Functional expression and CNS distribution of a β-alanine-sensitive neuronal GABA transporter

Neuron,

Vol. 9, 337-348, August,

1992, Copyright

0 1992 by Cell Press

Functional Expression and CNS Distribution of a P-AlanineSensitive Neuronal GABA Transporter Janet A. Clark,* Ariel Y. Deutch,*+ Patricia Z. Gallipoli,*+ and Susan G. Amara* *Department of Pharmacology +Department of Psychiatry Yale University School of Medicine New Haven, Connecticut 06510 Wollum Institute Oregon Health Sciences University Portland, Oregon 97201

Summary The synaptic action of y-aminobutyric acid (CABA) is terminated by high affinity, Na+-dependent transport processes in both neurons and glia. We have isolated a novel GABA transporter cDNA, CAT-B, which encodes a high affinity (K, = 2.3 PM), Na+- and Cl--dependent CABA transport protein that is potently blocked by @alanine, a compound generally considered a selective inhibitor of glial transport. However, in situ hybridization studies indicate that CAT-B mRNA is expressed predominantly within neurons. These data indicate that the neuronal-glial distinction of GABA transporters based on inhibitor sensitivities must be reconsidered and suggest a greater diversity of GABA transporters than has been predicted by previous pharmacologic studies. Introduction y-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the mammalian brain, with exceptionally high levels found in the substantia nigra, globus pallidus, and hypothalamus (Fahn and C&+,1968; Perry et al., 1971). Rapid and efficient termination of GABA neurotransmission is achieved, in part, by a high affinity, Na+-dependent, presynaptic GABA transport (reuptake) process that has been studied extensively (Iversen and Neal, 1968; Iversen, 1971; lversen and Johnston, 1971; Gottesfeld and Elliott, 1971). A similar GABA transport process has been identified in glia and is thought to function in signal termination as well as in control of neuronal excitability (Schrier and Thompson, 1974; Schousboe et al., 1977; Wilkin et al., 1983). GABA transport systems have often been classified as being either glial or neuronal on the basis of their pharmacological sensitivities to a few specific GABA uptake inhibitors. Neuronal GABA transport is effectively inhibited by cis-3-aminocyclohexanecarboxylic acid (ACHC) (Boweryet al., 1976) and L-2,4diaminobutyric acid (L-DABA) (Iversen and Kelly, 1975; Larsson et al., 1983). GABA transport processes in both central and peripheral glia (Schon and Kelly, 1974,1975; Gavrilovic et al., 1984) are generally characterized by their ability to transport p-alanine and their sensitivity to inhibition by this GABA analog. This neuron-glia dichotomy

of GABA reuptake mechanisms has proven a useful heuristic in the study of central GABA function. However, a number of discrepancies in the literature suggest that GABA transport exhibits a complexity not explained by the above pharmacological classification. Studies of a variety of glial cell types in primary culture, including rat retinal Miiller cells (Iversen and Kelly, 1975), cerebellar stellate astrocytes (Cummins et al., 1982; Levi et al., 1983), and oligodendrocytes (Reynolds and Herschowitz, 1986), have shown that not all glial GABA transport is sensitive to b-alanine, nor is p-alanine a substrate for all glial GABA transport systems. In fact, GABA transport in rat retinal Miiller cells and cerebellar stellate astrocytes is sensitive to the putative neuronal transport-selective agents ACHC and L-2,Qdiaminobutyric acid (Iversen and Kelly, 1975; Levi et al., 1983). Molecular cloning studies have resulted in the isolation and characterization of cDNAs encoding two transporters, CAT-A and BCT-1, for which GABA is a substrate. CAT-A is an ACHC-sensitive GABA transporter that is found exclusively in the CNS (Guastella et al., 1990), while BGT-1, a carrier for the osmolyte betaine in the kidney (Yamauchi et al., 1992), is absent from the CNS. Molecular characterization of these and other genes for GABA transport activities is essential for understanding the diversity of GABA carriers suggested by previous studies of inhibitor and substrate specificities. Here we report the isolation and characterization of a cDNA clone from rat midbrain encoding a novel high affinity, Na+- and Cl--dependent GABA transport protein, designated CAT-B for its fi-alanine sensitivity. GAT-B shares significant amino acid homology with GAT-A and BGT-1 and with the other members of the Na+-dependent neurotransmitter transporter gene family. The pharmacologic sensitivity of GAT-B transport to b-alanine is similar to that reported for “glial” GABA transporters. Despite this similarity, the mRNA for GAT-B is predominantly localized to neurons within the mammalian brain.

Results Nucleotide of CAT-B

Sequence and Amino Acid Sequence

A GABA transporter cDNA, GAT-B, was isolated from a ratmidbrainlibraryusingasequencesimilarity-based polymerase chain reaction strategy as described in Experimental Procedures. The 2063 base insert of the CAT-B clone (Figure 1) includes a 1897 baseopen reading frame defined by an initial ATG within a region that agrees well with the consensus sequence for translational start sites (Kozak, 1984). The cDNA sequence predicts a protein of 627 aa with a relative molecular mass of 70,000. Sequence similarity and hydropathy profiles (Kyte

Neuron 338

1

GGCGGCAGGGCGGCCATGACTGC~GAGCGCTGCCCC

MT

1

A

E

10~’

Q

A

LPLGNGKAAEEARG

SEALGGG'1

2h

101 29

GGGGCGCGGCGGGGACGCGCGAGGCGCGCGAC~GGCAGTCCACGAGCGCGGTCACTGGAACAACAAGGTGGAGTTCGTGTTGAGCGTAGCGGGAGAGAT L):', G GA A G TR EAR DKA V H E R G H W N N K V ELF ') 2 V L S VA G E,

201 63

CATCGGTCTGGGCAACGTGTGGCGCTTCCCCTACCTGTGCTACAAGkACGGCGGAGGGGCATTCCTGATTCCTTACGTGGTGTTTTTCATCTGCTGTGGA 3 I, 0 8' IGLGNVWRFPYLlCYKNGG[.GAFLIPYVVFFlCC< Y:

301 96

ATCCCCGTCTTCTTCCTGGAAACGGCTCTGGGGCAGTTCACGAGCGAGGGCGGCATCACGTGCTGGAGGAGAGTCTGTCCTTTATTTGAAGGCA'rCGG;I 4 8: /' JPVFFIJETALGQFTSEGGITCWRRVCPLFEGI, 1:s

401 ATGCAACACAGGTGATCGAGGCGCATCTCAATGTCTACTACATCATCATCCTGGCGTGGGCCATCTTCTACTTAAGCAACTGCTTCACCACCGAGC?“ C! 129XATOVIEAHLNVYYIIIL1AWAI FYLSNCFT T E L i' (I t f 501 CTGGGCCACCTGTGGGCATGAGTGG~CACAGAG~~TGTGGAGTTCCAG~GCTG~CTTCAGC~CTACAGTCATGTGTCCCTGCAGAACG~~C~ 163 WA T C G HEWNTEKCVEFQ KLNFSNYS H V S I. ; N A rr

Cl:, i' 1 :. ._ 60. 13'

601 196

TCCCCGGTCATGGAGTTCTGGGAACGCCGGGTCTTGGCTATATCTGATGGCATTGARCACATCGGGAACCTCCGATCGGAGCTGGCACT~;TGTCTC-",~I; : II , T r SPVMEFWERRVLAI IGNLRWEILALCdr SDGIEH 38

701 229

CGGCTTGGACCATCTGCTACTTCTGCATCTGGRAGGGTACGAAGTCAACTGGAAAGGTCGTGTATGTCACTGCAACCTTCCCCTACATCATr,CTSCT;A~ a il L &a A W T I C Y F C I WIK G T K S T G K[YV Y V T A T F P Y I M L L: 262

801 263

CCTCCTGATCCGAGGGGT~A~GTTGCCAGGTGC~TCGG~GGCATC~GTT~TA~~TGTACCCTGACCTCT~CCGGCTCTCTGAT~CACAGGTGTGGGTI‘ LLIRGVTLIPGASEG IKFYLYPDLSR L S D P Q v w xr

90 ! 29:

901 296

GATG~TGGGACGCAGAT~TTTTTCTC~TATGC~ATCTGC~TGGGCTGC~TGACCG~T~TGGGCAGTTA~AACAACTATAA~AA~AACTGCTA~AGGGAC'~ D[AGTOIFFSYAICLGCLTALGSlYNNYNNNCYRt

:oui 328

1001 GTATTATGCTCTGCTGTCTGAACAGTGGCACCAGCTTCGTGGCTGGGTTTGCTATCTTCTCAGTCCTGGGCTTCATGGCGTACGAGCAGGGCGTGCCTA_ 1 1 0 '2 329C[~MLCCLNSGTSFVAGFAIFSVL,lGFMAYEQGVF'l 752 1101 363

TGCTGAGGTGGCAGAAT~AGGTCCTGGACTGGCTTTCATCGCCTACCCC~GGCTGT~A~TATGATGCCCCTGTCC~CATTGTG~GCCACC~~~TT~.~"' 1 A EVAESGPGLAF IAYPKAVT[BMPLSPLWATLEr

;;:

1201 396

ATGATGCTCATCTTC~TGGGCCTGGACAGTCAGTTTGTGTGTGTGGAGAG~~TTGTGACAGCCGTGGTTGACATGTACCCC~GGTCTTCI~~GCGG~~,.? M M L I F L G L,D S Q F" C V E S L V T A V V D MY P K V C R R -

42%

1301 429

ACCGGCGAGAACTGCTCATCCTGGCCCTGTCCATTGTCTCTTATTTCCTAGGCCTGGTGATGCTGACAGAGGGAGGCATGTACATTTTC~~AGCTTTTT!~~ i43ti Y R R E[L L I L A L S I V S Y F L G L V M I,lT E G G M Y I F Q 1, F LI 462

1401 463

CTCATACGCAGCCAGTGGCATGTGCTTGCTCTTCGTGGCCATCTTTGAGTGTGTCTGCATCGGCTGGGTGTATGGAAGTARCAGGTTCTATGACAATATT IiCl! S YAAS[CMCLLFVAIFECVCIGWVYGSlNRFY3N'4qc~

1501 496 1601 529

GAGGACATGATTGGATACCGGCCACTGTCACTCATCRAGTGGTGCTGGAAAGTTGTGACCCCTGGGATCTGTGCGGGCATCTTCATCTTCTTTCTGGTiA ; 5 82,I E DM I G Y R P L SLIK[YCWKVVTPGICAGIFIFFL 52s VI A AGTACARGCCGCTCAAGTACAACRATGTGTACACATATCCTGCTTGGGGCTACGGCATTGGCTGGCTCATGGCTCTGTCCTCCATGCTGTGCATCCCGC? i I:',', ,- IP KY K P L K Y NNV Y T Y PA WG 562 YGUGWLMALSSMLs 2

1701 563

CTGGATCTTCATCAAGCTGTGGAAGACAGAGGGCACCCTGCCCGAGAAATTACAGAAGTTGACAGTCCCCAGCGCTGATCTGAAAATGAGG~GCAAil‘TT 1851'. W I F LIK L W K T E G T L P E K I, Q K LTV P .$ A D L K M K G K L i q 5

1801 596 1901

GGGGCCAGCCCACGGATGGTGACCGTTAATGACTGACTGTGAGGCC~GGTC~GGCGACGGTACCATCTCTGCCATCACAGAG~GGAGAC~~'A~T'rCTGATl?iWl EAKVKGDGTI S A I T E K E T H F i G A S P 62" RMVzVNDC CCCCGCCAGCCACTTGGATGTGTCTCCAGCCTTCCTTCCCTGCGTGTAAATGAATCCTGAACCTTGTATTTCACCTAATGTTAGGGGCTTGCTCGTCTG; LOb'i

2001

ACAGGATTTATTAACAAGTTAACTCCCCAGCCGGCCACTGTG~CCCTGTACCCTCACCTCAC 2062

Figure 1. The cDNA

Sequence

and Deduced

Amino

Acid Sequence

i35'5

for Rat CAT-B

Putative transmembrane domains are bracketed and underlined. Potential N-linked glycosylation sites are designated by an asterisk over the appropriate residues. Residues potentially phosphorylated by Ca”-calmodulin-dependent protein kinase II are denoted by a triangle under the appropriate residue, and the residue potentially phosphorylated by PKC is double underlined.

and Doolittie, 1982) of the predicted protein demonstrate that it shares many structural features with representative members of the Na+-dependent transporter gene family shown in Figure 2 (CAT-A, Guastella et al., 1990; norepinephrine transporter, Pacholczyk et al., 1991; dopamine transporter, Kilty et al., 1991; serotonin transporter, Blakely et al., 1991a). Significant features in the GAT-B sequence include 11 or

12 putative transmembrane regions and a large extracellular loop between transmembrane domains 3 and 4 with three sites for N-linked glycosylation. The lack of a strong candidate for an amino-terminal signal sequence (von Heijne, 1983) suggests that for a protein with I2 membrane-spanning domains, both amino and carboxyl termini of the protein are located in the cytoplasm. Within predicted intracellular portions of

A B-Alanine-Sensitive

Neuronal

GABA Transporter

339

AEQALPLGNGKAAEEARGSELGGGGGGAA..GTREARD..........KA...VH.E TDNS.KVADGQIST~..SEAPVASDKPKTLWKVQK............KAG..DLPD RKVAVPEDGPPWSWLPE.E..GEKLD.......QEGED..........Q....VK.D KSKCSVG..PMSSWAPAKESNAVGPREVELILVKEQNGVQLTNSTLINPPQTPV~Q LARMNPQVQPENNGADTGPEQPLRARKTAELLWKERNGVQ...C.LLAPRDG..DAQ FYRRVSP..PQR....TGQSLAKYPMGTLQSPGTSAGDEASHSIPAATTTLVAEIRQG

GAT-B GAT-A BGT-1 DAT NET SHT-T GAT-B GAT-A BGT-1 DAT NET SHT-T GAT-B GAT-A BGT-1 DAT NET SHT-T

19 78 70

94 90 90

E D E P P G

AICIIAFYIAS VEFQKLNFSNYSHVS...LQNATS . . . . . . . FSNYSLVNTTNM...TS MDFLN.HSGARTATS...SENFTS SDAHASNSSlXLGLNDTFG...TT TDPKLLNGS.VLGNHTKYSKYKFT T.....NYFAQ.DNITWTL.HSTS

175 173 166

189 185 186

266 257 256 282 280 275

GAT-B GAT-A BGT-1 DAT NET SHT-T

362 353 352 378 376 371

GAT-B GAT-A BGT-1 DAT NET 5HT-T

457 446 447 470 468 465

GAT-B GAT-A BGT-1 DAT NET SHT-T GAT-B GAT-A BGT-1 DAT NET SHT-T

F F F F

.. 552 540 542 563 561 558

YATYKFCSL YVIYKFLST

FREKLAYAI LWERLAYGI

ADLKMRGKLGASPRMVTVNDCEAKVKGDGTISAITEKETHF 627 DIVRPENGPEQPQAGSSASKEAYI................ 599 SLP.QPKQHLYLDGGTSQDCGPSPTKEGLI..VGEKETHL614 DHQLVDRGEVRQFTLRHWLLL................... 613 EHHLVAQRDIRQFQLQHWLIA................... 61'7 PTEIPCGDIRMNAV.......................... 60'7

Figure 2. Alignment of the Amino Acid Sequences of Rat CAT-B, CAT-A, Dopamine Canine BGT-1, and Human Norepinephrine Transporter (NET)

Transporter

(DAT), Serotonin

Boxes designate amino acids identical in all transporter sequences or those unique to the three sequences proteins. Transmembrane domains for pCAT-B are bracketed above the appropriate residues.

the carboxyl terminus of CAT-B, potential sites for phosphorylation by Ca *+-calmodulin-dependent protein kinase II and protein kinase C (PKC) (Kemp and Pearson, 1990) are observed (Figure 1). Another feature that GAT-B shares with other members of the neurotransmitter transporter family is a cluster of charged residues in the intracellular loop immediately preceding transmembrane domain IX. Clusters of charged residues are found in a similar location in members of another symporter family, which includes the Na’/glucose and Na’/proline transporters (Hediger et al., 1989), raising the possibility that these residues are involved in a Na’ cotransport process common to all Na+-dependent transporters. Comparison of the amino acid sequence for CAT-B with that of CAT-A shows that these two transporters

Transporter

that encode

(5HT-T),

CABA transport

are 50% identical with respect to amino acid residues, with an overall 61% similarity when conservative amino acid substitutions are considered (Figure 2). The CAT-B protein is most similar to another member of this transporter family, the Na’-dependent betaine carrier from canine kidney (Yamauchi et al., 1992), which transports GABA as well as betaine, with relatively lower affinities (K, = 93 t.rM and 398 PM, respectively; Yamauchi et al., 1992) than are generally observed for amino acid neurotransmitter carriers in brain. Alignment of GAT-B with BGT-1 shows a striking degree of conservation between the two sequences, exhibiting 63% identity, increasing to 72% with conservativeaminoacid substitutions. As indicated in Figure 2, 50 residues unique to the three GABA transporter sequences are interspersed throughout the

teins, including receptors, ion channels, porters, or facilitated carriers.

other

sym-

pCAT-B Encodes a P-Alanine-Sensitive GABA Transporter

Time (mins)

GABA

Figure 3. Characterization port in HeLa cells

concentration

@MI

of pCAT-B-Expressed

[3H]CABA Trans-

(A) Time course and Na+ and Cl- dependence of I’H]GABA transport by pCAT-B. (B) Saturation analyses of [3H]CABA transport. Inset: EadieHofstee plot of initial velocity data. Data for these experiments are representative of that obtained in at least three separate experiments, and each point is the mean of triplicate determinations with an SEM 6 10%.

and include a number of uncharged polar residues, which may be important for recognition of common substrates and inhibitors. In addition to residues uniquely conserved in the transporters that use GABA as a substrate, approximately one-fourth of the amino acid residues are identical in all members of the family (Figure 2). The greatest homology is found within the transmembrane domains, suggesting that these regions play an important role in functions common to all Na+-dependent neurotransmitter transporters. A search of the GenBank database showed that CAT-B has no extended regions of sequence similarity to other prosequence

Transient expression of CAT-B in HeLa cells resulted in a significant increase in cellular [3H]CABA accumulation above the background levels of cells transfected with vector alone (Figure 3A). Isotonic substitution of LiCl for NaCl in the assay buffer resulted in a decrease in GABA uptake below that seen with vector alone, revealing the presence of a low level, endogenous, Na+-dependent GABA transport in these cells. Reduction of Cl- concentration in the assay buffer by replacement of NaCl with sodium glucuronate decreased GABA transport to background levels. Therefore, the GABA transport resulting from pGAT-B expression is both Na+ and Cl- dependent, as has been shown for CAT-A (Cuastella et al., 1990). Transport of GABA by CAT-B is saturable, with an apparent MichaeIis constant (K,) of 2.3 P M (Figure 3B). No transport above background was detected when [jH]L-glutamate (100 nM), [“HID-aspartate (100 nM), [3H]L-glycine (100 nM), or [3H]fl-alanine (up to 500 P M with cold (3-alanine) was substituted for GABA in the uptake assay (data not shown). The differential sensitivity of CAT-B or CAT-A transport to a variety of agents was examined in HeLa cells expressing these proteins. The results of these studies, presented in Figure 4 and Table 1, indicate that these two transport processes have very different pharmacological profiles. Basedon itsabilityto inhibit GABA transport potently and to serve as a substrate for transport in gliaof rat sensory ganglia and cerebral cortical slices (Schon and Kelly, 1975), B-alanine has been assumed to be a glial-selective agent. Although p-alanine does not appear to be transported by CAT-B, it is a potent and selective inhibitor of CAT-B-mediated GABA transport. b-Alanine was three orders of magnitude more potent at inhibiting GABA transport by CAT-B than by CAT-A (K, = 6.7 P M and 2 mM, respectively). Nipecotic acid, a nonselective competitor for CABA transport, as well as guvacine and cis-4 hydroxynipecotic acid, compounds reported to be slightly more effective at competing GABA transport in astrocytes than in neurons (Krogsgaard-Larsen et al., 1987), was all an order of magnitude more potent at inhibiting CAT-A transport than CAT-B transport (Figure 4; Table 1). THPO (4,5,6,7-tetrahydro-isoxazolo[4,5-cl-pyridin-3-01) showed similar K, values for the inhibition of CAT-B and CAT-A (K, = 233 P M and 378 PM, respectively). Taurine has also been associated with GABA transport in both neurons and glia, although it is not clear whether taurine is transported by the same reuptake system (Kaczmarek and Davison, 1972; Schrier and Thompson, 1974; Martin and Shain, 1979; Borg et al., 1980; Holopainen and Kontro, 1986). Taurine was a weak competitor for GABA transport by CAT-B, but did not inhibit CAT-A transport. ACHC and L-2,4-diaminobutyric acid, both consid-

A P-Alanine-Sensitive 341

Neuronal

CABA Transporter

Table 1. Inhibitor Transfected with

Sensitivity of GABA Uptake in HeLa Cells pGAT-B or pGAT-A Transporter cDNA K CM)

Inhibitor

pCAT-B

b-Alanine Guvacine Nipecotic acid Hydroxynipecotic L-DABA THPO NO-711 Taurine ACHC

6.7 21.9 53.3 76.7 109 233 234 459 813

acid

+ + f + f f f * +

pCAT-A 1.5 6.2 14.4 15 28 37 44 219 177

2030 + 1060 3.2 * 1.9 6.6 + 4.8 8.8 f 2.9 28 f 21 378 f 104 0.0058 f 0.0032 >10,000 15.4 + 3.7

K, values are presented as means + standard errors of the means and represent the average of at least three separate experiments. Values were calculated from IC, determinations: K, = (IC&l

(Cheng

Concentration (M) Figure 4. Pharmacological Characterization A-Expressed PHIGABA Transport

of CAT-B- and GAT-

Sensitivity of PHJGABA uptake by cells expressing CAT-B (upper panel) or CAT-A (lower panel) to various transport inhibitors. Curves are representative of data obtained from three separate experiments; each point is the mean of triplicatedeterminations.

ered selective competitors of neuronal GABA transport (Bowery et al., 1976; Larsson et al., 1983; lversen and Kelly, 1975; Krogsgaard-Larsen et al., 1987), were more potent at inhibiting CABA transport by CAT-A than by CAT-B (Ki = 15.4 PM and 28 PM versus 813 PM and 109 PM, respectively). NO-711, a highly potent GABA uptake inhibitor with anticonvulsant properties (Suzdak et al., 1992), showed the most marked selectivity between the two transporters: GAT-Amediated transport was nearly five orders of magnitude more sensitive to inhibition by NO-711 than was CAT-B. Expression of CAT-B mRNA in Rat CNS Northern blot analyses performed using a polymerase chain reaction fragment corresponding to CAT-B as a specific probe demonstrated a 4.7 kb mRNA species that is expressed in tissues in the nervous system (Figure 5). In situ hybridization studies demonstrated that CAT-B mRNA is heterogeneously distributed in brain (Figure 6) and is expressed predominantly in neurons (Figure 7). Examination of emulsion-coated sections revealed the presence of silver grains over neurons (Figures 7A and 7B); there did not appear to be glial labeling. To confirm the neuronal expression of

+ S) where

and Prusoff,

S = (concentration of radioligand) (K, of radioligand)

1973).

CAT-B mRNA, ibotenic acid lesions were made by infusing the excitotoxin into the mesencephalon. Such lesions result in severe neuronal loss with a concomitant reactive gliosis (KGhler and Schwartz, 1983); thus, if the transporter is expressed in glia one should observe high signal over the lesion, whereas neuronal localization would be reflected by the absence of specific hybridization. Five days following the lesion animals were perfused and brains sectioned for in situ hybridization analyses. These lesions resulted in very dense glial accumulation in the area of the injection sites, as can be seen in the Nissl-stained section (Figure 7C), and the virtual absence of neurons; no CAT-B expression was seen in the injection locus, supporting a neuronal localization for GAT-B mRNA in these regions (Figure 7D). In situ hybridization revealed several areas of high

-

4.7kb

* Figure 5. CAT-B mRNA Is Expressed by Northern Blot Analysis

in the CNS as Determined

Poly(A)’ RNA (1 kg per lane) was blotted as described in Experimental Procedures and probed with KW13. C6 glioma, rat glial tumor cell line; PC12, rat adrenal pheochromocytoma cell line; SKNSH, human neuroblastoma cell line: ‘1’79, human retinoblastoma cell line.

Figure 6. Direct

Reverse

Prints

Showing

Localization

of CAT-B mRNA

by In Situ Hybridization

Histochemistry

The mediobasal aspects of the forebrain and many mid- and hindbrain regions express CAT-B. In contrast, CAT-B expression is absent in the striatum and very low in most cortical regions and in the cerebellum. Abbreviations: AON, anterior olfactory nucleus; BST, bed nucleus of the stria terminalis; CER, cerebellum; CC, central gray; CIN, cingulate cortex; CMA, corticomedial amygdala; DCN, deep cerebellar nuclei; DBB, diagonal band of Broca; DHP, dentate gyrus of hippocampus; DMN, dorsomedial hypothalamic nucleus; ENT, entorhinal cortex; IC, inferior colliculus; IO, inferior olivary nucleus; IPN, interpeduncular nucleus; LHB, lateral habenula; LSN, lateral septal nucleus; MB, mammillary body; MSN, medial septal nucleus; NRP, nucleus reticularis paragigantocellularis; POA, preoptic area; PFC, prefrontal cortex; PHP, pyramidal cell layer of hippocampus; PN, pontine nuclei; PVN, thalamic paraventricular nucleus; PYR, pyriform cortex; SC, superior colliculus; SN, substantia nigra; VN, vestibular nuclei; VP, ventral pallidum; VPL, ventroposterolateral thalamus; ZI, zona incerta.

A l3-Alanine-Sensitive 343

Figure 7. Localization

Neuronal

GABA Transporter

of CAT-B

mRNA to Neurons

Silver grains were present over individual neurons in the brain (arrows in [A] nucleus interpositusand [B] nucleus in [B] indicates an unlabeled neuron). A large unilateral ibotenic acid lesion in the mesencephalon resulted in of neurons and very dense gliosis (X in [C] and [D]) in the substantia nigra (SN) and overlying mesencephalic more medial ventral tegmental area (VTA) and the mammilary body (MB) were intact on both the lesioned and was the myelinated medial lemniscus (ml). The loss of specific hybridization in the gliotic zone (D) indicates that expressed in neurons.

CAT-B expression. Prominent areas of labeling were seen in mediobasal structures in the forebrain, contrasting with the much weaker expression of CAT-B in the cortex and the virtual absence of expression in the neostriatum. CAT-B mRNA was extensively distributed in the brain stem, but absent in the cerebellum (see Figure 6). In the telencephalon, cortical CAT-B expression was relativelyweak and was most prominent in thetemporoparietal regions (where a bilaminar appearance of CAT-B mRNA could be detected) and in the pyriform and entorhinal cortices. No neurons expressing CAT-B were seen in the neostriatum, and therewasveryweak expression in the ventral striatum (nucleus accumbens-olfactory tubercle). GAT-B expression was very high along the entire rostrocaudal extent of the medial septum-diagonal band complex; in contrast, there was weaker labeling of neurons in the lateral septum. Relatively high CAT-B expression was seen in the ventral pallidum and extended into the medial and lateral preoptic regions. GAT-6 expression was moderate in the bed nucleus of the stria terminalis. In the amygdala, CAT-B labeling was essentially restricted to the anterior cortical and medial amygdaloid nuclei. GAT-6 mRNA was expressed throughout the thalamus and epithalamus. Thus, a high density of CAT-B transcripts was seen in the lateral habenula and medially adjacent paraventricular thalamic nucleus. Moderate labeling was observed in the ventroposteromedial and ventroposterolateral nuclei of the thalamus. In the posterior thalamus, moderate labeling was present in the medial geniculate nucleus. The zona incerta was strongly labeled. More ventrally, CAT-B was expressed in a variety of hypothalamic nuclei. The anterior, dorsomedial, ventromedial, arcuate, and lateral hypothalamic nuclei all expressed GAT-B mRNA. Hippocampal labeling was relatively low; CAT-B mRNA

cuneiformts; arrowhead a virtually complete loss reticular formation; the contralateral (E) sides, as GAT-B is predominantly

was expressed in neurons of both the dentate gyrus and pyramidal cell layer. In the brain stem, CAT-B labeling was seen in a large number of structures in moderate to high density. In the mesencephalon, labeling was present in neurons of the superior colliculus, central gray, and substantia nigra; in the latter structure, CAT-B mRNA was expressed both in the pars reticulata and pars compacta, with the dopamine-rich pars compacta being more densely labeled in the rostra1 substantia nigra. CAT-B expression was very high along the entire rostrocaudal extent of the interpeduncular nucleus. In the pons, GAT-B was strongly expressed in the pontine nuclei, with moderate to high degree of expression in the paralemniscal area, central gray, cuneiform nucleus, and dorsal nucleus of the lateral lemniscus. The dorsal pontinetegmentum(particularlythedorsaltegmental nucleus) was strongly labeled, as was the parabrachial region, the trapezoid area, and the superior and inferior olivary nuclei. The vestibular nuclei were prominently labeled. The absence of GAT-B mRNA in neurons of the cerebellar cortex and vermis was in striking contrast to the more ventral brain stem sites. However, the deep cerebellar nuclei were strongly labeled. Discussion The predominant mechanism of terminating GABAergic neurotransmission in the CNS appears to be the Na+-dependent reuptake of GABA into presynaptic terminals and adjacent glia (Iversen and Kelly, 1975; Balcar et al., 1979). Glial transport activities may have the additional important role of limiting the diffusion of GABA into adjacent synapses present on GABAresponsive neurons widely distributed throughout the CNS. Because disrupted GABAergic neurotransmission has been implicated in anumberof neurolog-

ical and psychiatric disorders, including epilepsy (Meldrum, 1975; Ribak et al., 1979; Lijscher and Schwartz-Porsche, 1986) and schizophrenia (Reynolds et al., 1990; Benes et al., 1991,1992), it is important to acquire a detailed understanding of GABA transporters both as candidate etiologic sites of these disorders and as potential sites for therapeutic intervention. Previous studies classifying GABA reuptake systems solely on cellular or biochemical fractionation and pharmacological sensitivities have proven inconsistent. However, the molecular cloning of two high affinity neuronal GABA transporters-an ACHC-sensitive carrier, CAT-A (Guastella et al., 1990), and a p-alanine-sensitive carrier, CAT-B, reported hereshould allow the development of a new classification based on the identification of specific transporter gene products. Amino acid sequence comparisons reveal that GATB, like GAT-A, is a member of the recently described Na+-dependent neurotransmitter transporter gene family. Although these two nervous system-specific carriers have similar affinities for GABA, the overall similarity of their amino acid sequences is not significantly greater than that seen in other individual members of the family. In fact, CAT-B is most similar to the renal betaine transporter, which transports GABA with low affinity, although GABA is unlikely to be an endogenous substrate for this carrier. In contrast, the two catecholamine transporters (norepinephrine transporter and dopamine transporter) share a significantly greater number of common amino acids relative to other family members. The alignment of neurotransmitter transporter sequences reveals 50 aa that are uniquely conserved in the members capable of GABA transport. These residues are scattered throughout the sequences and include a number of uncharged polar amino acids that could interact with substrates or inhibitors through hydrogen bonding. The sensitivity of GABA transport to thiol reagents such as N-ethylmaleimide suggests that substrate interactions may also involve cysteine residues (Krogsgaard-Larsen, 1980) and points to several conserved cysteines as potentially important. A significant degree of homology exists in the transmembrane domains of the entire family of Na’dependent neurotransmitter transporter proteins, with considerable stretches of identical amino acids, while much less homology is apparent in the intracellular and extracellular loops connecting these domains. A notable region of divergence is found in the large, glycosylated extracellular loop that connects transmembrane regions 3 and 4 in each of the transporter sequences. Since this loop is unique for each transporter, this region may contribute to substrate and/or inhibitor specificities. Finally, GAT-B contains proline residues in 6 of its 12 hydrophobic domains; the majority of these residues are conserved in all members of the neurotransmitter transporter family. Proline residues have been found in the transmem-

brane domains of many transport proteins and are presumed to facilitate the conformational changes necessary for transport of substrates across a lipid bilayer (Brand1 and Deber, 1986). Proline residues also introduce bends in membrane-spanning regions and generate domains that traverse the membrane multiple times before exiting the bilayer, as has been suggested for the chicken erythroid anion transporter, band 3 (Cox and Lazarides, 1988). Thus, transmembranetopographycan differ significantly from predictions based solely upon hydropathy analyses (jennings, 1989). Mutational and chimeric analyses using cloned cDNAs can now be used to test formally structural predictions and to examine the roles that various regions play in the transport process. A variety of potential phosphorylation sites have been found in all the neurotransmitter transporter sequences reported thus far. GAT-B has potential phosphorylation sites for both Cal+-calmodulindependent protein kinase II and PKC. Studies using cultured primary astrocytes have suggested that activation of various second messenger systems is capable of acutely regulating GABA transport (Rhoads et al., 1984; Bouhaddi et al., 1988; Hansson and Riinnback, 1989,199l; Gomeza et al., 1991). The potent PKC activator phorbol 12-myristate 13-acetate decreases GABA transport in astrocytes; this can be prevented by the addition of the PKC inhibitor H7 (Gomeza et al., 1991). Such modulatory effects on astrocytic GABA transport, as well as the presence of potential phosphorylation sites in GAT-B and GAT-A, implythat intracellular signaling mechanisms and protein kinases may regulate GABA transport processes in neurons as well. Comparison of the regional distribution ot expression of GAT-A and GAT-B mRNAs using Northern blot analyses suggests that there are striking divergences between the patterns of expression of the two GABA transporters (Figure 5; Xia et al., 1991, Sot. Neurosci., abstract). For example GAT-A mRNA is very abundant in striatum and cerebellar cortex, areas where GAT-B mRNA is virtually absent. Similarly, CAT-A mRNA levels are high in cortex and hippocampus where there are only low levels of GAT-B. Some areas such as midbrain contain significant levels of both species. In situ hybridization studies demonstrate that GAT-B mRNA is distributed predominantly in neurons. It is conceivable that GAT-B transcripts are present in very low abundance in certain types of glia, but at the present time there is no indication of glial expression of this GABA transporter. The distribution of GAT-B is in many cases consistent with the localization of well-characterized populations of GABAergic cell bodies. For example, GABAergic neurons are present in high density in the medial septum-diagonal band complex and in the ventral pallidum (Onteniente et al., 1986), sites in which GAT-B mRNA is very high. There are similar convergences of areas containing GAT-B and glutamic acid decarboxylase-like

A P-Alanine-Sensitive 345

Neuronal

GABA Transporter

immunoreactivity (see Mugnaini and Oertel, 1985). However, there are also dissimilarities between the distribution of CAT-B and GABA- or glutamic acid decarboxylase-like immunoreactive neurons, such as the cerebellum (Aoki et al., 1989). A more detailed comparison of the regional distribution of CAT-A and GAT-B is difficult because CAT-B-positive cell bodies have been identified by in situ hybridization, while reports on the localization of GABA transport proteins to date have been limited to immunohistochemistry using antibodies generated against purified brain GABA transporter (Radian et al., 1990). However, preliminary GAT-A in situ hybridization studies (N. Brecha, personal communication) along with our data suggest that the pattern of expression of these two GABAtransporters isoften complementary. For example, in the cerebellum CAT-A is expressed in cerebellar Purkinje cells while CAT-B is found in neurons of the deep nuclei. It is not yet clear whether the distribution of CAT-A- and CAT-B-positive neurons accounts for all known GABAergic neurons or whether there could be additional neuronal GABA transporters. In addition, it is possible that GABA transport may occur in non-GABAergic neurons (Bonnano and Raiteri, 1987). The characterization of GABA transport processes as either glial or neuronal has typically been based on the ability of a few selective agents to inhibit transport. However, these pharmacologic distinctions were largely based on studies of uptake in cortical tissue or in specialized glial populations, which would have excluded most CAT-B activity. The identification of a novel GABA transport activity encoded by CAT-B provides evidence for a greater heterogeneity of CNS GABA transport systems than hitherto realized and suggests that additional transporters must exist to account for glial GABA transport activities. There are clear pharmacological distinctions between GAT-B transport and glial GABA transport. Although p-alanine is a potent inhibitor of GABA uptake by GAT-B, it is not transported by the carrier. This finding distinguishes GAT-B from glial GABA transport because b-alanine transport and inhibition of GABA transport by fi-alanine have usually been associated with the same transport process in both brain slices and cultured astrocytes (Iversen and Kelly, 1975; Schon and Kelly, 1975). Further characterization of the inhibitor sensitivities of GAT-B transport with THPO, guvacine, and hydroxynipecotic acid, all relatively weak but selective inhibitors of glial GABA transport (Krogsgaard-Larsen et al., 1987), shows that GAT-B transport is even less sensitive to these agents than GAT-A transport, again suggesting that GAT-B has a unique pharmacolgic profile. It is clear from these data that GABA transporters should not be classified as glial or neuronal based solelyon their pharmacological characteristics. Other investigators have alluded to such difficulties in the classification of GABA transport processes in studies using primary cultures of glia, which exhibit B-ala-

nine-insensitive but ACHC- and L-2,4diaminobutyrlc acid-sensitive GABA transport (Iversen and Kelly, 1975; Levi et al., 1983; Reynolds and Herschowitz, 1986). These findings have led at least one group to conclude that (3-alanine transport should not be considered aglial transport marker (Cumminset al., 1982). The use of molecular biological techniques will enable the study of individual transporters in isolated systems and define how these proteins act in concert to regulate the extracellular levels of GABA. It is anticipated that future studies will reveal additional novel members of the GABA transporter family. The specific distributions of GAT-B and CAT-A in the CNS suggest that the development and use of highly selective transport agents may provide a means to manipulate extracellular GABA concentrations in discrete brain regions. Experimental Procedures Cloning of CAT-B CAT-B was cloned using standard molecular biology techniques (Sambrook et al., 1989). Degenerate oligonucleotide primers, corresponding to transmembrane domains II and VI of norepinephrine transporter and CAT-A, were generated with the following sequences: sense, CCGCTCGAGAAGAACGC(C~)GG(C/ T)GG(CIl)GC(CIT)lTC(C/T)T(G/A)AT(C/T)CC(A/G)TA and antisense, CCTCTAGAAA(G/A)AACATCTG(G/A)GT(G/T)GC(G/A)GC (G/A)TC (G/A/C)A(G/T)CCA (Yale Medical School Protein and Nucleic Acid Facility; New Haven, CT). Oligonucleotide primersand rat midbrain cDNA were used in polymerase chain reactions performed with Taq polymerase (Promega) for 25 cycles at 94OC for 1 min, 45OC for2 min, 72OC for 3 min, with the final extension lengthened to 12 min. KW13, a 681 bp fragment, was isolated from the polymerase chain reaction products, labeled with [‘LP]dCTP (Amersham) using a random primer kit (Boehringer Mannheim), and used to screen a random primed rat midbrain cDNA library constructed in the vector Bluescript SKII (pBSSKII(-)) (Stratagene), as previously described (Pacholczyk et al., 1991). The cDNA library was derived from a single fraction of sizefractionated poly(A)’ RNA that was previously shown to have both GABA and L-glutamate transport aciivity when injected into Xenopus oocytes (Blakely et al., 1991b). Nitrocellulose filter lifts were made from ten plates of -lOOO--5000 individual clones each, resulting in a total library size of 30,000 clones. Filter lifts were processed following standard protocols (Sambrook et al., 1989). Screening was performed in hybridization buffer (50% formamide, 6x SSPE, 0.05x BLOTTO) at 42°C. Between 5 x 1Or and 1 x IO6 cpm of 32P-labeled KW13 was added per ml of hybridization buffer, and filters were incubated until 1 x C,,tI,z was achieved. Filters were washed four times for 5 min in 2x SSPE/ 0.1% SDS at 25OC, air dried, and exposed to XAR-5 film (Kodak) for 16 hr. Eight positive clones were isolated from this screen, 5 of which proved to be unique by restriction enzyme mapping and sequencing of both the 5’and 3’ends of each clone. Positive clones were confirmed by Southern blot analysis using KW13 labeled with [‘*P]dCTP as described above. A single clone was found to encode a potential start site with no stop codon downstream and a stretch of bases with homology to the Kozak consensus sequence (Kozak, 1984) preceding an in-frame methionine. Restriction digest analyses of this clone, 4C4a-1, and a second clone, 2C4-lc-2, revealed that they share a unique Pstl site that is located within an 889 bp overlap in the KW13 region of these 2clones.AfterdigestingwithPst1, the3’endof4C4a-1 wasligated to the 5’end of 2c4-lc-5, resulting in the c:onstruction of p&mid GAT-B (pGAT-B) with a complete open reading frame as deduced by functional expression studies.

NfXl,O” 346

DNA Sequencing and Analysis Sequencingwas performed bydideoxy chain termination, using the Sequenase 2.0 kit (United States Biochemical Corporation) on a set of overlappingexonuclease Ill-digested EcoRI, Sal1 unidirectional deletions (Erase-a-Base; Promega). Double-stranded templates were sequenced with the vector primers T7 and 73. Sequence of the antisense strand was compiled from sequencing both partial clones 4C4a-1 and 2C4lc-2. To confirm this sequence, pGAT-B was excised from the Xhol site of pBSSKII(-), religated in the opposite orientation, and sequenced as described above. A region of poor resolution on the 5’ end of the sense strand was unambiguously resolved on the other strand. MacVector DNA analysis software (IBI) was used for sequence assembly and analysis. Transport Assay Intestine407cells, human embryonic intestinecells that possess the four HeLa markers as well as the A subtype of giucose-hphosphate dehydrogenase (Int 407; ATCC) (1.5-2.0 x IO1 per well), were infected with recombinant vaccinia virus strain VTF-7 encoding a T7 RNA polymerase (Fuerst et al., 1986). Infection was followed 30 min later by liposome-mediated transfection with pGAT-B or pGAT-A (also referred to as pGAT-1, Guastella et al., 1990, isolated as described previously, Blakely et al., 1991~) diluted in pESSKII(-) (100 ng of pGAT-A or pCAT-B + 900 ng of pBSSKII(-)) (Lipofectin; BRL) (see Blakely et al., 1991~). Control transfections were done with equivalent amounts of vector alone. Assays were performed 8 hr following transfection in a modified Krebs-Ringer-HEPES buffer (Blakelyet al., 1991~). Cells were incubated with [‘HIGABA (15-30 nM) (Du Pont/NEN;Amersham) and the CABA transaminase inhibitoraminooxyacetic acid (100 PM), with or without cold GABA transport inhibitors, for 20 min (pGAT-B) or 30 min (pCAT-A) at 37’C. Uptake was stopped by placing the cells on ice and washing twice with 1 ml of ice-cold assaybuffer.Cellsweresolubilized inl% SDS,and theamountof radioactivity accumulated was determined by liquid scintillation counting, Na’ and Cl- dependence were determined by isotonic substitutionof lithiumchlorideand sodiumglucuronate, respectively, for sodium chloride in the assay buffer. Background levels of CABA transport were determined using control transfections with pBSSKII(-) for each assay, and the values obtained were subtracted from thesignalsdetermined for pCAT-A and pGAT-B. Northern Analysis The KW13 cDNA was labeled with [‘lP]dCTP using random primersynthesisandusedasaprobetoexaminethetissuedistribution of CAT-B mRNA by Northern blot analysis. Poly(A)’ RNA was isolated from various rat brain regions using the Mini RiboSep (Collaborative Research, Inc.) and Fast Track kits (Invitrogen) and from cell lines using standard protocols (Sambrook et al., 1989). Brain dissections were performed as previously described (Blakely et al., 1991b; Deutch et al., 1985). Approximately 1 pg of poly(A)’ RNA was separated on a 1% formaldehyde agarose gel (Ogden and Adams, 1987) and vacuum transferred (LKB) to a nylon membrane (Zeta-probe, Bio-Rad). Hybridization was carried out at 42OC in 50% formamide (Sambrook et al., 1989). Blots were washed twice at room temperature for 20 min each in 2x SSPE/O.l% SDS, followed by an hour wash at 65OC in 0.2x SSPEi 0.1% SDS. In Situ Hybridization Histochemistry Serial coronal sections through the rat neuraxis were cut at 16 brn and thaw mounted onto subbed slides. Sections were fixed by immersion in 4% paraformaldehydel0.1 M sodium phosphate buffer (pH 7.4) for 10 min and then washed twice in phosphate buffer. Sections were then acetylated in 0.25% acetic anhydridei 0.25% triethanolamine (pH 8.0) for 10 min and subsequently dehydrated and delipidated, then rehydrated and dried at room temperature. Sections were prehybridized for 2 hr at 37’C. The prehybridization buffer consisted of 50% formamide, 10 m M Tris (pH 8.0). 1 m M EDTA, 0.1 M dithiothreitol, 0.3 M NaCI, Ix Denhardt’s solution, and 10% dextran sulfate. Sections were then spotted

with theabove hybridization solution containing ‘3s-endlabeled antisense or sense 45-mer oligonucleotides (antisense: CATTGAGATCCGCCTCGATCACCTGTCTTGCATACCCGATCCGATGCCTT, tense: AAGGCATCCCCTATGCAACACAGGTGATCGAGGCGCATCTCAAT G) at 500,000 cpm per 50 ~1, coverslipped with parafilm, and hybridized overnight at 37OC. The slides were washed fourtimesforl5minin2x SSC/50%formamideat40°C,followt:d by two times for 30 min in 1 x SSC at 42OC; sections were then dipped briefly in water followed by 70% ethanol and allowed to air dry before beingapposed to Hyperfilm (Amersham): the film\ were exposed for 7 weeks. Separate sets of slides were dipped in 50% Kodak NTB-2 emulsion, placed in light-tight boxes, and exposed for 7 weeks prior to development. To determine whether CAT-B mRNA is expressed In neuron\ or glia, the midbrain of male Sprague-Dawley rats (CAMM) was infused with ibotenic acid (7.5 pg in 1.0 pl of 0.1 M phosphatebuffered saline [pH 7.41, delivered over 10 min) to lesion neurons and increase glial cells. Five days later, animals were pertused with 4% paraformaldehyde, and sections were prepared for localization of CAT-B mRNA; adjacent sections were stained with neutral red. Acknowledgments Wewish tothankM. Lernerforhisgeneroussupportandencouragement, W. Clark for his valued suggesttons and critical review of the manuscript, and R. Blakely and T. Pacholczyk for their helpful discussions. ACHC was generously provided by Research Biochemicals Incorporated as part of the Chemical Synthesis Program of the National Institute of Mental Health, contract 278-90-0007 (BS). J. A. C. is a recipient of an Advanced Predoctoral Fellowship from the Pharmaceutical Manutacturer’s Association Foundation. This work was supported by the Howard Hughes Medical Institute (S. G. A.) and MH-14092 and the National Parkinson Foundation (A. Y. D.). 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 USC Section 1734 solely to indicate this fact. Received

March

11, 1992; revised

June 10, 1992

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