borden et al., 1994), SKF 89976-A (Swinyard et al., 199 1), and NNC-711 (Suzdak et al., 1992).

Although TmGAT- 1 is most similar to rnammalian GABA transporter GAT-1 in kinetic propenies, TmGAT-1 can be distinguished from rnammalian GAT-1 by the lack of inhibition by cyclic GABA analogue ACHC and low inhibition by nipecotic acid. Most striking is the lack of inhibition by the lipophilic nipecotic acid analogue SKF899764, a potent blocker (Ki's in the 0.1 pM range) of high affinity GABA uptake by rnammalian

GAT-1.This suggests that TmGAT-1 has an exceptionally high ability to discrirninate asainst GABA analogues that act as GABA transport substrates in rnammalian nervous tissue. The unique phamacology of TrnGAT-L suggests that GABA transport in the lepidopteran CNS may be a selective target for intervention by insect-specific neuroactive compounds.

With the significant advances in molecuIar and pharmacological characterization and localiation, researchers are now turning their attention toward identifying the aructural components of high-affinity GABA transporters which are essential for function.

Of particular interest are the amino acid residues which are important for subarate specificity and affinity; substrate and ion translocation, transport inhibition; and protein synthesis, targeting, and conformation (Miller et al., 1997). Site-directed mutagenesis studies of mammalian GAT-1 showed that tryptophan residue 222 is strictly involved in substrate binding through the amino group of GABA (Kleinberger-Doron et al., 1994), whereas arginine 69, which is conserved in al1 members of the GABA family, is involved in

Cl- coupling (Pantanowitz et al., 1993, lursky, et al., 1994). Recently, Yu et al. (1998) reported that cysteine 74 in the mammalian GABA transporter GAT-1 is important in Na' binding and pemeation. Amino acid residues in corresponding positions (W234, R60 and

C65) are conserved in TrnGAT- L. My findings suggest that the differentiaf sensitivity of

TmGAT-1 and mammalian GAT-1 to inhibitors such as nipecotic acid and its lipophilic analogues is not caused by amino acid differences at these important substrate-binding domains, altematively, flanking arnino acids may play a role, specifically at the GABA binding domain associated with a less conserved region around W234.

Finally, several compounds unrelated to GABA in structure blocked GABA- uptake in a (apparently) non-competitive rnanner. Of these, riluzole was the most effective. Rilwole (3-amino-6-tnfluoromethoxythiaz01e)is marketed by Rhone-Poulenc Rorer under the trade name ~ilutek' as treatment for the motoneuron disease amyotrophic lateral sclerosis, or ALS. Riluzole also has anti-convulsant propenies and reduces excitatocy amino acid neurotoxkity (Eaevez et ai., 1995;). Its primary mode of action is to block voltage- dependent Na* channels in the inactivated state. One of many neuroprotective drus acting on

Na' channels (Taylor and Meldrurn, 1995), rilwole is funher reported to block glycinergic excitatov postsynaptic potentials (Umemiya and Berger, 1985) and MPTP-induced parkinsonism (Benazzouz et al., 1995). Its use in this study was rnotivated by an (uncited) statement in the Tocris Cookson cataloçue that riluzole blocks GABA uptake into stnatal synaptosomes. This was confirmed here, aithough the effective riluzole concentration used was greater than that reported to aQ on the membrane targets mentioned above.

In conclusion, my findings on the structure and pharrnacology of the Na'- dependent GABA transporter in the caterpillar CNS show that the characteristics of

GABA uptake in the insect CNS differ from those in the rnarnmalian CNS. Consequently, insect-selective blockers of GABA uptake may sewe as future compounds for insect control. REFEREES

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